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NZ705184B2 - Yeast-based therapeutic for chronic hepatitis b infection - Google Patents
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NZ705184B2 - Yeast-based therapeutic for chronic hepatitis b infection - Google Patents

Yeast-based therapeutic for chronic hepatitis b infection Download PDF

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Publication number
NZ705184B2
NZ705184B2 NZ705184A NZ70518412A NZ705184B2 NZ 705184 B2 NZ705184 B2 NZ 705184B2 NZ 705184 A NZ705184 A NZ 705184A NZ 70518412 A NZ70518412 A NZ 70518412A NZ 705184 B2 NZ705184 B2 NZ 705184B2
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New Zealand
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seq
positions
hbv
amino acid
acid sequence
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NZ705184A
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NZ705184A (en
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David Apelian
Claire Coeshott
Zhimin Guo
Thomas H King
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Globeimmune Inc
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Publication of NZ705184B2 publication Critical patent/NZ705184B2/en

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Abstract

Disclosed is an immunotherapeutic composition comprising: a) a yeast vehicle; and b) a fusion protein comprising HBV antigens, wherein the HBV antigens consist of: i) an HBV surface antigen having an amino acid sequence that is at least 90% identical to positions 1 to 399 of SEQ ID NO:118, positions 1 to 399 SEQ ID NO:116, positions 1 to 399 SEQ ID NO: 114 or positions 1 to 399 SEQ ID NO:112; and ii) an HBV core antigen having an amino acid sequence that is at least 90% identical to positions 400 to 581 of SEQ ID NO:118, positions 400 to 581 SEQ ID NO:116, positions 400 to 581 SEQ ID NO: 114 or positions 400 to 581 SEQ ID NO:112; wherein the composition elicits an HBV-specific immune response, wherein the fusion protein is expressed by the yeast vehicle, and wherein the sequences are as defined in the complete specification. itions 1 to 399 SEQ ID NO:116, positions 1 to 399 SEQ ID NO: 114 or positions 1 to 399 SEQ ID NO:112; and ii) an HBV core antigen having an amino acid sequence that is at least 90% identical to positions 400 to 581 of SEQ ID NO:118, positions 400 to 581 SEQ ID NO:116, positions 400 to 581 SEQ ID NO: 114 or positions 400 to 581 SEQ ID NO:112; wherein the composition elicits an HBV-specific immune response, wherein the fusion protein is expressed by the yeast vehicle, and wherein the sequences are as defined in the complete specification.

Description

YEAST-BASED THERAPEUTIC FOR CHRONIC TIS B INFECTION CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority under 35 U.S.C. § 119(e) from each of US ional Application No. 61/442,204, filed February 12, 2011, US.
Provisional Application No. 61/496,945, filed June 14, 2011, and US. ional ation No. 61/507,361, filed July 13, 2011. The entire disclosure of each of US.
Provisional Application No. 61/442,204, US. ional Application No. 61/496,945, and US. Provisional Application No. 61/507,361 is incorporated herein by reference.
REFERENCE TO A SEQUENCE LISTING This application contains a Sequence Listing submitted electronically as a text file by EFS-Web. The text file, named "3923PCT_ST25", has a size in bytes of 476 KB, and was recorded on February 7, 2012. The information ned in the text file is incorporated herein by reference in its entirety pursuant to 37 CFR § 1.52(e)(5).
FIELD OF THE INVENTION The present invention generally relates to imrnunotherapeutic compositions and methods for preventing and/or treating hepatitis B virus (HBV) infection.
BACKGROUND OF THE INVENTION Hepatitis B virus (HBV) is a member of the hepadnavirus family and is a causative agent of acute and chronic hepatitis worldwide. HBV epidemics have been prevalent in Asia and Afiica, and HBV infection is endemic in China ams, R. (2006), "Global challenges in liver disease", Hepatology (Baltimore, Md.) 44 (3): 521—526). More than 2 billion people have been infected with the virus, and it is estimated that there are 350 million chronically HBV-infected individuals worldwide ("Hepatitis B", World Health Organization, 2009; "FAQ About Hepatitis B", Stanford School of Medicine. 200810). Routes of infection are through blood and bodily fluid t, including blood transfusions and IV drug use, sexual transmission, bites and lesions, and al transmission (e. g., childbirth).
HBV is found as one of four major pes (adr, adw, ayr, ayw) that are determined based on antigenic epitopes within its pe proteins. There are eight ent genotypes (A-H) based on the nucleotide sequence variations in the genome.
Genotype differences impact disease severity, disease course and likelihood of complications, response to treatment and possibly response to vaccination (Kramvis et al., , Vaccine 23 (19): 2409—2423; Magnius and Norder, (1995), Intervirology 38 (1-2): 24—34).
The clinical incubation period for HBV is usually 2-3 months; approximately two thirds of those acutely infected are asymptomatic or have mild, subclinical symptoms.
The remaining one third of acutely infected duals may experience jaundice, inflammation of the liver, vomiting, aches and/or mild fever, but the disease is eventually resolved in most adults and rarely leads to liver failure. Indeed, approximately 95% of adults r completely from HBV infection and do not become chronically infected.
However, approximately 90% of s and 25%—50% of children aged 1—5 years will remain chronically infected with HBV rs for Disease Control and Prevention as of September 2010). imately 25% of those who become chronically infected during childhood and 15% of those who become chronically infected after childhood die prematurely from cirrhosis or hepatocellular carcinoma, and the majority of chronically infected individuals remain asymptomatic until onset of cirrhosis or end—stage liver disease (CDC as of September 2010). 1 million deaths per year ide (about 2000-4000 deaths per year in the US.) result from chronic HBV infection. Chronically infected individuals have elevated serum alanine aminotransferase (ALT) levels (a marker of liver damage), liver inflammation and/or fibrosis upon liver biopsy. For those patients who develop cirrhosis, the 5 year survival rate is about 50%.
HBV infection and its treatment are typically monitored by the detection of viral antigens and/or antibodies t the antigens. Upon infection with HBV, the first detectable antigen is the hepatitis B surface antigen (HBsAg), followed by the hepatitis B "e" antigen (HBeAg). Clearance of the virus is ted by the appearance of IgG antibodies in the serum against HBsAg and/or against the core antigen (HBcAg), also known as seroconversion. Numerous studies indicate that viral replication, the level of viremia and progression to the chronic state in HBV-infected individuals are influenced directly and indirectly by HBV-specific ar immunity mediated by CD4+ helper (TH) and CD8+ cytotoxic T lymphocytes . ts progressing to chronic disease tend to have absent, weaker, or narrowly focused HBV-specific T cell responses as compared to patients who clear acute infection. See, e.g., i, 1997, J Clin Invest 99: 1472— 1477; Maini et al., 1999, enterology 117:1386-1396; Rehermann et al., 2005, Nat Rev Immunol 2005; 229; Thimme et al., 2001, J Virol 75: 3984-3987; Urbani et al., 2002, J Virol 76: 12423-12434; Wieland and Chisari, 2005, J Virol 79: 9369-9380; Webster et al., 2000, Hepatology 32:1117-1124; Penna et al., 1996, J Clin Invest 98: 1185- 1194; Sprengers et al., 2006, JHepatoZ 2006; 45: 182-189.
Vaccines for the prevention of HBV have been commercially available since the early 1980’s. Current commercial es are fectious, subunit viral vaccines providing purified recombinant hepatitis B virus surface n (HBsAg), and can be administered beginning at birth. The vaccines have been effective at reducing the incidence of infection in countries where the e is ely administered. While a few immunotherapeutics are in development, ing various HBV protein or epitope vaccines and cytokines, there are currently no approved immunotherapeutics for the treatment of active HBV infection in the United States.
Current rd of care (SOC) therapy for HBV infection includes primarily antiviral drugs, such as tenofovir (VIREAD®), lamivudine (EPIVIR®), adefovir (HEPSERA®), telbivudine (TYZEKA®) and entecavir (BARACLUDE®), as well as eron-0t2a and pegylated interferon-0L2a (PEGASYS®). These drugs, and particularly the ral drugs, are typically administered for long periods of time (e.g., daily or weekly for one to five years or ), and although they slow or stop viral replication, they typically do not provide a complete "cure" or eradication of the virus. Interferon— based approaches are toxic and have modest remission rates. The antiviral therapies inhibit viral replication and are better tolerated than interferon, but as ned above, these drugs typically do not provide a complete viral cure, and in some cases long term remission rates are not achieved. Moreover, in some cases, development of drug resistance ensues. For example, lamivudine is a potent oral antiviral that inhibits HBV reverse transcriptase (Pol). As lamivudine is well tolerated, and because it is now a generic drug, lamivudine is an option for HBV antiviral therapy in developing countries.
However, a 20% annual viral resistance rate from point mutations in the P01 sequence limits the y of lamivudine for HBV. Moreover, response to current anti-viral and interferon treatment is differently effective among HBV genotypes (Cao, World Journal of Gastroenterology 2009;15(46):5761—9) and in some patients, because the hepatitis B virus DNA can persist in the body even after infection clears, reactivation of the virus can occur over time.
Accordingly, while rd of care (SOC) therapy provides the best currently approved treatment for patients suffering from chronic HBV, the length of time for therapy and the significant adverse effects of the regimens can lead to pliance, dose reduction, and treatment tinuation, combined with viral escape, reactivation of 304153833 TIS510927NZPR the virus, and patients who still fail to respond or sustain response to therapy. Therefore, there remains a need in the art for ed therapeutic treatments for HBV infection. [0010A] It is an object of the ion to provide an immunotherapeutic composition as described herein, or at least to provide the public with a useful choice.
SUMMARY OF THE INVENTION One embodiment of the invention relates to an therapeutic composition for the treatment and/or prevention of hepatitis B virus (HBV) infection and/or a symptom of HBV infection. The immunotherapeutic composition comprises: (a) a yeast vehicle; and (b) one or more HBV antigens. In one aspect, the HBV antigens are provided as one or more fusion proteins, although single protein HBV antigens may also be provided. The HBV antigens consist of: (i) an HBV surface n comprising at least one immunogenic domain of a full-length HBV large (L), medium (M) and/or small (S) surface antigen; (ii) an HBV polymerase antigen comprising at least one immunogenic domain of a full-length HBV polymerase or domain f (e.g., a reverse riptase (RT) domain); (iii) an HBV core antigen or HBV gen comprising at least one immunogenic domain of a full-length HBV core n and/or a full-length HBV eantigen , respectively; and/or (iv) an HBV X antigen comprising at least one genic domain of a full-length HBV X antigen. The ition elicits an HBV-specific immune response against one or more HBV antigens in the composition and/or against one or more antigens in a hepatitis B virus that has infected, or may infect, an individual. [011A] In a particular embodiment, the invention provides an immunotherapeutic composition comprising: a) a yeast vehicle; and b) a fusion protein comprising HBV antigens, wherein the HBV antigens consist of: i) an HBV surface antigen having an amino acid sequence that is at least 90% identical to positions 1 to 399 of SEQ ID NO:118, positions 1 to 399 SEQ ID NO:116, positions 1 to 399 SEQ ID NO: 114 or positions 1 to 399 SEQ ID NO:112; and ii) an HBV core antigen having an amino acid sequence that is at least 90% identical to positions 400 to 581 of SEQ ID NO:118, positions 400 to 581 SEQ ID NO:116, ons 400 to 581 SEQ ID NO: 114 or ons 400 to 581 SEQ ID NO:112; 304153833 TIS510927NZPR wherein the composition s an HBV-specific immune response and wherein the fusion protein is expressed by the yeast vehicle. [011B] In another embodiment, the invention es a yeast expressing a fusion protein comprising HBV antigens, wherein the HBV antigens consist of: a) an HBV surface antigen having an amino acid sequence that is at least 90% identical to positions 1 to 399 of SEQ ID NO:118, positions 1 to 399 of SEQ ID NO:116, positions 1 to 399 of SEQ ID NO: 114 or positions 1 to 399 of SEQ ID NO:112; and b) an HBV core antigen having an amino acid sequence that is at least 90% identical to positions 400 to 581 of SEQ ID NO:118, positions 400 to 581 of SEQ ID , positions 400 to 581 of SEQ ID NO: 114 or positions 400 to 581 of SEQ ID NO:112. [011C] In another embodiment, the invention provides use of: a) a yeast vehicle comprising a fusion protein; b) the fusion protein comprising HBV antigens, wherein the HBV antigens consist of: i) an HBV surface antigen having an amino acid ce that is at least 90% identical to positions 1 to 399 of SEQ ID NO:118, positions 1 to 399 of SEQ ID , positions 1 to 399 of SEQ ID NO: 114 or positions 1 to 399 of SEQ ID NO:112; and ii) an HBV core n having an amino acid sequence that is at least 90% identical to positions 400 to 581 of SEQ ID , ons 400 to 581 of SEQ ID NO:116, positions 400 to 581 of SEQ ID NO: 114 or positions 400 to 581 of SEQ ID NO:112, in the manufacture of an immunotherapeutic ition for the treatment of hepatitis B virus (HBV) infection or at least one symptom resulting from HBV infection in a subject. [011D] In another embodiment, the invention provides use of: a) a yeast comprising a fusion protein; b) the fusion protein comprising an amino acid sequence that is at least 90% cal to SEQ ID NO:118, SEQ ID NO:116, SEQ ID NO: 114 or SEQ ID NO:112, in the manufacture of an immunotherapeutic composition for the treatment of hepatitis B virus (HBV) infection in a subject. [011E] In another embodiment, the ion provides use of: a) a whole, heat-inactivated yeast from Saccharomyces cerevisiae comprising a fusion protein; b) the fusion protein expressed by the yeast, and sing SEQ ID NO:118, SEQ ID NO:116, SEQ ID NO: 114 or SEQ ID NO:112, 304153833 TIS510927NZPR in the manufacture of an immunotherapeutic composition for the treatment of chronic hepatitis B virus (HBV) infection in a subject. [011F] In another embodiment, the invention provides use of: a) a yeast vehicle comprising a fusion protein b) the fusion protein sing HBV ns, wherein the HBV antigens consist of: i) an HBV surface antigen having an amino acid sequence that is at least 90% identical to positions 1 to 399 of SEQ ID NO:118, positions 1 to 399 of SEQ ID NO:116, positions 1 to 399 of SEQ ID NO: 114 or positions 1 to 399 of SEQ ID NO:112; and ii) an HBV core antigen having an amino acid sequence that is at least 90% identical to positions 400 to 581 of SEQ ID NO:118, positions 400 to 581 of SEQ ID NO:116, positions 400 to 581 of SEQ ID NO: 114 or positions 400 to 581 of SEQ ID NO:112, in the manufacture of an immunotherapeutic composition for the treatment of hepatitis B virus (HBV) infection in a subject, n the immunotherapeutic composition is ated for administration with an anti-viral nd. [011G] In another embodiment, the invention provides use of: a) a yeast vehicle comprising a fusion protein b) the fusion n comprising HBV antigens, wherein the HBV antigens consist of: i) an HBV surface antigen having an amino acid sequence that is at least 90% identical to ons 1 to 399 of SEQ ID NO:118, positions 1 to 399 of SEQ ID NO:116, positions 1 to 399 of SEQ ID NO: 114 or positions 1 to 399 of SEQ ID ; and ii) an HBV core antigen having an amino acid sequence that is at least 90% identical to ons 400 to 581 of SEQ ID NO:118, positions 400 to 581 of SEQ ID NO:116, positions 400 to 581 of SEQ ID NO: 114 or positions 400 to 581 of SEQ ID NO:112, in the cture of an therapeutic composition for the treatment of hepatitis B virus (HBV) ion in a subject receiving anti-viral treatment. [011H] In another embodiment, the invention provides use of: a) a yeast comprising a fusion protein b) the fusion protein comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:118, SEQ ID NO:116, SEQ ID NO: 114 or SEQ ID NO:112, in the manufacture of an immunotherapeutic composition for the treatment of hepatitis B virus (HBV) infection in a subject wherein the composition is formulated for administration to a subject with an anti-viral compound.
TIS510927NZPR [011I] In another ment, the invention provides use of: a) a yeast comprising a fusion protein b) the fusion protein comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: SEQ ID NO:118, SEQ ID NO:116, SEQ ID NO: 114 or SEQ ID NO:112, in the manufacture of an immunotherapeutic composition for treating hepatitis B virus (HBV) infection in a subject receiving anti-viral treatment. [011J] In another embodiment, the invention provides use of: a) a whole, heat-inactivated yeast from Saccharomyces cerevisiae sing a fusion protein b) the fusion protein expressed by the yeast, and comprising SEQ ID NO:118, SEQ ID NO:116, SEQ ID NO: 114 or SEQ ID NO:112, in the manufacture of an immunotherapeutic composition for treating chronic hepatitis B virus (HBV) infection in a subject receiving anti-viral treatment. [011K] In another embodiment, the invention provides use of: a) a whole, heat-inactivated yeast from Saccharomyces cerevisiae comprising a fusion protein b) the fusion protein expressed by the yeast, and comprising SEQ ID , SEQ ID NO:116, SEQ ID NO: 114 or SEQ ID NO:112, in the manufacture of an immunotherapeutic composition for treating chronic hepatitis B virus (HBV) ion in a subject wherein the composition is formulated for administration with an anti-viral compound.
In any of the embodiments of the invention described herein, including any embodiment related to an immunotherapeutic ition, HBV n, fusion protein or use of such composition, HBV antigen or fusion n, in one aspect, the amino acid sequence of the HBV large surface antigen (L) can include, but is not limited to, an amino acid sequence ented by SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO:23, SEQ DI NO:27 or SEQ ID NO:31, or a corresponding sequence from another HBV strain/isolate. The amino acid sequence of HBV polymerase can include, but is not limited to, an amino acid sequence represented by SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:26 or SEQ ID NO:30, a domain of these ces, such as the e transcriptase (RT) domain, or a corresponding sequence from another HBV /isolate. The amino acid sequence of HBV precore protein, which includes both HBV core protein sequence and HBV gen sequence, can include, but is not limited 304153833 TIS510927NZPR to, an amino acid sequence represented by SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21, SEQ ID NO:25, or SEQ ID NO:29, or a corresponding sequence from r HBV strain/isolate. The amino acid sequence of an W0 2012/109404 HBV X antigen can include, but is not limited to, an amino acid sequence represented by SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:l6, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:28, or SEQ ID NO:32, or a corresponding sequence from another HBV strain/isolate.
In any of the embodiments of the invention described herein, including any embodiment d to an immunotherapeutic composition, HBV n, filsion protein or use of such composition, HBV antigen or fusion protein, in one aspect, an amino acid of an HBV surface antigen useful as an HBV antigen or in a fiJsion protein or an immunotherapeutic composition ofthe invention can include, but is not limited to, SEQ ID NO:3, SEQ ID N027, SEQ ID NO:ll, positions 21—47 of SEQ ID NO2ll, positions 176— 400 of SEQ ID NO:ll, SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO:23, SEQ ID NO:27, SEQ ID N023l, positions 9-407 of SEQ ID NO:34, positions 6-257 of SEQ ID NO:36, ons 6-257 of SEQ ID NO:4l, positions 92-343 of SEQ ID NO:92, positions 90-488 of SEQ ID NO:93, SEQ ID NO:97, ons 90-338 of SEQ ID , positions 7-254 of SEQ ID N02102, positions 1-249 of SEQ ID , positions 1-249 of SEQ ID NO:lO8, positions 1-249 of SEQ ID NO:109, positions 1-249 of SEQ ID NO:llO, positions 1-399 of SEQ ID NO2ll2, positions 1-399 of SEQ ID N021 14, or positions 1— 399 of SEQ ID NO:1 16, positions 1—399 of SEQ ID NO:1 18, ons 1-399 of SEQ ID N02120, positions 1-399 of SEQ ID NO:122, positions 1—399 of SEQ ID N02124, positions 1-399 of SEQ ID NO:126, positions 231-629 of SEQ ID NO:128, ons 63- 461 of SEQ ID NO:l30, positions 289-687 of SEQ ID NO:l32, positions 289-687 of SEQ ID NO: 134, or a corresponding sequence from a ent HBV strain.
In any of the embodiments of the invention described herein, including any embodiment related to an immunotherapeutic composition, HBV antigen, fusion protein or use of such composition, HBV antigen or fusion protein, in one aspect, an amino acid of an HBV polymerase antigen useful as an HBV antigen or in a fusion protein or an immunotherapeutic composition of the invention can include, but is not limited to, positions 383-602 of SEQ ID NO:2, positions 0 of SEQ ID N026, positions 381- 600 of SEQ ID NO:IO, positions 453 to 680 of SEQ ID NO:lO, positions 370-589 of SEQ ID NO:l4, positions 380-599 of SEQ ID NO:l8, positions 381-600 of SEQ ID NO:22, positions 380-599 of SEQ ID NO:26, positions 381—600 of SEQ ID NO:30, ons 260 to 604 of SEQ ID NO:36, positions 7-351 of SEQ ID NO:38, positions 7—351 of SEQ ID NO:40, 260 to 604 of SEQ ID NO:4l, positions 346 to 690 of SEQ ID NO:92, positions 90-434 of SEQ ID NO:94, SEQ ID NO:98, positions 339 to 566 of SEQ ID NO:lOl, positions 255 to 482 of SEQ ID NO:102, positions 250—477 of SEQ ID , ons 250-477 of SEQ ID NO:108, positions 250-477 of SEQ ID NO:109, positions 250-477 of SEQ ID NO:110, positions 582 to 809 of SEQ ID NO:120, positions 582 to 809 of SEQ ID NO:124, positions 642 to 869 of SEQ ID NO:126, positions 1 to 228 of SEQ ID NO:128, positions 1 to 228 of SEQ ID NO:132, positions 61 to 288 of SEQ ID NO:134, or a corresponding sequence from a different HBV strain.
In any of the ments of the invention described herein, including any embodiment related to an immunotherapeutic composition, HBV antigen, fusion protein or use of such composition, HBV antigen or fusion protein, in one aspect, an amino acid of an HBV core antigen useful as an HBV antigen or in a fusion protein or an immunotherapeutic composition of the ion can e, but is not limited to, positions 31—212 of SEQ ID NO:1, positions 31—212 of SEQ ID NO:5, positions 31—212 of SEQ ID NO:9, positions 37 to 188 of SEQ ID NO:9, positions 31-212 of SEQ ID NO:13, positions 31-212 of SEQ ID NO:17, positions 31-212 of SEQ ID NO:21, positions 14-194 of SEQ ID NO:25, positions 31-212 of SEQ ID NO:29, positions 408—589 of SEQ ID NO:34, positions 605 to 786 of SEQ ID NO:36, positions 352-533 of SEQ ID NO:38, positions 160-341 of SEQ ID N0239, positions 605-786 of SEQ ID NO:41, ons 691— 872 of SEQ ID NO:92, positions 90-271 of SEQ ID NO:95, SEQ ID NO:99, positions 567 to 718 of SEQ ID , positions 483 to 634 of SEQ ID NO:102, positions 2—183 of SEQ ID , positions 184-395 of SEQ ID NO:105, positions 396-578 of SEQ ID NO:105, positions 579—761 of SEQ ID NO:105, positions 2-183 of SEQ ID NO:106, 338- 520 of SEQ ID NO:106, positions 478-629 of SEQ ID NO:107, positions 478-629 of SEQ ID NO:108, positions 478-629 of SEQ ID NO: 109, positions 478-629 of SEQ ID NO:110, positions 400-581 of SEQ ID NO:112, positions 400—581 of SEQ ID NO:114, positions 1 of SEQ ID NO:116, ons 1 of SEQ ID NO:118, positions 400 to 581 of SEQ ID NO:120, ons 400 to 581 of SEQ ID NO:122, positions 400 to 581 of SEQ ID NO:124, positions 400 to 581 of SEQ ID NO:126, positions 630 to 811 of SEQ ID NO:128, positions 462 to 643 of SEQ ID NO:130, positions 688 to 869 of SEQ ID NO:132, ons 688 to 869 of SEQ ID NO:134, or a corresponding sequence from a different HBV .
In any of the embodiments of the invention described herein, including any embodiment related to an immunotherapeutic composition, HBV antigen, fiasion protein or use of such composition, HBV antigen or fusion protein, in one aspect, an amino acid of an HBV X n useful as an HBV antigen or in a fusion protein or an therapeutic composition of the invention can include, but is not limited to, SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, positions 2 to 154 of SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID N0232, positions 52—68 followed by positions 84-126 of SEQ ID NO:4, positions 52-68 followed by positions 84- 126 of SEQ ID NO:8, ons 52-68 followed by positions 84-126 of SEQ ID NO:12, positions 52-68 followed by positions 84-126 of SEQ ID NO:16, positions 52-68 followed by positions 84-126 of SEQ ID NO:20, positions 52-68 followed by positions 84-126 of SEQ ID NO:24, positions 52—68 followed by ons 84—126 of SEQ ID NO:28, positions 52-68 followed by positions 84-126 of SEQ ID NO:32, ons 787 to 939 of SEQ ID N0236, positions 7-159 of SEQ ID NO:39, positions 873-1025 of SEQ ID NO:92, ons 90-242 of SEQ ID NO:96, SEQ ID NO:100, ons 719-778 of SEQ ID NOlel, positions 635-694 of SEQ ID NO:102, positions 184-337 of SEQ ID NO:106, positions 521-674 of SEQ ID NO:106, positions 630-689 of SEQ ID , positions 630-689 of SEQ ID NO:108, positions 630-689 of SEQ ID NO:109, positions 630—689 of SEQ ID NO:110, positions 582-641 of SEQ ID NO:122, positions 810-869 of SEQ ID NO:124, positions 582-641 of SEQ ID , positions l-60 of SEQ ID NO:130, positions 229 to 288 of SEQ ID NO:132, positions 1 to 60 of SEQ ID NO:134, or a corresponding ce from a different HBV strain.
In one embodiment, the present invention includes an immunotherapeutic composition comprising: (a) a yeast vehicle; and (b) a fusion protein comprising HBV antigens, wherein the HBV ns t of: (i) an HBV X antigen comprising at least one immunogenic domain of a full-length HBV X antigen; (ii) an HBV surface antigen comprising at least one immunogenic domain of a full-length HBV large surface antigen (L), and; (iii) an HBV core antigen comprising at least one immunogenic domain of a full— length HBV core protein. In one aspect of this embodiment, the immunotherapeutic composition comprises: (a) a yeast vehicle; and (b) a fusion protein comprising HBV ns, wherein the HBV antigens consist of: (i) an HBV X antigen having an amino acid sequence that is at least 80% identical to ons 52 to 126 of a full-length HBV X antigen; (ii) an HBV surface antigen having an amino acid sequence that is at least 95% identical to an amino acid sequence of a full-length HBV large surface antigen (L), and; (iii) an HBV core antigen having an amino acid sequence that is at least 95% identical to an amino acid sequence of a full-length HBV core protein. The composition elicits an HBV-specific immune response.
In one aspect of this embodiment of the invention, the amino acid sequence of HBV X antigen is at least 95% identical, or at least 96% identical, or at least 97% cal, or at least 98% cal, or at least 99% identical, or is identical, to an amino acid ce selected from: positions 1-60 of SEQ ID NO:130, positions 630-689 of SEQ ID NO:110, ons 582-641 of SEQ ID NO:122, ons 630-689 of SEQ ID NO:107, positions 630-689 of SEQ ID NO:108, positions 630-689 of SEQ ID , positions 52-68 followed by positions 84-126 of SEQ ID NO:4, positions 52-68 followed by ons 84-126 of SEQ ID NO:8, positions 52-68 ed by positions 84-126 of SEQ ID NO:12, positions 52-68 followed by positions 84-126 of SEQ ID NO:16, positions 52- 68 followed by positions 84-126 of SEQ ID NO:20, positions 52—68 followed by positions 84-126 of SEQ ID NO:24, positions 52-68 followed by positions 84-126 of SEQ ID NO:28, positions 52-68 followed by positions 84-126 of SEQ ID NO:32, SEQ ID NO:100, positions 719—778 of SEQ ID NO:101, positions 4 of SEQ ID NO:102, positions 810-869 of SEQ ID NO:124, positions 582—641 of SEQ ID NO:126, positions 229 to 288 of SEQ ID NO:132, positions 1 to 60 of SEQ ID , or a corresponding sequence from a different HBV strain. In one aspect, the amino acid sequence of HBV X antigen is selected from: positions 1-60 of SEQ ID NO:130, positions 630-689 of SEQ ID NO:110, positions 582-641 of SEQ ID NO:122, ons 630-689 of SEQ ID NO:109, positions 630-689 of SEQ ID NO:108, positions 630-689 of SEQ ID NO:107, SEQ ID NO:100, or a corresponding sequence from a different HBV strain.
In one aspect of this embodiment of the invention, the amino acid sequence of the HBV surface antigen is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% cal, or is identical, to an amino acid sequence ed from: positions 63-461 of SEQ ID NO:130, positions 1-399 of SEQ ID NO:118, positions 1-399 of SEQ ID NO:122, positions 9-407 of SEQ ID N034, positions 1-399 of SEQ ID NO:112, positions 1-399 of SEQ ID NO:114, positions 1-399 of SEQ ID NO:116, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO:23, SEQ ID NO:27, SEQ ID NO:31, positions 90-488 of SEQ ID NO:93, positions 1-399 of SEQ ID NO:120, positions 1-399 of SEQ ID NO:124, positions 1-399 of SEQ ID NO:126, positions 231-629 of SEQ ID NO:128, positions 7 of SEQ ID NO:132, positions 289-687 of SEQ ID NO:134, or a corresponding sequence from a different HBV strain. In one , the amino acid sequence of the HBV surface antigen is selected from: positions 63-461 of SEQ ID NO:130, positions 1-399 of SEQ ID NO:118, positions 1-399 of SEQ ID NO:122, positions 9-407 of SEQ ID NO:34, positions 1—399 of SEQ ID NO:112, ons 1-399 of SEQ ID , positions 1-399 of SEQ ID NO:116, or a corresponding sequence from a different HBV strain.
In one aspect of this embodiment of the invention, the amino acid sequence of the HBV core antigen is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% cal, or is identical, to an amino acid sequence selected from: positions 462 to 643 of SEQ ID NO:130, positions 400-581 of SEQ ID NO:118, positions 400 to 581 of SEQ ID NO:122, positions 408-589 of SEQ ID NO:34, positions 400-581 of SEQ ID NO:112, ons 1 of SEQ ID NO:114, positions 400-581 of SEQ ID NO:116, positions 31-212 of SEQ ID NO:1, positions 31- 212 of SEQ ID N025, positions 31-212 of SEQ ID NO:9, ons 31-212 of SEQ ID NO:13, positions 31-212 of SEQ ID NO:17, ons 31-212 of SEQ ID NO:21, positions 14-194 of SEQ ID NO:25, positions 31-212 of SEQ ID NO:29, ons 605 to 786 of SEQ ID NO:36, positions 352-533 of SEQ ID NO:38, positions 160-341 of SEQ ID NO:39, positions 605—786 of SEQ ID NO:41, positions 691-872 of SEQ ID NO:92, positions 90-271 of SEQ ID NO:95, positions 2-183 of SEQ ID NO:105, positions 184- 395 of SEQ ID NO:105, positions 396-578 of SEQ ID NO:105, ons 579-761 of SEQ ID NO:105, positions 2-183 of SEQ ID NO:106, 338-520 of SEQ ID NO:106, positions 400 to 581 of SEQ ID NO:120, positions 400 to 581 of SEQ ID NO:124, positions 400 to 581 of SEQ ID NO:126, positions 630 to 811 of SEQ ID , positions 688 to 869 of SEQ ID NO:132, positions 688 to 869 of SEQ ID , or a corresponding sequence from a different HBV strain. In one aspect, the amino acid sequence of the HBV core antigen is ed from: positions 462 to 643 of SEQ ID NO:130, positions 1 of SEQ ID NO:118, positions 400 to 581 of SEQ ID NO:122, positions 408-589 of SEQ ID NO:34, positions 400-581 of SEQ ID , positions 400-581 of SEQ ID NO:112, positions 400-581 of SEQ ID NO:114, or a corresponding sequence from a different HBV strain.
In one aspect of this embodiment of the invention, the HBV antigens are arranged in the following order, from N- to C-terminus, in the fusion protein: HBV X antigen, HBV surface antigen, HBV core antigen. In one aspect of this embodiment of the invention, the HBV antigens are arranged in the following order, from N— to C-terminus, in the fusion protein: HBV surface antigen, HBV core antigen, HBV X antigen.
In one aspect of this embodiment of the invention, the fusion protein comprises an amino acid sequence that is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, or is identical, to an amino acid sequence selected from SEQ ID NO:130, SEQ ID NO:122, or SEQ ID Yet another embodiment of the invention relates to an immunotherapeutic composition comprising: (a) a whole, heat-inactivated yeast from Saccharomyces siae; and (b) an HBV fusion protein expressed by the yeast, wherein the fusion protein comprises SEQ ID NO:130.
Another embodiment of the invention relates to an therapeutic composition sing: (a) a whole, heat-inactivated yeast from Saccharomyces cerevisiae; and (b) an HBV fusion protein sed by the yeast, n the fusion protein comprises SEQ ID NO:150.
Yet another embodiment of the invention relates to an immunotherapeutic composition comprising: (a) a whole, nactivated yeast from Saccharomyces cerevisiae; and (b) an HBV fusion protein expressed by the yeast, wherein the fusion protein comprises SEQ ID NO:122. In one aspect, the fusion protein is a single polypeptide with the following sequences fused in frame from N- to C-terminus: (1) an amino acid sequence of SEQ ID N037; (2) a two amino acid linker peptide of threonine- serine; (3) an amino acid sequence of SEQ ID NO:122; and (4) a stidine peptide.
In another embodiment of the invention, the immunotherapeutic composition includes: (a) a yeast vehicle; and (b) a fusion protein comprising HBV antigens consisting of: (i) at least one immunogenic domain of HBV large surface antigen (L) and (ii) at least one immunogenic domain of HBV core protein or HBV e-antigen. The composition elicits an HBV-specific immune response, such as an immune response against HBV large e antigen (L) and/or HBV core n or HBV e-antigen.
In one embodiment, the present invention includes an immunotherapeutic composition comprising: (a) a yeast e; and (b) a fusion n comprising HBV antigens consisting of: (i) an HBV surface n having an amino acid sequence that is at least 95% identical to an amino acid sequence of a full-length HBV large surface antigen (L), and; (ii) an HBV core antigen having an amino acid sequence that is at least 95% identical to an amino acid sequence of a full-length HBV core protein. The composition elicits an HBV-specific immune response. In one aspect of this embodiment, the HBV antigens consist of an amino acid sequence comprising at least 95% of a full— length HBV large surface antigen (L) fused to an amino acid sequence comprising at least 95% of a full-length HBV core protein or HBV e-antigen. In one aspect of this embodiment, the HBV antigens consist of an amino acid sequence comprising at least 95% of a full—length HBV large surface antigen (L) fused to the N—terminus of an amino acid sequence comprising at least 95% of a full-length HBV core protein. In one aspect, the HBV antigens consist of: amino acids 2 to 400 of HBV large surface antigen (L); and amino acids 31 to 212 of the HBV precore protein comprising HBV core protein and a portion ofHBV e-antigen.
In one aspect of this ment of the ion, the amino acid sequence of the HBV surface antigen is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% cal, or is identical, to an amino acid ce selected from: positions 1-399 of SEQ ID N0:118, positions 9-407 of SEQ ID N0234, positions 1-399 of SEQ ID N0:116, positions 1-399 of SEQ ID N02112, positions 1-399 of SEQ ID N0:114, SEQ ID N023 or ons 2-400 of SEQ ID N023, SEQ ID N027 or positions 2—400 of SEQ ID N017, SEQ ID N0:1l or positions 2—400 of SEQ ID N0:11, SEQ ID N0:15 or positions 2-389 of SEQ ID N0:15, SEQ ID N0:19 or positions 2-399 of SEQ ID N0:19, SEQ ID NO:23 or positions 2-400 of SEQ ID NO:23, SEQ ID NO:27 or positions 2—399 of SEQ ID NO:27, SEQ ID N03] or positions 2-400 of SEQ ID N0231, positions 90-488 of SEQ ID N0293, positions 1-399 of SEQ ID N0:120, positions 1-399 of SEQ ID N02122, positions 1—399 of SEQ ID N02124, positions 1-399 of SEQ ID N0:126, ons 9 of SEQ ID , positions 63- 461 of SEQ ID N0:130, positions 289-687 of SEQ ID N0:132, positions 289-687 of SEQ ID N0:134, or a corresponding sequence from a different HBV strain. In one aspect, the amino acid sequence of the HBV surface antigen is selected from: positions 1-399 of SEQ ID N0:118, positions 9-407 of SEQ ID N0z34, positions 1-399 of SEQ ID N0:112, positions 1-399 of SEQ ID N0:114, positions 1-399 of SEQ ID N0:116, or a corresponding sequence from a different HBV strain.
In one aspect of this embodiment of the invention, the amino acid sequence of the HBV core antigen is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, or is identical, to an amino acid sequence selected from: positions 1 of SEQ ID , positions 408—589 of SEQ ID N0234, positions 400-581 of SEQ ID N0:116, positions 400-581 of SEQ ID N0:112, ons 400-581 of SEQ ID N0:114, positions 31-212 of SEQ ID N021, positions 31—212 of SEQ ID N025, positions 31—212 of SEQ ID N029, positions 31—212 of SEQ ID N0:13, positions 31-212 of SEQ ID N0:17, positions 31-212 of SEQ ID NO:21, positions 14—194 of SEQ ID N0225, positions 31-212 of SEQ ID N0229, positions 605 to 786 of SEQ ID N0:36, positions 352-533 of SEQ ID N0z38, positions 160-341 of SEQ ID NO:39, positions 605-786 of SEQ ID NO:41, positions 691-872 of SEQ ID NO:92, positions 90-271 of SEQ ID NO:95, positions 2—183 of SEQ ID NO:105, positions 184- 395 of SEQ ID NO:105, positions 396-578 of SEQ ID NO:105, positions 579-761 of SEQ ID NO:105, ons 2—183 of SEQ ID NO:106, 338-520 of SEQ ID NO:106, positions 400 to 581 of SEQ ID NO:120, positions 400 to 581 of SEQ ID NO:122, positions 400 to 581 of SEQ ID NO:124, ons 400 to 581 of SEQ ID NO:126, positions 630 to 811 of SEQ ID NO:128, positions 462 to 643 of SEQ ID NO:130, ons 688 to 869 of SEQ ID NO:132, positions 688 to 869 of SEQ ID , or a ponding sequence from a different HBV strain. In one aspect, the amino acid sequence of the HBV core n is selected from: positions 400—581 of SEQ ID NO:118, positions 408—589 of SEQ ID NO:34, positions 400-581 of SEQ ID NO:116, positions 400-581 of SEQ ID NO:112, positions 400-581 of SEQ ID NO:114, or a corresponding sequence from a different HBV strain.
In one aspect of this embodiment of the invention, the HBV antigens consist of amino acids 9 to 589 of SEQ ID NO:34, or a ponding sequence from a different HBV strain. In one aspect, the HBV antigens t of an amino acid sequence that is at least 95% cal, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, or is identical, to an amino acid sequence selected from: SEQ ID NO:118, SEQ ID NO:116, positions 9—589 of SEQ ID N034, SEQ ID NO:112, SEQ ID NO:114, or a corresponding sequence for a different HBV . In one aspect, the HBV antigens consist of a full—length or near ength HBV large surface antigen (L) and a ength or near filll-length HBV core protein.
In one aspect of this embodiment of the invention, any of the fusion proteins can include an N—terminal amino acid sequence (appended to the N—terminus of the fusion protein) of SEQ ID NO:37. In another , any of the fusion proteins can include an N- terminal amino acid sequence selected from SEQ ID NO:89 or SEQ ID NO:90. In one aspect, the fusion protein comprises an amino acid sequence of SEQ ID NO:151.
Yet another embodiment of the invention relates to an immunotherapeutic composition comprising: (a) a Whole, heat-inactivated yeast from Saccharomyces cerevisiae; and (b) an HBV fusion protein expressed by the yeast, wherein the fusion protein comprises SEQ ID NO:118.
Another embodiment of the invention relates to an immunotherapeutic composition comprising: (a) a Whole, heat-inactivated yeast from Saccharomyces cerevisiae; and (b) an HBV fusion protein expressed by the yeast, wherein the fusion n comprises SEQ ID .
Yet another ment of the invention relates to an immunotherapeutic composition comprising: (a) a whole, heat-inactivated yeast from Saccharomyces cerevisiae; and (b) an HBV fusion protein expressed by the yeast, wherein the fusion protein ses the amino acid sequence of SEQ ID NO:34.
In another embodiment, the present invention includes an immunotherapeutic composition comprising: (a) a yeast vehicle; and (b) a fusion protein comprising HBV antigens. The HBV antigens consist of: (i) an HBV surface antigen consisting of at least one immunogenic domain of full—length HBV large (L), medium (M) or small (S) surface n; (ii) an HBV rase antigen consisting of at least one immunogenic domain of full-length HBV polymerase or of the reverse transcriptase (RT) domain of HBV polymerase; (iii) an HBV core antigen consisting of at least one immunogenic domain of full-length HBV core protein or of full-length HBV e-antigen; and (iv) an HBV X antigen consisting of at least one immunogenic domain of full-length HBV X antigen. The composition elicits an HBV-specific immune response. In one aspect of this embodiment, the HBV surface antigen comprises at least one genic domain of hepatocyte receptor region of Pre-Sl of the HBV large surface antigen (L) and at least one immunogenic domain ofHBV small surface antigen (S).
In one aspect of this embodiment, the HBV antigens consist of: at least 95% of the full-length hepatocyte receptor of Pre-Sl of the HBV large e antigen (L), at least 95% of the full-length HBV small surface antigen (S), at least 95% of the e transcriptase domain of HBV polymerase, at least 95% of the full-length HBV core protein or HBV gen, and at least 95% of the full-length X antigen. In one aspect, the HBV antigens consist of: an HBV large surface antigen (L) sing at least 95% of amino acids 120 to 368 of HBV large surface antigen (L); an RT domain of HBV polymerase comprising at least 95% of amino acids 453 to 680 of the RT domain of HBV polymerase; an HBV core protein sing at least 95% of amino acids 37 to 188 of HBV core protein; and an HBV X n comprising at least 80% of amino acids 52 to 127 of HBV X antigen. In one aspect, the HBV ns consist of: amino acids 21 to 47 of HBV large surface antigen (L) comprising the hepatocyte receptor domain of Pre-Sl; amino acids 176 to 400 of HBV large surface antigen (L) comprising HBV small surface antigen (S); amino acids 247 to 691 of HBV polymerase comprising the reverse transcriptase domain; amino acids 31 to 212 of HBV precore protein comprising HBV W0 09404 core n and a portion of HBV e—antigen; and amino acids 2 to 154 of HBV X antigen.
In one aspect, the HBV antigens consist of: an amino acid sequence at least 95% identical to amino acids 120 to 368 of HBV large surface antigen (L); an amino acid sequence at least 95% identical to amino acids 453 to 680 of the RT domain of HBV polymerase; an amino acid sequence at least 95% identical to amino acids 37 to 188 of HBV core protein; and an amino acid sequence at least 80% identical to amino acids 52 to 127 of HBV X antigen. In one aspect, the HBV antigens have been modified to incorporate one or more T cell epitopes set forth in Table 5 and represented herein by SEQ ID NOsz42 to 88 or SEQ ID NOs:135-140. In one aspect, the HBV large e antigen (L) comprises an amino acid sequence of SEQ ID NO:97 or a sequence that is 95% identical to SEQ ID NO:97. In one aspect, the RT domain of an HBV polymerase comprises an amino acid sequence of SEQ ID NO:98 or a sequence that is 95% identical to SEQ ID NO:98. In one aspect, the HBV core protein comprises an amino acid sequence of SEQ ID NO:99 or a sequence that is 95% identical to SEQ ID NO:99. In one aspect, the HBV X antigen comprises an amino acid sequence of SEQ ID NO:100 or a sequence that is 95% identical to SEQ ID NO:100.
In one aspect of this embodiment of the invention, the amino acid sequence of the HBV surface antigen is at least 95% cal to an amino acid sequence of a full- length HBV large surface antigen (L). In one aspect, the amino acid sequence of the HBV surface antigen is at least 95% identical, or at least 96% identical, or at least 97% cal, or at least 98% identical, or at least 99% cal, or is identical, to an amino acid ce selected from: positions 1-399 of SEQ ID NO:124, positions 1-399 of SEQ ID NO:126, positions 289-687 of SEQ ID NO:132, positions 289-687 of SEQ ID NO:134, SEQ ID N023, SEQ ID N027, SEQ ID NO:11, SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO:23, SEQ ID NO:27, SEQ ID NO:31, ons 9-407 of SEQ ID NO:34, positions 90- 488 of SEQ ID NO:93, positions 1-399 of SEQ ID , positions 1-399 of SEQ ID NO:114, positions 1-399 of SEQ ID NO:116, positions 1-399 of SEQ ID , positions 1-399 of SEQ ID NO:120, positions 1-399 of SEQ ID NO:122, positions 231- 629 of SEQ ID NO:128, positions 63-461 of SEQ ID NO:130, or a corresponding sequence from a different HBV strain.
In one aspect of this embodiment, the amino acid sequence of the HBV surface antigen is at least 95% cal, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, or is identical, to an amino acid sequence selected from: SEQ ID NO:97, positions 1-249 of SEQ ID NO:107, positions 1- WO 09404 249 of SEQ ID NO:108, positions 1-249 of SEQ ID NO:109, positions 1—249 of SEQ ID NO:110, ons 21-47 of SEQ ID NO:11, positions 176-400 of SEQ ID NO:1], positions 6-257 of SEQ ID NO:36, positions 6-257 of SEQ ID NO:41, positions 92—343 of SEQ ID NO:92, positions 90-338 of SEQ ID NO:101, positions 7-254 of SEQ ID NO:102, or a corresponding ce from a different HBV strain.
In one aspect of this ment, the HBV polymerase antigen consists of at least one immunogenic domain of the RT domain of HBV polymerase. In one , the amino acid sequence of the HBV rase antigen is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, or is identical, to an amino acid sequence selected from: SEQ ID NO:98, positions 582 to 809 of SEQ ID NO:124, positions 642 to 869 of SEQ ID NO:126, positions 1 to 228 of SEQ ID NO:132, positions 61 to 288 of SEQ ID NO:134, positions 250-477 of SEQ ID NO:107, positions 250-477 of SEQ ID NO:108, positions 250-477 of SEQ ID NO:109, positions 250-477 of SEQ ID , positions 383-602 of SEQ ID NO:2, positions 381- 600 of SEQ ID NO:6, positions 381-600 of SEQ ID NO:10, ons 453 to 680 of SEQ ID NO:10, positions 370-589 of SEQ ID NO:14, positions 380-599 of SEQ ID NO:18, positions 381-600 of SEQ ID N0222, ons 380-599 of SEQ ID NO:26, positions 381— 600 of SEQ ID NO:30, positions 260 to 604 of SEQ ID NO:36, positions 7-351 of SEQ ID NO:38, positions 7-351 of SEQ ID NO:40, 260 to 604 of SEQ ID NO:41, positions 346 to 690 of SEQ ID NO:92, positions 90-434 of SEQ ID NO:94, positions 339 to 566 of SEQ ID NO:101, positions 255 to 482 of SEQ ID NO:102, positions 582 to 809 of SEQ ID NO:120, positions 1 to 228 of SEQ ID NO:128, or a corresponding sequence from a different HBV .
In one aspect of this embodiment, the amino acid sequence of the HBV core antigen is at least 95% identical to an amino acid sequence of a full—length HBV core n. In one aspect, the amino acid sequence of the HBV core antigen is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, or is identical, to an amino acid sequence selected from: positions 400 to 581 of SEQ ID NO:124, ons 400 to 581 of SEQ ID NO:126, positions 688 to 869 of SEQ ID NO:132, positions 688 to 869 of SEQ ID NO:134, positions 408-589 of SEQ ID NO:34, positions 400—581 of SEQ ID NO:112, positions 400—581 of SEQ ID NO:114, positions 400-581 of SEQ ID NO:116, positions 400-581 of SEQ ID NO:118, ons 31-212 of SEQ ID NO:1, positions 31-212 of SEQ ID NO:5, positions 31-212 of SEQ ID N029, positions 31-212 of SEQ ID NO:13, positions 31-212 of SEQ ID NO:17, positions W0 2012/109404 31-212 of SEQ ID NO:2l, positions 14-194 of SEQ ID NO:25, positions 31-212 of SEQ ID NO:29, ons 605 to 786 of SEQ ID NO:36, positions 352-533 of SEQ ID NO:38, positions 160-341 of SEQ ID NO:39, positions 605-786 of SEQ ID NO:4l, positions 691— 872 of SEQ ID NO:92, positions 90-271 of SEQ ID NO:95, positions 2-183 of SEQ ID NO:105, positions 5 of SEQ ID NO:105, positions 396-578 of SEQ ID NO:105, positions 579—761 of SEQ ID NO:105, positions 2-183 of SEQ ID NO:106, 338-520 of SEQ ID , positions 400 to 581 of SEQ ID NO:120, positions 400 to 581 of SEQ ID NO:l22, positions 630 to 811 of SEQ ID NO:l28, positions 462 to 643 of SEQ ID NO:130, or a corresponding sequence from a different HBV .
In one aspect of this embodiment, the amino acid sequence of the HBV core antigen is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, or is identical, to an amino acid sequence selected from: positions SEQ ID NO:99, 37 to 188 of SEQ ID NO:9, positions 567 to 718 of SEQ ID NO:101, positions 483 to 634 of SEQ ID NO:102, positions 478-629 of SEQ ID NO:107, positions 478—629 of SEQ ID NO: 108, positions 9 of SEQ ID NO:109, positions 478-629 of SEQ ID NO:110, or a corresponding sequence from a different HBV In one aspect of this embodiment, the HBV X antigen consists of an amino acid sequence that is at least 95% identical to a full-length HBV X antigen. In one aspect, the HBV X antigen is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, or is identical, to an amino acid sequence selected from: SEQ ID NO:4, SEQ ID N028, SEQ ID NO:12, positions 2 to 154 of SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:32, positions 787 to 939 of SEQ ID NO:36, positions 7—159 of SEQ ID NO:39, ons 25 of SEQ ID NO:92, ons 90-242 of SEQ ID NO:96, positions 184-337 of SEQ ID NO:106, positions 521-674 of SEQ ID , or a corresponding sequence from a different HBV strain.
In one aspect, the HBV X n consists of an amino acid sequence that is at least 80% identical to positions 52 to 126 of a full-length HBV X antigen. In one aspect, the amino acid sequence of HBV X antigen is at least 95% cal, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, or is identical, to an amino acid sequence selected from: SEQ ID NO: 100, positions 9 of SEQ ID NO:124, positions 582-641 of SEQ ID NO:126, positions 229 to 288 of SEQ ID NO:132, positions 1 to 60 of SEQ ID NO:134, positions 630-689 of SEQ ID NO:107, ons 630—689 of SEQ ID NO:108, positions 630-689 of SEQ ID , positions 630-689 of SEQ ID NO:110, positions 52-68 followed by positions 84-126 of SEQ ID NO:4, ons 52-68 followed by positions 84—126 of SEQ ID N028, positions 52—68 followed by positions 84-126 of SEQ ID NO:12, ons 52-68 followed by positions 84-126 of SEQ ID NO:16, ons 52-68 followed by positions 84-126 of SEQ ID NO:20, positions 52-68 followed by positions 84-126 of SEQ ID NO:24, positions 52-68 followed by positions 84-126 of SEQ ID NO:28, positions 52-68 followed by positions 84-126 of SEQ ID NO:32, ons 719—778 of SEQ ID NO:101, positions 635-694 of SEQ ID NO:102, positions 582-641 of SEQ ID NO:122, positions 1-60 of SEQ ID NO:130, or a corresponding ce from a different HBV strain.
In one aspect of this embodiment, the HBV antigens have an amino acid sequence that is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, or is identical, to an amino acid sequence selected from: positions 6 to 939 of SEQ ID NO:36, positions 92 to 1025 of SEQ ID NO:92, positions 90 to 778 of SEQ ID , positions 7 to 694 of SEQ ID NO:102, or a corresponding sequence from a different HBV strain.
In one aspect of this embodiment, the fusion protein comprises an amino acid sequence that is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, or is identical, to an amino acid sequence selected from: SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:1 10, SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:132 or SEQ ID NO: 134.
Any of the fusion proteins may, in one aspect, comprise an N-terminal sequence selected from SEQ ID NO:37, SEQ ID NO:89, or SEQ ID NO:90.
In one aspect of this embodiment, the fusion protein comprises an amino acid sequence that is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, or is identical, to an amino acid ce selected from: SEQ ID NO:36, SEQ ID NO:92, SEQ ID , or SEQ ID NO:102.
Another embodiment of the invention relates to an immunotherapeutic composition comprising: (a) a yeast vehicle; and (b) a fusion protein comprising HBV antigens, wherein the HBV antigens consist of: (i) an HBV surface antigen consisting of at least one immunogenic domain of hepatocyte receptor region of Pre-Sl of the HBV large surface antigen (L) and at least one immunogenic domain of HBV small surface n (S); (ii) an HBV rase antigen consisting of at least one immunogenic domain of reverse transcriptase domain of HBV polymerase; and (iii) an HBV core antigen consisting of at least one imrnunogenic domain of HBV core n. The composition elicits an HBV-specific immune se. In one aspect, the HBV antigens consist of at least 95% of full-length hepatocyte receptor of Pre-Sl of HBV large surface antigen (L), at least 95% of full-length HBV small e antigen, at least 95% of full-length reverse transcriptase domain of HBV rase, and at least 95% of ength HBV core protein. In one aspect, the HBV antigens consist of at least 95% of full-length HBV large surface antigen (L), at least 95% of full—length reverse transcriptase domain of HBV polymerase, and at least 95% of full-length HBV core protein.
In one aspect of this embodiment, the amino acid sequence of the HBV surface antigen is at least 95% identical, or at least 96% identical, or at least 97% cal, or at least 98% identical, or at least 99% identical, or is identical, to an amino acid ce selected from: positions 1-399 of SEQ ID NO:120, positions 231-629 of SEQ ID NO:128, positions 1-399 of SEQ ID NO:112, positions 1-399 of SEQ ID NO:114, positions 1-399 of SEQ ID N0:ll6, positions 1-399 of SEQ ID N0:ll8, positions 6-257 of SEQ ID NO:4l, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:ll, ons 21-47 of SEQ ID NO:11, positions 176—400 of SEQ ID NO:11, SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO:23, SEQ ID N0:27, SEQ ID N0:31, positions 9-407 of SEQ ID N0:34, positions 6- 257 of SEQ ID N0:36, positions 92-343 of SEQ ID NO:92, positions 90-488 of SEQ ID NO:93, SEQ ID NO:97, positions 90-338 of SEQ ID NO:101, positions 7-254 of SEQ ID NO:102, positions 1-249 of SEQ ID NO:107, positions 1-249 of SEQ ID NO:108, positions 1-249 of SEQ ID NO:109, positions 1-249 of SEQ ID NO:110, positions 1-399 of SEQ ID NO:l22, positions 1-399 of SEQ ID NOzl24, ons 1-399 of SEQ ID NO:126, positions 63-461 of SEQ ID NO:130, positions 289-687 of SEQ ID NO:132, positions 289-687 of SEQ ID NO: 134, or a corresponding sequence from a different HBV strain.
In one aspect of this embodiment of the invention, the amino acid sequence of the HBV polymerase antigen is at least 95% cal, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, or is identical, to an amino acid sequence selected from: positions 582 to 809 of SEQ ID NO:120, ons 1 to 228 of SEQ ID NO:128, ons 250-477 of SEQ ID NO:107, positions 250—477 of SEQ ID NO:108, positions 250-477 of SEQ ID NO:109, positions 250-477 of SEQ ID NO:110, 260 to 604 of SEQ ID N0241, positions 383-602 of SEQ ID N022, positions 381- 600 of SEQ ID N0:6, ons 381-600 of SEQ ID N0:lO, positions 453 to 680 of SEQ W0 09404 ID NO:10, positions 370-589 of SEQ ID NO:14, positions 9 of SEQ ID NO:18, positions 381-600 of SEQ ID NO:22, positions 380-599 of SEQ ID N0226, positions 381- 600 of SEQ ID NO:30, positions 260 to 604 of SEQ ID NO:36, positions 7—351 of SEQ ID NO:38, positions 7—351 of SEQ ID N0240, positions 346 to 690 of SEQ ID NO:92, positions 90—434 of SEQ ID N0294, SEQ ID NO:98, positions 339 to 566 of SEQ ID NO:101, positions 255 to 482 of SEQ ID NO:102, positions 582 to 809 of SEQ ID NO:124, positions 642 to 869 of SEQ ID NO:126, ons 1 to 228 of SEQ ID NO:132, positions 61 to 288 of SEQ ID NO:134, or a corresponding sequence from a different HBV strain.
In one aspect of this embodiment of the invention, the amino acid ce of the HBV core antigen is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, or is identical, to an amino acid sequence selected from: positions 400 to 581 of SEQ ID , positions 630 to 811 of SEQ ID NO:128, positions 400-581 of SEQ ID NO:112, positions 1 of SEQ ID , positions 400-581 of SEQ ID NO:116, positions 400-581 of SEQ ID NO:118, positions 605-786 of SEQ ID N0241, positions 31-212 of SEQ ID NO:1, positions 31-212 of SEQ ID N025, positions 31-212 of SEQ ID N029, positions 37 to 188 of SEQ ID N029, positions 31-212 of SEQ ID NO:13, positions 31—212 of SEQ ID NO:17, positions 31-212 of SEQ ID NO:21, positions 14-194 of SEQ ID NO:25, positions 31-212 of SEQ ID NO:29, positions 408-589 of SEQ ID NO:34, ons 605 to 786 of SEQ ID NO:36, positions 352-533 of SEQ ID NO:38, positions 160-341 of SEQ ID NO:39, positions 691- 872 of SEQ ID NO:92, positions 90-271 of SEQ ID NO:95, SEQ ID NO:99, positions 567 to 718 of SEQ ID NO:101, positions 483 to 634 of SEQ ID NO:102, positions 2-183 of SEQ ID NO:105, positions 184-395 of SEQ ID NO:105, positions 396-578 of SEQ ID NO:105, positions 579-761 of SEQ ID NO:105, positions 2-183 of SEQ ID NO:106, 338- 520 of SEQ ID NO:106, positions 478-629 of SEQ ID NO:107, positions 478-629 of SEQ ID NO:108, ons 478-629 of SEQ ID NO: 109, positions 9 of SEQ ID NO:110, positions 400 to 581 of SEQ ID NO:122, positions 400 to 581 of SEQ ID NO:124, positions 400 to 581 of SEQ ID NO:126, ons 462 to 643 of SEQ ID NO:130, positions 688 to 869 of SEQ ID NO:132, positions 688 to 869 of SEQ ID NO:134, or a ponding sequence from a ent HBV strain.
In one aspect of this embodiment of the invention, the fusion protein has an amino acid sequence that is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, or is identical, to an amino acid sequence ed from: SEQ ID NO:120, SEQ ID NO:128, positions 6-786 of SEQ ID NO:4l, or SEQ ID NO:4l, or a corresponding sequence from a different HBV strain.
Another embodiment of the ion relates to an immunotherapeutic composition comprising: (a) a yeast vehicle; and (b) a fusion protein comprising HBV antigens, wherein the HBV antigens consist of: (i) an HBV polymerase n consisting of at least one immunogenic domain of the reverse transcriptase (RT) domain of HBV polymerase; and (ii) an HBV core antigen ting of at least one immunogenic domain of HBV core protein. The composition elicits an ecific immune response. In one aspect of this embodiment of the ion, the HBV antigens consist of: an amino acid ce that is at least 95% identical to full-length RT domain of HBV polymerase and an amino acid sequence that is at least 95% identical to full-length HBV core protein.
In one aspect of this embodiment of the invention, the amino acid sequence of the HBV polymerase antigen is at least 95% cal, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, or is identical, to an amino acid sequence selected from: positions 7-351 of SEQ ID NO:38, positions 383-602 of SEQ ID N022, positions 381-600 of SEQ ID NO:6, positions 0 of SEQ ID NO:10, positions 453 to 680 of SEQ ID NO:10, positions 370-589 of SEQ ID NO:14, positions 380-599 of SEQ ID NO:18, positions 381-600 of SEQ ID NO:22, positions 380— 599 of SEQ ID NO:26, positions 381-600 of SEQ ID NO:30, positions 260 to 604 of SEQ ID NO:36, positions 7-351 of SEQ ID NO:40, 260 to 604 of SEQ ID NO:41, positions 346 to 690 of SEQ ID NO:92, positions 90-434 of SEQ ID NO:94, SEQ ID NO:98, positions 339 to 566 of SEQ ID NO:101, positions 255 to 482 of SEQ ID NO:102, positions 250- 477 of SEQ ID NO:107, ons 250-477 of SEQ ID NO:108, positions 250-477 of SEQ ID NO:109, positions 250-477 of SEQ ID , positions 582 to 809 of SEQ ID , positions 582 to 809 of SEQ ID NO:124, positions 642 to 869 of SEQ ID NO:126, positions 1 to 228 of SEQ ID NO:128, positions 1 to 228 of SEQ ID NO:132, positions 61 to 288 of SEQ ID NO:134, or a corresponding sequence from a different HBV .
In one aspect of this embodiment of the invention, the amino acid sequence of the HBV core antigen is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, or is identical, to an amino acid sequence selected from: positions 352-533 of SEQ ID NO:38, positions 31-212 of SEQ ID NO:1, positions 31-212 of SEQ ID NO:5, positions 31-212 of SEQ ID NO:9, WO 09404 2012/024409 positions 37 to 188 of SEQ ID NO:9, positions 31-212 of SEQ ID NO:13, positions 31- 212 of SEQ ID NO:17, positions 31-212 of SEQ ID NO:21, ons 14-194 of SEQ ID NO:25, positions 31-212 of SEQ ID NO:29, positions 9 of SEQ ID NO:34, positions 605 to 786 of SEQ ID NO:36, positions 160-341 of SEQ ID NO:39, positions 605-786 of SEQ ID N024l, ons 691-872 of SEQ ID NO:92, positions 90-271 of SEQ ID NO:95, SEQ ID NO:99, positions 567 to 718 of SEQ ID NO:101, positions 483 to 634 of SEQ ID NO:102, positions 2-183 of SEQ ID NO:105, positions 184-395 of SEQ ID NO:105, positions 396—578 of SEQ ID NO:105, positions 579-761 of SEQ ID NO:105, positions 2-183 of SEQ ID NO:106, 338-520 of SEQ ID NO:106, positions 478-629 of SEQ ID , positions 478-629 of SEQ ID NO:108, positions 478-629 of SEQ ID NO:109, positions 478-629 of SEQ ID NO:110, positions 400-581 of SEQ ID NO:112, positions 400-581 of SEQ ID NO:114, positions 400-581 of SEQ ID NO:116, positions 400-581 of SEQ ID NO:118, positions 400 to 581 of SEQ ID NO:120, positions 400 to 581 of SEQ ID NO:122, positions 400 to 581 of SEQ ID NO:124, positions 400 to 581 of SEQ ID NO:126, positions 630 to 811 of SEQ ID NO:128, ons 462 to 643 of SEQ ID NO:130, ons 688 to 869 of SEQ ID , positions 688 to 869 of SEQ ID NO:134, or a corresponding sequence from a different HBV strain.
In one aspect of this embodiment of the invention, the fusion protein has an amino acid sequence that is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, or is cal, to an amino acid sequence of SEQ ID NO:38, or a corresponding sequence from a different HBV strain.
Yet another embodiment of the invention relates to an immunotherapeutic composition sing: (a) a yeast vehicle; and (b) a fusion protein sing HBV antigens, wherein the HBV antigens consist of: (i) an HBV X antigen consisting of at least one immunogenic domain of HBV X n; and (ii) an HBV core antigen consisting of at least one immunogenic domain of HBV core protein. The composition elicits an HBV- specific immune response. In one aspect of this embodiment, the HBV antigens consist of: an amino acid sequence that is at least 95% identical to full-length HBV X antigen and an amino acid sequence that is at least 95% identical to full-length HBV core protein.
In one aspect of this ment of the invention, the amino acid sequence of the HBV core antigen is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, or is identical, to an amino acid sequence selected from: positions 160-341 of SEQ ID N0239, positions 31-212 of SEQ ID N0:1, positions 31-212 of SEQ ID N0:5, positions 31-212 of SEQ ID NO:9, positions 37 to 188 of SEQ ID N0:9, positions 31-212 of SEQ ID N0:13, positions 31- 212 of SEQ ID N0:17, positions 31-212 of SEQ ID N0:21, positions 14-194 of SEQ ID N0:25, positions 31-212 of SEQ ID N0:29, positions 408-589 of SEQ ID N0:34, positions 605 to 786 of SEQ ID N0236, ons 352-533 of SEQ ID N0238, positions 605-786 of SEQ ID N0:41, positions 691-872 of SEQ ID N0:92, positions 90-271 of SEQ ID N0:95, SEQ ID N0:99, positions 567 to 718 of SEQ ID N0:101, positions 483 to 634 of SEQ ID N0:102, positions 2-183 of SEQ ID N0:105, positions 184-395 of SEQ ID N0:105, positions 8 of SEQ ID N0:105, positions 579-761 of SEQ ID N0:105, positions 2-183 of SEQ ID N0:106, 338-520 of SEQ ID N0:106, positions 478-629 of SEQ ID N0:107, positions 478—629 of SEQ ID , positions 478-629 of SEQ ID N0:109, positions 478-629 of SEQ ID N0:110, positions 400-581 of SEQ ID N0:112, positions 400-581 of SEQ ID N0:114, positions 400-581 of SEQ ID N0:116, positions 400-581 of SEQ ID N0:118, positions 400 to 581 of SEQ ID NO:120, positions 400 to 581 of SEQ ID N0:122, positions 400 to 581 of SEQ ID N0:124, positions 400 to 581 of SEQ ID N0:126, positions 630 to 811 of SEQ ID , positions 462 to 643 of SEQ ID N0:130, positions 688 to 869 of SEQ ID N0:132, positions 688 to 869 of SEQ ID , or a corresponding sequence from a different HBV strain.
In one aspect of this ment of the invention, the amino acid sequence of the HBV X antigen is at least 95% identical, or at least 96% cal, or at least 97% identical, or at least 98% identical, or at least 99% cal, or is identical, to an amino acid sequence selected from: positions 7-159 of SEQ ID N0239, SEQ ID N024, SEQ ID N0:8, SEQ ID N0:12, positions 2 to 154 of SEQ ID N0:12, SEQ ID N0:16, SEQ ID N0:20, SEQ ID N0:24, SEQ ID N0:28, SEQ ID N0232, positions 52-68 followed by positions 84-126 of SEQ ID N0:4, ons 52-68 followed by positions 84-126 of SEQ ID N0:8, positions 52-68 followed by positions 84-126 of SEQ ID N0:12, positions 52-68 followed by positions 84-126 of SEQ ID N0:16, positions 52-68 ed by positions 84-126 of SEQ ID N0:20, positions 52-68 followed by positions 84-126 of SEQ ID N0:24, positions 52-68 followed by positions 84-126 of SEQ ID N0:28, positions 52-68 followed by positions 84-126 of SEQ ID N0:32, positions 787 to 939 of SEQ ID N0:36, positions 873-1025 of SEQ ID N0292, positions 90-242 of SEQ ID N0296, SEQ ID N0:100, positions 719-778 of SEQ ID , positions 635-694 of SEQ ID N0:102, positions 184-337 of SEQ ID N0:106, positions 521-674 of SEQ ID N0:106, positions 630-689 of SEQ ID N0:107, positions 630-689 of SEQ ID NO:108, positions 630-689 of W0 2012/109404 SEQ ID NO:109, positions 630—689 of SEQ ID NOleO, positions 582—641 of SEQ ID NO:122, positions 810-869 of SEQ ID NO:124, positions 582-641 of SEQ ID NO:126, positions 1-60 of SEQ ID NO:130, positions 229 to 288 of SEQ ID NO:132, ons 1 to 60 of SEQ ID NO:134, or a corresponding sequence from a different HBV strain.
In one aspect of this embodiment of the invention, the fusion protein has the amino acid sequence that is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, or is identical, to an amino acid sequence to SEQ ID NO:39, or a corresponding sequence from a different HBV .
Another embodiment of the invention relates to an immunotherapeutic composition sing: (a) a yeast vehicle; and (b) a fusion protein comprising an HBV surface antigen consisting of at least one immunogenic domain of an HBV large surface antigen (L), wherein the composition elicits an HBV-specific immune response. In one aspect of this embodiment, the HBV surface n consists of at least 95% of full-length HBV large surface antigen (L) In one aspect, the amino acid sequence of the HBV surface antigen is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, or is identical, to an amino acid sequence ed from: positions 90-488 of SEQ ID NO:93, SEQ ID NO:3, SEQ ID N027, SEQ ID NO:11, positions 21-47 of SEQ ID NO:11, positions 176-400 of SEQ ID NO:11, SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO:23, SEQ ID NO:27, SEQ ID NOz3l, positions 9-407 of SEQ ID N034, positions 6-257 of SEQ ID N036, positions 6—257 of SEQ ID NO:4l, positions 92-343 of SEQ ID NO:92, ons 90-488 of SEQ ID NO:93, SEQ ID NO:97, positions 90-338 of SEQ ID , positions 7-254 of SEQ ID NO:102, positions 1-249 of SEQ ID NO:107, positions 1-249 of SEQ ID NO:108, positions 1-249 of SEQ ID NO:109, positions 1-249 of SEQ ID NO:110, ons l-399 of SEQ ID NO:1 12, ons 1-399 of SEQ ID NO:1l4, positions 1—399 of SEQ ID NO:116, positions l-399 of SEQ ID NO:118, positions l-399 of SEQ ID NO:120, positions 1-399 of SEQ ID NOzl22, positions 1-399 of SEQ ID NO:124, ons l-399 of SEQ ID NO:126, ons 231-629 of SEQ ID NO:128, positions 63-461 of SEQ ID NO:130, positions 289-687 of SEQ ID NO:132, positions 289-687 of SEQ ID NO:134, or a corresponding sequence from a different HBV strain. In one , the fusion protein has the amino acid sequence that is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, or is identical, to an W0 2012/109404 amino acid sequence of SEQ ID NO:93, or a corresponding sequence from a different HBV strain.
Yet another embodiment of the ion relates to an immunotherapeutic composition comprising: (a) a yeast vehicle; and (b) a fusion protein comprising an HBV polymerase antigen consisting of at least one immunogenic domain of a reverse transcriptase domain of HBV polymerase, wherein the ition elicits an HBV- specific immune response. In one aspect of this embodiment of the invention, the HBV polymerase n consists of at least 95% of full—length reverse transcriptase domain of HBV polymerase. In one aspect, the amino acid sequence of the HBV polymerase antigen is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, or is identical, to an amino acid sequence selected from: positions 7-351 of SEQ ID NO:40, positions 90-434 of SEQ ID NO:94, positions 383-602 of SEQ ID NO:2, positions 0 of SEQ ID NO:6, positions 381-600 of SEQ ID NO:lO, positions 453 to 680 of SEQ ID NO:lO, ons 370-589 of SEQ ID NO:l4, positions 380-599 of SEQ ID N0:l8, positions 381-600 of SEQ ID N0:22, positions 380- 599 of SEQ ID NO:26, ons 381-600 of SEQ ID NO:30, positions 260 to 604 of SEQ ID N0236, positions 7-351 of SEQ ID N0:38, 260 to 604 of SEQ ID N024l, positions 346 to 690 of SEQ ID NO:92, SEQ ID NO:98, positions 339 to 566 of SEQ ID NO:lOl, ons 255 to 482 of SEQ ID NO:102, positions 7 of SEQ ID NO:107, positions 250-477 of SEQ ID NO:lO8, positions 250-477 of SEQ ID NO:lO9, positions 250-477 of SEQ ID NO:llO, positions 582 to 809 of SEQ ID NO:l20, positions 582 to 809 of SEQ ID N01124, ons 642 to 869 of SEQ ID N0:l26, positions I to 228 of SEQ ID , positions 1 to 228 of SEQ ID NO:l32, positions 61 to 288 of SEQ ID NO:l34, or a corresponding sequence from a different HBV strain. In one aspect, the fusion protein has the amino acid sequence that is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, or is identical, to an amino acid sequence of SEQ ID NO:40 or SEQ ID NO:94, or a corresponding sequence from a different HBV strain.
Another embodiment of the invention relates to an immunotherapeutic composition comprising: (a) a yeast vehicle; and (b) a fusion protein comprising an HBV core n consisting of at least one immunogenic domain of an HBV core protein, wherein the composition elicits an HBV-specific immune response. In one aspect of this embodiment of the ion, the HBV antigens consist of at least 95% of full-length HBV core protein. In one , the amino acid sequence of the HBV core antigen is at least W0 2012/109404 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, or is identical, to an amino acid sequence selected from: positions 90—271 of SEQ ID NO:95, positions 31—212 of SEQ ID NO:1, positions 31-212 of SEQ ID NO:5, positions 31-212 of SEQ ID NO:9, ons 37 to 188 of SEQ ID NO:9, positions 31—212 of SEQ ID NO:13, positions 31-212 of SEQ ID NO:17, positions 31-212 of SEQ ID NO:21, positions 14-194 of SEQ ID NO:25, positions 31-212 of SEQ ID NO:29, positions 408-589 of SEQ ID NO:34, positions 605 to 786 of SEQ ID NO:36, positions 352—533 of SEQ ID NO:38, positions 160-341 of SEQ ID NO:39, ons 605- 786 of SEQ ID NO:41, positions 2 of SEQ ID NO:92, SEQ ID NO:99, positions 567 to 718 of SEQ ID NO:101, positions 483 to 634 of SEQ ID NO:102, positions 2-183 of SEQ ID NO:105, positions 184—395 of SEQ ID NO:105, positions 396-578 of SEQ ID NO:105, positions 579—761 of SEQ ID NO:105, positions 2-183 of SEQ ID NO:106, 338— 520 of SEQ ID NO:106, positions 478-629 of SEQ ID NO:107, positions 478-629 of SEQ ID NO:108, positions 478-629 of SEQ ID NO: 109, positions 478-629 of SEQ ID NO:110, positions 400-581 of SEQ ID NO:112, positions 400-581 of SEQ ID NO:114, positions 400-581 of SEQ ID NO:116, positions 400-581 of SEQ ID NO:118, positions 400 to 581 of SEQ ID NO:120, positions 400 to 581 of SEQ ID NO:122, ons 400 to 581 of SEQ ID NO:124, positions 400 to 581 of SEQ ID NO:126, positions 630 to 811 of SEQ ID NO:128, positions 462 to 643 of SEQ ID , positions 688 to 869 of SEQ ID , positions 688 to 869 of SEQ ID NO:134, or a corresponding sequence from a different HBV strain. In one aspect, the protein has the amino acid sequence that is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% cal, or is identical, to an amino acid sequence of SEQ ID NO:95, or a corresponding sequence from a different HBV .
Yet another embodiment of the ion relates to an therapeutic composition comprising: (a) a yeast vehicle; and (b) a fusion n sing an HBV X antigen consisting of at least one immunogenic domain of a full-length HBV X antigen, wherein the composition elicits an HBV-specific immune response. In one aspect, the HBV antigen consists of at least 95% of full-length HBV X n. In one aspect, the amino acid sequence of the HBV X antigen is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% cal, or at least 99% identical, or is identical, to an amino acid sequence selected from: positions 90-242 of SEQ ID NO:96, SEQ ID N024, SEQ ID NO:8, SEQ ID NO:12, positions 2 to 154 of SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:32, positions 52- 68 followed by positions 84—126 of SEQ ID NO:4, positions 52-68 followed by positions 84-126 of SEQ ID NO:8, positions 52-68 followed by positions 84-126 of SEQ ID NO:12, positions 52—68 followed by positions 84-126 of SEQ ID NO:16, ons 52-68 followed by positions 84-126 of SEQ ID NO:20, positions 52-68 followed by positions 84-126 of SEQ ID NO:24, positions 52-68 followed by positions 84-126 of SEQ ID NO:28, positions 52-68 followed by positions 84-126 of SEQ ID NO:32, ons 787 to 939 of SEQ ID NO:36, positions 7-159 of SEQ ID NO:39, positions 873-1025 of SEQ ID NO:92, SEQ ID NO:100, positions 719—778 of SEQ ID , positions 635-694 of SEQ ID NOle2, positions 184-337 of SEQ ID NO:106, positions 521-674 of SEQ ID NO:lO6, positions 630-689 of SEQ ID NO:107, positions 9 of SEQ ID NO:108, positions 630-689 of SEQ ID NO:109, positions 630-689 of SEQ ID NO:110, positions 582-641 of SEQ ID NO:l22, positions 9 of SEQ ID NO:l24, positions 582-641 of SEQ ID NO:l26, positions 1-60 of SEQ ID NO:130, positions 229 to 288 of SEQ ID NO:132, positions 1 to 60 of SEQ ID NO:l34, or a corresponding sequence from a different HBV strain. In one aspect, the protein has the amino acid sequence that is at least 95% identical or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, or is identical, to an amino acid sequence of SEQ ID NO:96, or a ponding sequence from a different HBV strain.
Another embodiment of the invention relates to an immunotherapeutic composition comprising any two, three or four of the immunotherapeutic compositions described above, or elsewhere herein and in particular, any two, three, or four of the immunotherapeutic compositions described above that relate to single HBV proteins.
Yet r embodiment of the invention relates to an immunotherapeutic composition comprising: (a) a yeast vehicle; and (b) a fusion protein comprising HBV antigens, n the HBV antigens consist of at least one immunogenic domain of two, three or four HBV surface antigen proteins, wherein each of the HBV surface antigen ns is from a ent HBV genotype. The composition elicits an ecific immune response.
Yet another embodiment of the ion relates to an immunotherapeutic composition comprising: (a) a yeast vehicle; and (b) a fusion protein comprising HBV antigens, wherein the HBV antigens consist of at least one immunogenic domain of two, three or four HBV rase proteins, wherein each of the HBV polymerase proteins is from a different HBV genotype. The composition s an HBV-specific immune response .
W0 2012/109404 Yet another embodiment of the ion relates to an immunotherapeutic composition sing: (a) a yeast vehicle; and (b) a fusion protein comprising HBV antigens, wherein the HBV antigens t of at least one immunogenic domain of two, three or four HBV X antigens, wherein each of the HBV X antigens is from a different HBV genotype. The composition elicits an HBV-specific immune response.
Yet another embodiment of the invention relates to an therapeutic composition comprising: (a) a yeast vehicle; and (b) a fusion protein comprising HBV antigens, wherein the HBV antigens consist of at least one immunogenic domain of two, three or four HBV core proteins, wherein each of the HBV core proteins is from a ent HBV genotype. The composition elicits an HBV-specific immune response. In one , each of the HBV core proteins consists of at least 95% of a full-length HBV core protein. In one aspect, each of the HBV core proteins consists of amino acids 31 to 212 of HBV core protein. In one aspect, the HBV genotypes include pe C, and in one aspect, the HBV genotypes include genotype D, and in one aspect, the HBV genotypes include genotype A, and in one aspect, the HBV genotypes e genotype B.
In one aspect, each of the HBV core proteins ts of amino acids 37 to 188 of HBV core protein. In one aspect, the fusion protein comprises four HBV core proteins from genotype A, genotype B, genotype C and genotype D.
In one aspect of this embodiment of the invention, the amino acid sequence of any one or more of the HBV core antigens is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, or is identical, to an amino acid sequence selected from: positions 90-271 of SEQ ID NO:95, positions 31-212 of SEQ ID NO:1, positions 31-212 of SEQ ID NO:5, positions 31-212 of SEQ ID N029, positions 37 to 188 of SEQ ID N029, positions 31-212 of SEQ ID NO:13, positions 31-212 of SEQ ID NO:17, positions 31—212 of SEQ ID NO:21, positions 14-194 of SEQ ID NO:25, positions 31-212 of SEQ ID NO:29, positions 408-589 of SEQ ID NO:34, positions 605 to 786 of SEQ ID NO:36, positions 352-533 of SEQ ID NO:38, positions 160-341 of SEQ ID NO:39, positions 605-786 of SEQ ID NO:41, ons 691- 872 of SEQ ID NO:92, SEQ ID NO:99, positions 567 to 718 of SEQ ID NO:101, positions 483 to 634 of SEQ ID NO:102, positions 2-183 of SEQ ID NO:105, ons 184-395 of SEQ ID NO:105, positions 396-578 of SEQ ID NO:105, positions 579-761 of SEQ ID NO:105, positions 2-183 of SEQ ID NO:106, 338-520 of SEQ ID NO:106, positions 478- 629 of SEQ ID NO:107, ons 9 of SEQ ID NO:108, positions 478-629 of SEQ ID NO:109, positions 478-629 of SEQ ID N011 10, positions 400-581 of SEQ ID NO:112, WO 09404 positions 400-581 of SEQ ID NO:114, positions 400-581 of SEQ ID NO:116, positions 400-581 of SEQ ID NO:118, positions 400 to 581 of SEQ ID NO:120, positions 400 to 581 of SEQ ID NO:122, positions 400 to 581 of SEQ ID NO:124, positions 400 to 581 of SEQ ID NO:126, positions 630 to 811 of SEQ ID , positions 462 to 643 of SEQ ID NO:130, positions 688 to 869 of SEQ ID NO:132, positions 688 to 869 of SEQ ID NO:134, or a corresponding sequence from a different HBV strain. In one aspect, the HBV antigens have an amino acid ce that is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% cal, or at least 99% identical, or is identical, to an amino acid sequence of SEQ ID NO:105, or a corresponding ce from a different HBV strain.
Yet another embodiment of the invention relates to an immunotherapeutic composition comprising: (a) a yeast vehicle; and (b) a fusion n comprising at least two HBV Core proteins and at least two HBV X antigens, where each of the HBV Core proteins is from a different HBV pe and where each of the HBV X antigens is from a different HBV genotype. The composition elicits an HBV-specific immune response. In one aspect, the HBV genotypes e genotype C; in one aspect, the HBV genotypes include pe D; in one aspect, the HBV genotypes include genotype A; and in one aspect, the HBV pes include genotype B. In one aspect, each of the HBV core ns consists of at least 95% of a ength HBV Core protein. In one aspect, each of the HBV core proteins comprises amino acids 31 to 212 of HBV Core protein. In one aspect, each of the HBV core proteins comprises amino acids 37 to 188 of HBV Core protein. In one aspect, each of the HBV X antigens comprises at least 95% of a full-length of HBV X antigen. In one aspect, each of the HBV X antigens comprises amino acids 52 to 127 of HBV X antigen.
In one aspect, the amino acid sequence of the HBV core antigen is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, or is identical, to an amino acid sequence selected from: positions 90—271 of SEQ ID NO:95, positions 31-212 of SEQ ID NO:1, positions 31-212 of SEQ ID NO:5, ons 31-212 of SEQ ID NO:9, positions 37 to 188 of SEQ ID NO:9, positions 31-212 of SEQ ID NO:13, positions 31-212 of SEQ ID NO:17, positions 31-212 of SEQ ID NO:21, positions 14-194 of SEQ ID NO:25, positions 31—212 of SEQ ID NO:29, positions 408-589 of SEQ ID NO:34, positions 605 to 786 of SEQ ID NO:36, positions 352-533 of SEQ ID NO:38, positions 160-341 of SEQ ID NO:39, positions 605- 786 of SEQ ID NO:41, positions 691-872 of SEQ ID NO:92, SEQ ID NO:99, positions WO 09404 567 to 718 of SEQ ID NO:101, positions 483 to 634 of SEQ ID NO:102, positions 2-183 of SEQ ID NO:105, positions 184-395 of SEQ ID , positions 396-578 of SEQ ID NO:105, positions 579—761 of SEQ ID NO:105, positions 2-183 of SEQ ID NO:106, 338— 520 of SEQ ID NO:106, positions 478-629 of SEQ ID NO:107, positions 478-629 of SEQ ID NO:108, positions 478-629 of SEQ ID NO: 109, positions 478-629 of SEQ ID NO:110, positions 1 of SEQ ID NO:112, positions 400-581 of SEQ ID NO:114, positions 400-581 of SEQ ID NO:116, positions 400—581 of SEQ ID NO:118, positions 400 to 581 of SEQ ID NO:120, positions 400 to 581 of SEQ ID NO:122, positions 400 to 581 of SEQ ID NO:124, positions 400 to 581 of SEQ ID NO:126, positions 630 to 811 of SEQ ID NO:128, positions 462 to 643 of SEQ ID NO:130, positions 688 to 869 of SEQ ID NO:132, positions 688 to 869 of SEQ ID NO:134, or a corresponding sequence from a different HBV strain.
In one aspect, the amino acid sequence of the HBV X n is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, or is identical, to an amino acid sequence selected from: positions 90- 242 of SEQ ID NO:96, SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, positions 2 to 154 of SEQ ID NO:12, SEQ ID NO:16, SEQ ID N0220, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:32, positions 52-68 followed by positions 84-126 of SEQ ID NO:4, positions 52-68 ed by positions 84—126 of SEQ ID NO:8, positions 52-68 followed by ons 84— 126 of SEQ ID NO:12, positions 52—68 followed by positions 84-126 of SEQ ID NO:16, positions 52-68 followed by positions 84-126 of SEQ ID NO:20, positions 52-68 followed by positions 84-126 of SEQ ID NO:24, positions 52-68 followed by positions 84-126 of SEQ ID NO:28, positions 52-68 followed by positions 84-126 of SEQ ID NO:32, positions 787 to 939 of SEQ ID NO:36, positions 7-159 of SEQ ID NO:39, positions 873— 1025 of SEQ ID NO:92, SEQ ID , positions 719-778 of SEQ ID NO:101, positions 635-694 of SEQ ID NO:102, positions 184-337 of SEQ ID NO:106, positions 521-674 of SEQ ID NO:106, positions 630-689 of SEQ ID NO:107, positions 630-689 of SEQ ID NO:108, positions 9 of SEQ ID NO:109, positions 630-689 of SEQ ID NO:110, positions 1 of SEQ ID , positions 810-869 of SEQ ID NO:124, ons 582-641 of SEQ ID NO:126, positions 1-60 of SEQ ID NO:130, ons 229 to 288 of SEQ ID NO:132, positions 1 to 60 of SEQ ID NO:134, or a ponding sequence from a different HBV strain.
In one aspect of this embodiment of the invention, the fusion protein has an amino acid sequence that is at least 95% cal, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, or is identical, to an amino acid sequence of SEQ ID , or a corresponding sequence from a different HBV strain.
In any of the embodiments described herein, including above and below, related to a fusion protein, HBV ns, or immunotherapeutic composition comprising such a fusion protein or HBV ns, in one further embodiment, the fusion protein can be appended at its N-terminus to add an additional sequence. In one aspect, the N- terminal ce is selected from an amino acid sequence that is 95% identical to SEQ ID NO:37, an amino acid sequence that is 95% identical to SEQ ID NO:89, or an amino acid sequence that is 95% identical to SEQ ID NOZ90. In one aspect, the N—terminal ce is selected from SEQ ID NO:37, positions 1 to 5 of SEQ ID NO:37, SEQ ID NO:89, or SEQ ID NO:90, or a ponding sequence from a different HBV strain.
In one aspect of any of the embodiments of the invention described above or elsewhere herein, the fusion protein is expressed by the yeast vehicle. In another aspect of any of the embodiments of the ion described above or elsewhere herein, the yeast e is a whole yeast. The whole yeast, in one aspect is killed. In one aspect, the whole yeast is heat-inactivated.
In one aspect of any of any of the embodiments of the invention described above or elsewhere , the yeast vehicle can be from a yeast genus selected from: Saccharomyces, Candida, Cryptococcus, Hansenula, romyces, Pichia, Rhodotorula, Schizosaccharomyces and Yarrowz'a. In one aspect, the yeast vehicle is from Saccharomyces. In one aspect, the yeast vehicle is from Saccharomyces cerevisiae.
In one aspect of any of the embodiments of the invention described above or elsewhere herein, the composition is formulated for administration to a subject or t.
In one aspect, the composition is formulated for administration by ion of a subject or patient (e.g., by a parenteral route, such as subcutaneous or intraperitoneal or intramuscular injection). In one aspect, the composition is formulated in a ceutically acceptable excipient that is suitable for administration to a human. In one aspect, the composition contains greater than 90% yeast protein. In one aspect, the composition contains greater than 90% yeast protein and is formulated for administration to a patient.
In one aspect of any of the embodiments of the invention described above or elsewhere herein, the fusion protein is not aggregated in the yeast. In one aspect, the fusion protein does not form inclusion bodies in the yeast. In one aspect, the fusion protein does not form VLPs or other large antigen particles in the yeast. In one aspect, the fusion protein does form VLPs or other large antigen particles in the yeast.
In one aspect of any embodiment of the invention described above or elsewhere herein, in one aspect, the HBV sequences are from HBV genotype A. In another aspect, the HBV sequences are from HBV genotype B. In another aspect, the HBV sequences are from HBV genotype C. In r aspect, the HBV sequences are from HBV genotype D. In another aspect, the HBV sequences are from HBV genotype E.
In another aspect, the HBV sequences are from HBV genotype F. In another , the HBV sequences are from HBV genotype G. In another aspect, the HBV sequences are from HBV genotype H. In one aspect, the HBV sequences are from a combination of any of the above-referenced HBV genotypes or of any known HBV genotypes or sub- genotypes.
Another embodiment of the invention s to any of the fusion proteins described above as part of an immunotherapeutic composition of the invention, or ere herein. In one aspect of this embodiment, a fusion protein comprises HBV antigens, the HBV ns selected from, but not limited to: (a) HBV antigens consisting of: HBV large surface antigen (L), HBV core protein and HBV X n; (b) HBV antigens consisting of: HBV large surface antigen (L) and HBV core protein; (c) HBV antigens consisting of: hepatocyte receptor of Pre—Sl of the HBV large surface antigen (L), HBV small surface antigen (S), the reverse transcriptase domain of HBV polymerase, HBV core protein or HBV gen, and HBV X antigen; (d) HBV antigens consisting of: HBV large surface n (L), the reverse riptase domain of HBV polymerase, HBV core protein or HBV gen, and HBV X antigen; (e) HBV antigens consisting of: HBV large surface antigen (L), the reverse transcriptase domain of HBV polymerase, and HBV core protein; (f) HBV antigens consisting of: HBV polymerase (RT domain) and HBV core protein; (g) HBV antigens consisting of: HBV X antigen and HBV core protein; (h) HBV antigens consisting of: hepatocyte or of Pre—Sl of the HBV large e n (L), HBV small surface antigen (S), the reverse transcriptase domain of HBV polymerase, and HBV core protein or HBV e-antigen; (i) HBV antigens consisting of HBV large surface antigen (L); (j) HBV antigens consisting of HBV core antigen; (k) HBV antigens ting of: HBV polymerase including the reverse transcriptase domain; (1) HBV antigens consisting of HBV X antigen; (m) HBV antigens consisting of n two and four HBV surface antigens, HBV polymerase antigens, HBV core antigens, or HBV X antigens, where each of the between two and four HBV antigens is from a 304153833 TIS510927NZPR different HBV genotype; and (n) HBV antigens consisting of two HBV core antigens and two HBV X antigens, wherein each of the two HBV core antigens and each of the two HBV X antigens are from a different HBV genotype. Aspects of the invention related to each of the HBV ns, including a variety of sequences useful in these antigens, have been described above. [0081A] In one aspect of this embodiment, the invention provides a fusion protein comprising HBV antigens, wherein the fusion protein comprises an amino acid sequence that is at least 90% identical to an amino acid sequence of SEQ ID NO:118, SEQ ID NO:116, SEQ ID NO: 114, SEQ ID NO:112 or SEQ ID NO:151.
In one aspect of this embodiment of the invention, the fusion protein comprises an amino acid ce that is at least 95% identical, or at least 96% cal, or at least 97% identical, or at least 98% identical, or at least 99% identical, or is identical, to an amino acid sequence selected from: SEQ ID NO:130, SEQ ID NO:150, SEQ ID , SEQ ID NO:151, SEQ ID NO:34, SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:132, SEQ ID NO:134, SEQ ID NO:112, SEQ ID NO:114, SEQ ID , SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, and SEQ ID NO:110.
Another embodiment of the ion relates to a recombinant nucleic acid molecule encoding any of the fusion proteins described herein. In one aspect, the inant nucleic acid molecule comprises a nucleic acid sequence selected from, but not limited to: SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:91, SEQ ID NO:111, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:119, SEQ ID , SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131, or SEQ ID NO:133. [0083A] Another embodiment of the invention s to a recombinant nucleic acid molecule encoding a fusion protein comprising HBV antigens, wherein the HBV ns consist of: i) an HBV surface antigen having an amino acid sequence that is at least 95% cal to ons 1 to 399 of SEQ ID NO:118, positions 1 to 399 of SEQ ID NO:116, positions 1 to 399 of SEQ ID NO: 114 or positions 1 to 399 of SEQ ID NO:112; and 304153833 TIS510927NZPR ii) an HBV core antigen having an amino acid sequence that is at least 95% identical to positions 400 to 581 of SEQ ID NO:118, positions 400 to 581 of SEQ ID NO:116, positions 400 to 581 of SEQ ID NO: 114 or positions 400 to 581 of SEQ ID NO:112. [0083B] Another embodiment of the invention provides an isolated cell ected with the recombinant nucleic acid molecule of the previous paragraph. [0083C] Another embodiment of the invention provides an isolated cell transfected with a inant nucleic acid molecule encoding a fusion protein wherein the fusion protein consists of an amino acid sequence of SEQ ID NO:118, SEQ ID NO:116, SEQ ID NO: 114 or SEQ ID NO:112.
Yet another embodiment of the invention relates to an isolated cell transfected with any of the recombinant nucleic acid molecules described herein. In one aspect, the cell is a yeast cell.
Another embodiment of the invention relates to a ition comprising any of the fusion proteins described herein. Yet another embodiment of the ion s to a composition comprising any of the recombinant nucleic acid les described herein. Another embodiment of the invention s to a composition comprising any of the ed cells described herein.
Yet another embodiment of the invention relates to a method to treat hepatitis B virus (HBV) infection or at least one symptom resulting from HBV infection in a subject, comprising administering to a subject that is infected with HBV at least one of 2012/024409 any of the immunotherapeutic compositions, including any HBV antigen, fusion protein, or yeast-based immunotherapeutic composition, described herein. The administration of the composition to the subject reduces HBV infection or at least one symptom resulting from HBV infection in a subject.
Yet another embodiment of the invention relates to a method to elicit an antigen-specific, cell-mediated immune response against an HBV antigen, comprising administering to a subject any one or more of the compositions, including any HBV antigen, fusion protein, or yeast-based immunotherapeutic composition, described herein.
Yet another embodiment of the invention relates to a method to prevent HBV infection in a subject, comprising administering to a subject that has not been infected with HBV, any one or more of the compositions, including any HBV antigen, fusion protein, or based therapeutic composition, described herein.
Another embodiment of the invention relates to a method to immunize a population of individuals against HBV, comprising stering to the tion of duals any one or more of the compositions, including any HBV antigen, fusion protein, or yeast-based immunotherapeutic ition, described herein.
Another embodiment of the invention relates to any one or more of the compositions, including any HBV antigen, fusion protein, or yeast-based immunotherapeutic composition, bed herein, for use to treat HBV infection or a symptom thereof.
Another embodiment of the ion relates to any one or more of the compositions, including any HBV antigen, fusion protein, or yeast-based immunotherapeutic composition, described herein, for use to prevent HBV infection or a m thereof.
Yet another embodiment of the invention relates to the use of any one or more of the compositions, including any HBV antigen, fusion protein, or yeast-based immunotherapeutic composition, described herein in the ation of a ment to treat HBV infection or a symptom thereof.
Yet another embodiment of the ion relates to the use of any one or more of the compositions, including any HBV antigen, filSlOIl protein, or yeast-based immunotherapeutic composition, described herein in the preparation of a medicament to prevent HBV ion or a symptom thereof.
In one aspect of any of the embodiments related to s or uses of the invention described above or elsewhere herein, the method can include administration of at least two, three, four or more of the compositions, ing any HBV antigen, fusion protein, or yeast-based immunotherapeutic composition, described herein. In one aspect, additional compositions or compounds useful for the prevention or treatment of HBV infection can be administered (e.g., iral compounds, interferons, other immunotherapeutic compositions, or combinations thereof). In one aspect, the various compositions or compounds are administered concurrently to an individual. In one aspect, the various itions or compounds are administered sequentially to an individual. In one aspect, each of the various compositions is administered by injection to a different site on the individual. In one aspect, a single dose of a yeast-based HBV immunotherapeutic composition of the invention is between 40 Y.U. total and 80 Y.U. total, administered in equal parts at two, three or four different sites on an individual, per close.
In one aspect of any of the embodiments related to methods or uses of the invention described above or elsewhere herein, administration of the composition to the subject causes seroconversion in the subject or improves seroconversion rates in a population of subjects. In one aspect, administration of the ition to the subject reduces serum HBsAg or results in loss of serum HBsAg in the subject or improves rates of loss of serum HBsAg in a population of subjects. In one aspect, stration of the composition to the subject reduces serum HBeAg or results in loss of serum HBeAg in the subject or improves rates of loss of serum HBeAg in a population of subjects. In one aspect, administration of the composition to the subject reduces HBV viral load in the subject or es rates in reduction of HBV viral load in a population of ts. In one aspect, administration of the composition to the subject results in undetectable HBV DNA in infected cells in the subject or results in higher rates of HBV DNA negativity in a population of subjects. In one aspect, administration of the composition to the t reduces liver damage or improves liver function in the subject or reduces the rate of liver damage or increases the rate of ed liver function in a population of subjects. In one aspect, administration of the composition to the subject improves ALT ization in the subject or in a population of ts.
In any of the embodiments related to an HBV antigen, fusion protein, therapeutic composition, or any method of use of the HBV antigen, fiision protein or immunotherapeutic ition described herein, in one aspect, the composition further comprises, or is used in conjunction with, at least one ical se modifier.
In one aspect, the composition further comprises, or is used in conjunction with, one or more onal compounds useful for treating or ameliorating a symptom of HBV infection. In one aspect, the composition r comprises, or is used in conjunction with, at least one anti-viral compound. In one aspect, the anti-viral is a nucleotide ue reverse riptase inhibitor. An anti—viral compound can include, but is not limited to, tenofovir, lamivudine, adefovir, telbivudine, entecavir, and combinations f. In one aspect, the anti-viral compound is tenofovir. In one aspect, the anti-viral compound is entecavir. In one aspect, the composition further comprises, or is used in ction with, at least one interferon. In one aspect, the interferon is interferon-0L. In one aspect, the interferon is pegylated interferon-(12a. In one aspect, the eron is interferon-X.
BRIEF DESCRIPTION OF THE DRAWINGS Fig. l is a schematic g showing the hepatitis B virus genome arrangement.
Fig. 2 is a schematic drawing showing the basic structure of a inant nucleic acid molecule encoding an HBV surface antigen/core fusion protein useful in a yeast-based immunotherapeutic composition of the invention.
Fig. 3 is a schematic drawing showing the basic structure of a recombinant nucleic acid molecule ng an HBV surface antigen/polymerase/core/X fiision protein useful in a yeast-based therapeutic composition of the invention.
Fig. 4 is a schematic drawing showing the basic ure of a recombinant nucleic acid le encoding an HBV polymerase/core fusion protein useful in a yeast- based immunotherapeutic composition of the invention.
Fig. 5 is a schematic g showing the basic structure of a recombinant nucleic acid molecule encoding an HBV X/core fusion protein useful in a based immunotherapeutic composition ofthe invention.
Fig. 6 is a schematic drawing showing the basic structure of a recombinant nucleic acid molecule encoding an HBV polymerase fusion protein useful in a yeast-based immunotherapeutic composition ofthe ion.
Fig. 7 is a schematic drawing showing the basic structure of a recombinant nucleic acid molecule encoding an HBV surface antigen/polymerase/core fusion protein useful in a yeast-based immunotherapeutic composition of the invention.
Fig. 8 is a schematic drawing showing the basic structure of a recombinant nucleic acid molecule encoding an HBV surface antigen/core/polymerase fusion protein usefiJl in a yeast-based immunotherapeutic composition of the invention.
Fig. 9 is a schematic drawing showing the basic structure of a recombinant nucleic acid molecule encoding an HBV surface antigen/core/X fusion protein useful in a yeast-based immunotherapeutic ition of the ion.
Fig. 10 is a schematic drawing showing the basic structure of a recombinant nucleic acid molecule encoding an HBV surface antigen/core/polymerase/X fusion protein useful in a yeast-based immunotherapeutic composition of the invention.
] Fig. 11 is a schematic drawing showing the basic structure of a recombinant nucleic acid molecule encoding an HBV surface antigen/core/X/polymerase fusion protein usefiJl in a yeast-based immunotherapeutic composition of the invention.
Fig. 12 is a schematic drawing showing the basic structure of a recombinant nucleic acid molecule encoding an HBV polymerase/surface antigen/core fusion n useful in a yeast-based immunotherapeutic composition of the invention.
Fig. 13 is a schematic drawing showing the basic structure of a recombinant nucleic acid molecule encoding an HBV X/surface antigen/core fusion protein useful in a yeast—based immunotherapeutic composition of the invention.
] Fig. 14 is a schematic drawing showing the basic structure of a recombinant nucleic acid molecule encoding an HBV polymerase/X/surface antigen/core fiasion protein usefiJl in a yeast-based immunotherapeutic composition of the invention.
Fig. 15 is a schematic drawing showing the basic structure of a inant nucleic acid molecule encoding an HBV X/polymerase/surface antigen/core fusion protein usefiJl in a yeast-based immunotherapeutic composition of the invention.
Fig. 16 is a digital image of a Western blot showing expression of l yeast-based immunotherapeutic compositions expressing an HBV e antigen/Core fusion n (heat-killed, whole yeast).
Fig. 17 is a digital image of a Western blot showing expression of several yeast-based immunotherapeutic itions sing an HBV Surface antigen/Core fusion protein (live, whole yeast).
Fig. 18 is a digital image of a Western blot showing expression of several yeast-based immunotherapeutic compositions sing an HBV surface antigen/polymerase/core/X fusion protein.
Fig. 19 is a digital image of a Western blot g sion of several yeast-based immunotherapeutic compositions expressing an HBV surface antigen/polymerase/core/X fusion protein.
Fig. 20 is a digital image of a Western blot showing expression of several yeast-based immunotherapeutic compositions expressing HBV antigens comprising surface-core fusion ns (Sc) or surface-polymerase-core—X fusion proteins (Sp).
Fig. 21 is a digital image of a n blot showing expression of HBV antigens from l based HBV immunotherapeutic compositions cultured in UL2 medium.
Fig. 22 is a bar graph showing the average expression of HBV antigens from several based HBV immunotherapeutic compositions cultured in UL2 medium or U2 medium (error bars are rd Deviation).
Fig. 23 is a graph showing the proliferation of splenic CD4+ T cells from mice immunized with a yeast-based immunotherapeutic product expressing an HBV Surface- Core antigen ) to an S/Core antigen mix or to a MHC Class II SAg mimetope peptide (error bars are Standard Deviation).
Fig. 24 is a graph showing the proliferation of lymph node T cells from mice immunized with a yeast-based immunotherapeutic t sing an HBV Surface- Core antigen (SCORE) to an S/Core antigen mix or to a MHC Class II SAg mimetope peptide (error bars are Standard Deviation).
Fig. 25 is a graph showing the interferon-y (IFN-y) ELISpot response of lymph node T cells from mice immunized with a yeast-based immunotherapeutic product expressing an HBV Surface-Core n (SCORE) to an S/Core antigen mix or to a MHC Class II SAg mimetope peptide.
Fig. 26 is a graph showing the proliferation of splenic CD4+ T cells from mice immunized with a yeast-based immunotherapeutic product expressing an HBV Surface- Pol-Core-X antigen (denoted a-Spex) to an S/Core antigen mix or to a MHC Class II SAg mimetope peptide (error bars are Standard Deviation).
Fig. 27 is a graph showing IL-lB production in splenocytes from mice immunized with: (a) a yeast-based immunotherapeutic product sing an HBV Surface-Pol-E/Core-X antigen (denoted Sp), left columns; or (b) a yeast-based immunotherapeutic product expressing an HBV e-Core antigen (denoted Sc) (error bars are Standard Deviation).
Fig. 28 is a graph showing 70 production in splenocytes from mice immunized with: (a) a yeast-based immunotherapeutic product expressing an HBV e-Pol-Core-X antigen (denoted Sp), left columns; or (b) a yeast-based 2012/024409 immunotherapeutic product expressing an HBV Surface-Core antigen (denoted Sc) (error bars are Standard Deviation).
Figs. 29A and 29B are graphs showing eron—y (IFN—y) production in splenocytes from mice immunized with: (Fig. 29A) a yeast-based immunotherapeutic product expressing an HBV Surface-Core antigen (denoted Sc) or (Fig. 29B) a yeast-based immunotherapeutic product expressing an HBV Surface-Pol-Core-X antigen (denoted Sp) (error bars are Standard Deviation).
Figs. 30A-D are graphs showing IL-lB (Fig. 30A), IL—6 (Fig. 30B), IL—l3 (Fig. 30C), and IL-12p70 (Fig. 30D) production in splenocytes from mice immunized with: (a) a yeast-based immunotherapeutic product expressing an HBV Surface-Pol-Core-X antigen (denoted Sp), left s; or (b) a yeast-based immunotherapeutic product expressing an HBV Surface-Core antigen ed Sc).
Fig. 31 is a bar graph showing that mice immunized with GI-l3002 or G]- 13002 + anti-CD40 antibody, but not YVEC, ed comparable protection from challenge with EL4 tumors expressing the target HBV antigen (error bars are Standard Deviation).
] Fig. 32 is a bar graph showing the results of an IFN-y ELISpot assay comparing T cell ses of mice immunized with GI-l3008 (SCORE-C) and GI-l3013 (SPEXVZ) as compared to YVEC using a variety ofHBV peptides and ns (error bars are Standard Deviation).
Fig. 33 is a bar graph showing IFN-y ELISpot responses to stimulation with 02 from a human subject pre- and post-immunization, and post-boost, with a prophylactic HBV vaccine.
Fig. 34 is a bar graph showing HBV antigen—specific IFN—y ELISpot responses from lymph node cells ed from HLA-A2 enic mice immunized with GI-l3009 (SCORE-D) or GI-l3020 (X-SCORE) as compared to mice immunized with a yeast control (YVEC) (error bars are Standard Deviation).
Fig. 35 is a bar graph showing HBV antigen—specific IFN—y ELISpot responses from spleen cells isolated from HLA-AZ transgenic mice immunized with (31-13009 (SCORE-D) as compared to mice immunized with a yeast control (YVEC) (error bars are Standard Error).
Fig. 36 is a bar graph showing HBV antigen-specific IFN—y ELISpot responses from lymph node cells isolated from C57BL/6 mice immunized with 09 (SCORE- D) or GI-l3020 RE) as compared to mice immunized with a yeast control (YVEC) or Naive mice (error bars are rd Deviation).
Fig. 37 is a line graph showing HBV n—specific CD8+ T cell responses to an MHC Class I-restricted HBV peptide in C57BL/6 mice immunized with GI-I3009 (SCORE-D) or GI-l3020 (X-SCORE) as compared to mice immunized with a yeast control (YVEC) or a yeast-based immunotherapeutic expressing ovalbumin (OVAX).
Fig. 38 is a bar graph showing HBV antigen-specific CD4+ T cell responses to an MHC Class II-restricted HBV peptide in C57BL/6 mice zed with (31-13009 (SCORE-D) or GI-13020 (X-SCORE) as compared to mice immunized with a yeast control (YVEC) or a yeast-based immunotherapeutic expressing ovalbumin (OVAX).
DETAILED PTION OF THE INVENTION This invention generally relates to compositions and methods for preventing and/or treating hepatitis B virus (HBV) infection. The invention includes a yeast-based immunotherapeutic composition (also referred to as "yeast-based HBV immunotherapy") comprising a yeast vehicle and HBV antigen(s) that have been designed to elicit a prophylactic and/or therapeutic immune se against HBV infection in an individual, and the use of such compositions to prevent and/or treat HBV ion and related symptoms f. The invention also includes the recombinant nucleic acid molecules used in the yeast-based compositions of the invention, as well as the proteins and fusion proteins encoded thereby, for use in any immunotherapeutic composition and/or any therapeutic or prophylactic protocol for HBV infection, including any therapeutic or prophylactic protocol that combines the HBV-specific yeast-based itions of the ion with any one or more other therapeutic or prophylactic compositions, agents, drugs, compounds, and/or protocols for HBV infection.
The yeast—based, HBV-specific immunotherapeutic compositions are unique among various types of immunotherapy, in that these compositions of the ion induce innate immune responses, as well as adaptive immune responses that specifically target HBV, including CD4-dependent THl7 and THI T cell ses and antigen-specific CD8+ T cell responses. The breadth of the immune response elicited by HBV-specific yeast-based immunotherapy is well-suited to target HBV. First, HBV is believed to evade the innate immune response early in ion by "hiding" from the innate response and thereby not inducing it, rather than by directly counteracting innate immunity nd and Chisari, 2005, J. Viral. 9—9380; Wieland, et al., 2004, PNAS USA 101:6669- 6674). Accordingly, it can be expected that HBV will be sensitive to innate immune responses if they are ted by another mechanism, z'.e., the yeast—based immunotherapeutic compositions of the invention. Second, HBV produces high-level antigen expression in infected host cells that is expected to be visible to the adaptive immune response (Guidotti, et al., 1999, Science 284:825—829; Thimme et al., 2003, J.
Viral. 77:68-76), and nce of acute infection has been associated with robust CD4+ and CD8+ T cell responses (Maini et al., 1999, Gastroenterol. 117:1386-1396; ann et al., 1995, J. Exp. Med. 181:1047-1058; Thimme et al., 2003, J. Viral. 77:68-76; Wieland and Chisari, 2005, J. Viral. 15:9369-9380). Therefore, yeast-based HBV immunotherapy, by activating the adaptive immune response, is expected to effectively target HBV-infected cells for destruction and/or is expected to effectively enhance viral nce. Moreover, the immune response generated by yeast-based immunotherapy is believed to be interferon-independent and interferon-dependent (Tamburini et al., 2012, J.
Immunoz‘her. 35(1):]4-22); accordingly, the ability, or lack f, of an individual to respond to interferon-based therapy, which is one standard of care treatment for HBV, is not believed to directly impact the ability of the subject to respond to yeast-based immunotherapy of the ion. In addition, the yeast-based HBV immunotherapy compositions described herein are designed to target genic and conserved regions of HBV, multiple CTL epitopes, and include regions of HBV that may be targeted for escape (allowing for modifications of the compositions as needed to target such escape mutations), making it a highly ble y for HBV that optimizes the opportunity for effective immune ses against this virus.
In addition, and without being bound by theory, yeast-based therapy for HBV is believed to induce an immune response that is not only directed cally against the target n carried by the yeast-based immunotherapeutic product, but that also evolves to be directed against other immunological epitopes on the Virus (i.e., other than those carried by the yeast-antigen ition). In other words, a primary cellular immune response to the antigen(s) and/or epitope(s) contained in the yeast-based immunotherapeutic can lead to ary cellular immune responses to antigen(s) and/or epitope(s) that are present in the infected cells in the treated subject but that are not present in the yeast-based immunotherapeutic, thereby leading to the evolution of complex and unpredictable immune response s that are unique to each treated subject. These secondary immune responses are specific to the molecular profile of the HBV ion in each subject treated, and the yeast-based immunotherapeutic may drive these downstream effects in a unique manner when compared to other treatment modalities, including other immunotherapy platforms. This phenomenon may also be generally referred to as "epitope spreading" and represents an age of using yeast-based HBV immunotherapy, because induction of an immune response against a ular HBV antigen or even against a particular HBV genotype (e.g., by providing that antigen in the context of the yeast immunotherapeutic), is expected to result in the cascading targeting of the immune system against a variety of additional HBV antigens, which may result in effective immune responses against antigens from ent HBV genotypes or strains than those represented in the yeast-based immunotherapeutic composition.
As discussed above, patients who become chronically infected with HBV tend to have weaker (or absent) and more narrow HBV-specific, T cell-mediated immunity.
Accordingly, the yeast-based HBV immunotherapy compositions of the invention address the need for therapeutic compositions to treat patients who are actively ed with HBV, including chronically infected patients, and further provide an additional vaccine for the prevention of HBV infection that may have advantages with respect to the production of durable memory immune responses. , the yeast-based HBV therapy compositions of the ion are expected to promote durable memory T cell responses against HBV, which can t infection, as well as provide long term s that can protect a chronically infected patient from viral reactivation. based HBV immunotherapy compositions as monotherapy or in combination with other therapeutic approaches for the ent of HBV (e.g., in combination with anti-viral compounds) are expected to increase the percentage of chronically infected ts who achieve clearance of HBsAg and HBeAg, who achieve complete seroconversion, and/or who e sustained viral clearance for at least 6 months after the completion of therapy.
Accordingly, yeast—based HBV immunotherapy can be combined with anti— viral drugs and/or interferon therapy, and/or with other therapies for HBV, in order to reduce the viral load in an individual to a level that can be more ively handled by the immune system. HBV Viral titers are typically very high (as many as 1011 hepatocytes may be infected) and thus may overwhelm an individual’s ability to mount an effective CTL se; accordingly, reduction of viral load using iral drugs in combination with induction of HBV-specific CTL activity using yeast-based immunotherapy is expected to be ial to the infected individual. In addition, reduction of viral load through the use of anti-viral drugs may also reduce negative effects, if any, of immune activation in the context of a high number of infected hepatocytes being targeted for destruction. Yeast-based HBV immunotherapy is also expected to play a role in reducing 2012/024409 and/or ating compartments of latent viral ion. For example, there are many tissues that have been shown to be HBV-positive by PCR, and that are considered potential sanctuaries for re-activation of HBV. HBV DNA can integrate into the host genome, which provides for a ent persistence of HBV, and cchNA is a supercoiled, dormant form of the HBV genome that also contributes to quiescence. Without being bound by theory, the inventors believe that yeast-based HBV immunotherapy described herein will play a role in eliminating all of these types of HBV "sanctuaries" that likely contribute to the low disease—free cure rate observed with the current anti-viral approaches.
In r scenario, use of a yeast-based HBV immunotherapeutic of the invention, alone or in combination with an anti—viral or other HBV therapeutic, if sufficient to achieve te clearance of HBsAg, but not sufficient to achieve anti-HB production, may be followed by, or further combined with, existing lactic t vaccines to achieve complete seroconversion. Alternatively, any of the fusion proteins described herein may also be used as subunit vaccines to achieve complete seroconversion, or to protect a subject from HBV infection, alone or in ation with a yeast-based HBV immunotherapeutic of the invention. Finally, the immunotherapeutic ition of the invention is well-suited for modification and/or combination with additional immunotherapeutic compositions, including any described herein, to treat escape mutations ofHBV that are elicited by treatment with anti-viral drugs.
Yeast-based immunotherapeutic itions are stered as biologics or pharmaceutically acceptable compositions. Accordingly, rather than using yeast as an antigen production system followed by purification of the antigen from the yeast, the entire yeast vehicle as described herein must be suitable for, and formulated for, administration to a patient. In contrast, existing commercial HBV vaccines as well as many in development, comprise recombinant HBV proteins (e.g, HBsAg proteins) that are produced in Saccharomyces cerevisiae, but are uently released from the yeast by disruption and purified from the yeast so that the final vaccine, combined with an adjuvant (e.g., aluminum hydroxyphosphate e or aluminum hydroxide), contains no detectable yeast DNA and contains no more than l-5% yeast protein. The HBV yeast- based immunotherapeutic compositions of the invention, on the other hand, contain readily able yeast DNA and contain substantially more than 5% yeast protein; generally, yeast-based immunotherapeutics of the invention contain more than 70%, more than 80%, or generally more than 90% yeast protein. based immunotherapeutic compositions are administered to a patient in order to immunize the patient for therapeutic and/or prophylactic purposes. In one embodiment of the ion, the yeast—based compositions are formulated for administration in a ceutically acceptable excipient or formulation. The composition should be formulated, in one aspect, to be suitable for administration to a human subject (e. g., the manufacturing conditions should be le for use in , and any excipients or formulations used to finish the composition and/or prepare the dose of the immunotherapeutic for administration should be suitable for use in ). In one aspect of the invention, based immunotherapeutic compositions are formulated for stration by injection of the patient or subject, such as by a parenteral route (e. g., by subcutaneous, intraperitoneal, intramuscular or intradermal injection, or another suitable parenteral route).
In one ment, the yeast express the antigen (e. g., detectable by a Western blot), and the antigen is not aggregated in the yeast, the antigen does not form inclusion bodies in the yeast, and/or does not form very large particles (VLPs) or other large antigen particles in the yeast. In one embodiment, the n is produced as a soluble protein in the yeast, and/or is not secreted from the yeast or is not substantially or primarily secreted from the yeast. In another embodiment, without being bound by theory, the present inventors believe that particular combinations and perhaps, arrangements, of antigens in an HBV fusion protein including surface antigen and core antigen, described in detail herein, may form VLPs or aggregate to some extent within the yeast expressing the antigens. As a , the antigen expressed by the yeast has immunogenic properties which appear to be related to its overall structure and form, as a separate characteristic from the immunogenic properties of the immune es (e.g., T cell epitopes) carried within the antigen. When the yeast expressing such fusion proteins are provided in a yeast-based HBV immunotherapeutic of the invention, the immunotherapeutic composition s properties that activate the innate immune system not only from the yeast vehicle as discussed above (as with all yeast-based immunotherapeutics described herein), but also in part from the fusion protein antigen structure (e.g, the surface-core fusion protein as expressed in the yeast also has nt-like properties); in on, the immunotherapeutic composition derives properties that activate the adaptive immune system in an antigen-specific manner from the fusion protein (via provision of various T cell epitopes), as with all of the yeast-based immunotherapeutics described herein. This specific combination of properties appears to be unique to yeast-based therapeutics expressing particular surface-core fusion proteins from HBV described herein. However, in all of the embodiments of the invention described herein, the yeast-based immunotherapeutics should be readily phagocytosed by tic cells of the immune system, and the yeast and antigens y processed by such dendritic cells, in order to elicit an ive immune response against HBV.
Compositions of the Invention One embodiment of the present invention s to a yeast-based immunotherapy composition which can be used to prevent and/or treat HBV infection and/or to alleviate at least one symptom resulting from the HBV infection. The composition comprises: (a) a yeast vehicle; and (b) one or more antigens comprising HBV protein(s) and/or immunogenic domain(s) thereof. In conjunction with the yeast e, the HBV proteins are most typically expressed as recombinant proteins by the yeast vehicle (eg, by an intact yeast or yeast spheroplast, which can optionally be further processed to a yeast cytoplast, yeast ghost, or yeast membrane t or fraction thereof), although it is an embodiment of the invention that one or more such HBV proteins are loaded into a yeast vehicle or ise complexed with, attached to, mixed with or administered with a yeast vehicle as described herein to form a ition of the present ion. According to the present invention, reference to a "heterologous" protein or "heterologous" antigen, including a heterologous fusion protein, in connection with a yeast vehicle of the invention, means that the protein or antigen is not a protein or antigen that is lly sed by the yeast, gh a fusion protein that es heterologous antigen or heterologous protein may also include yeast sequences or proteins or portions thereof that are also naturally expressed by yeast (e.g, an alpha factor prepro sequence as described herein).
One embodiment of the invention relates to various HBV fusion proteins. In one aspect, such HBV fusion proteins are useful in a yeast-based immunotherapeutic composition of the invention. Such fusion proteins, and/or the recombinant c acid molecules encoding such proteins, can also be used in, in combination with, or to produce, a non-yeast-based immunotherapeutic composition, which may include, without limitation, a DNA vaccine, a protein subunit vaccine, a recombinant viral-based immunotherapeutic composition, a killed or inactivated pathogen vaccine, and/or a tic cell vaccine. In another embodiment, such fusion proteins can be used in a diagnostic assay for HBV and/or to generate dies against HBV. Described herein are exemplary HBV fusion proteins providing selected portions of HBV antigens, including, for example, selected ns of and/or modified polymerase; selected portions of and/or d surface antigen; selected portions of and/or modified core (including at least portions of or most of e-antigen); selected portions of and/or modified X antigen; as well as selected portions of and/or arrangements of any one, two, three or all four of the antigens (surface antigen, core, X and polymerase), such as, but not limited to, selected portions and/or arrangements of surface antigen and core (including at least portions of or most of e—antigen); selected portions and/or arrangements of surface n, core (including at least ns of or most of e-antigen), polymerase and X antigen; selected ns and/or arrangements of surface antigen, core (including at least portions of or most of e-antigen), and polymerase; and selected portions and/or ements of surface antigen, core (including at least portions of or most of e-antigen), and X antigen.
In one embodiment, HBV antigens, including immunogenic domains of full- length proteins, as described herein, are fused to host proteins that are overexpressed in HBV infected, but not in fected, host cells. In one embodiment, HBV antigens, including immunogenic domains of ength proteins, as described herein, are fused to protein R2, a host factor required for HBV replication, which in one embodiment, is expressed in cytes. R2 is a protein component of ribonucleotide reductase (RNR), and is critical for the HBV life-cycle (see, e.g., Cohen et al., 2010, Hepatol. 51(5):1538- 1546). Other embodiments of the invention will be apparent in view of the disclosure provided herein.
Hepatitis B Virus, Genes, and Proteins. Hepatitis B virus (HBV) is a member of the Hepadnaviridae (hepadnavirus) family of s and causes transient and chronic infections of the liver in humans and the great apes. The aviruses that infect mammals have similar DNA sequences and genome organization, and are grouped in the genus Orthohepadnavirus. The hepatitis B virus particle has an outer envelope containing lipid and surface antigen particles known as HBsAg. A nucleocapsid core containing core protein (HBcAg) surrounds the viral DNA and a DNA polymerase with reverse transcriptase ty. As ed in Seeger and Mason, 2000, Microbiol. Mol. Biol. Rev. 64(1):51-68, HBV has a 3.2 kb partially double-stranded relaxed-circular DNA (rcDNA) genome that is converted into a ntly closed circular double—stranded DNA (cchNA) molecule upon delivery of the viral genome to the nucleus of an infected hepatocyte. The host cell RNA polymerase II transcribes four viral RNAs from the cchNA template which are transported to the host cell cytoplasm. The viral RNAs include mRNAs that are transcribed to e the viral core and envelope structural proteins and the precore, polymerase and X nonstructural viral proteins. The RNA that is translated to produce core and polymerase also serves as the pregenomic RNA ) which is the template for reverse ription. pgRNA and the polymerase are encapsulated by the core protein, producing the viral nucleocapsid where the pgRNA is reverse transcribed into rcDNA. These rcDNA-containing nucleocapsids are then enclosed by envelope ns and secreted from the host cell as mature virions or shuttled to the nucleus to amplify the viral cchNA.
The structural and non-structural proteins produced by the HBV genome are shown in Table 1. The partially double-stranded HBV genome contains four genes known as C, X, P, and S (see also Fig. 1).
Table 1. HBV genes and gene products Function(s) —HBcAg)—Formsviral capsidsurrounding viral pgRNA and polymerasecore protein suppressive factor for veImmune response Polymerase for viral DNA replication - Domain 1: terminal protein (TP) domain packages polymerase pgRNA and primes minus strand DNA - Domain2: reverse transcriptase (RT) domain RNase H; deoradespoRNA S HBsAg(s(urface Envelope protein and forms e antigen particles; may antI en small suporess immune function M HBsAg (surface _ Envelope protein and forms surface n particles antigen; middle— Pre-82 together WIth 8, may suppress Immune functIon. . .
Envelope protein and forms surface antigen particles L HBsAg (surface together with S; pre-S1 domain provides ligand for core antigen; large— Pre-81 + particles during assembly of viral envelope; hepatocyte pre-8—2 + S) or; ma suppress immune function Transcriptional transactivation; regulation of DNA repair ys; elevation of cytosolic calcium ; modulation X antigen (HBx) of protein degradation pathways; modulation of cell cycle progression and cell eration pathways in host cell; stimulation of HBV replication Gene C encodes two closely related antigens: a 21—kDa protein called "core protein" or "core antigen" (HBcAg) which forms the viral capsid, and a 17-kDa protein called gen (HBeAg) that forms dimers but that does not assemble into . Full- length core protein is an approximately 183 amino acid protein, comprising all but the N— terminal 10 amino acids of e-antigen and comprising approximately 34 additional amino acids at the C-terminus that are proteolytically cleaved in the tion of e-antigen. In other words, core n and e-antigen have 149 amino acid residues in common (this section sometimes being referred to as the hepatitis core antigen), but differ at the N- terminal and C—terminal s. Precore protein is a precursor protein comprising an amino acid sequence that includes sequence from both core and e-antigen, from which e- n is produced via proteolytic processing. Intracellular HBeAg includes precore residues -29 to -1 (the residue numbering in this particular description is provided with the first amino acid residue of core protein within the precore protein being denoted as position "1"), which ns a signal sequence that directs the protein to the endoplasmic reticulum at which point amino acids -29 to -11 are cleaved; another proteolytic cleavage between amino acids 149 and 150 removes the C—terminal portion of precore (which is present in full-length core protein), and the remaining HBeAg sting of amino acids - to -1 of precore plus amino acids 1—149 ofHBcAg or core) is then secreted as gen (Standing et al., 1988, PNAS USA 85: 8405-8409; Ou et al., 1986, PNAS USA 83:1578- 1582; Bruss and Gerlich, 1988, Virology 163:268-275; Takahashi etal., 1983, J. Immunol. 130:2903-2907). HBeAg consisting of the entire precore region has also been found in human sera (Takahashi et al., 1992, J. Immunol. 147:3156-3160). As mentioned, HBcAg (core) forms dimers that assemble into the viral capsid and n the polymerase and viral DNA or pgRNA. The function of HBeAg (e-antigen) is unknown, but it is not required for HBV replication or infection, and it is thought to be an immune suppressive factor that protects HBV against attack by the immune system (Milich et al., 1990, PNAS USA 87:6599-6603; Che et al., 2004, PNAS USA 101:14913-14918; Wieland and Chisari, 2005, J. Virol. 79:9369-9380). For clarity, in the HBV sequences described herein (e.g, see Table 3), the sequence for precore from representative HBV genotypes is provided, and the positions of core protein and e-antigen are denoted within the precore sequence, with the first amino acid of precore designated as on 1.
Gene P encodes the HBV DNA polymerase (Pol), which consists of two major domains linked by a spacer. The N—terminal domain of the polymerase (also referred to as "terminal protein" or TP) is involved in the packaging of pgRNA and in the priming of non-sense strand DNA. The C—terminal domain is a reverse riptase (RT) that has RNase H (RH) ty.
] Gene S has multiple start codons and encodes three envelope ns (also referred to herein generally as "surface protein" or ce n") denoted S, M and L, which are all components of the infectious viral particles, also known as Dane particles. S, by itself, and together with M and L, also form surface antigen particles (HBsAg) which can be secreted from infected cells in large quantities (Seeger and Mason, 2000, Microbiol.
Mol. Biol. Rev. 51-68; Beck, (2007), "Hepatitis B virus replication", World Journal WO 09404 of Gastroenterology .' WJG 13(1):48—64). The codons for M and L are located approximately 165 (M) and 489 (L) nucleotides, respectively, upstream of the initiation codon for S. S or "small" surface antigen is the smallest and most abundant of the surface antigens. Antibodies produced against this antigen represent seroconversion in infected individuals. M or "middle" surface antigen has an extra protein domain, as compared to S, known as pre—S2, and the protein domain that is unique to L or "large" surface antigen is known as pre-Sl (L therefore also contains pre—S2 and the additional sequence belonging to M and S). Pre-Sl contains the Viral hepatocyte receptor domain (hepatocyte receptor binding site), which is located approximately n amino acid positions 21 and 47 of Pre-Sl. Epitopes in pre-Sl can elicit Virus-neutralizing antibodies. In addition, the pre-Sl domain provides the ligand for core particles during the ly of the Viral envelope.
Surface antigen particles (HBsAg) may also ss immune elimination of ed cells by fianctioning as a high-dose toleragen (Reignat et al., 2002, J Exp. Med. 195:1089-1101; Webster et al., 2004, J. Viral. 78:5707-5719).
Gene X encodes X antigen (HBx) (which may also be referred to as "X protein") which is involved in riptional transactivation, regulation of DNA repair pathways, elevation of cytosolic calcium levels, modulation of protein degradation pathways, and modulation of cell cycle progression and cell proliferation ys in the host cell (Gearhart et al., 2010, J. Viral), which enhances stimulation ofHBV replication.
HBx is also ated with the development of liver cancer (Kim et al., Nature 1991, 351:317-320; illos et al., Oncogene 1997, 14:395-404).
HBV is found as one of four major serotypes (adr, adw, ayr, ayw) that are determined based on antigenic epitopes within its pe proteins. There are eight different HBV genotypes (A-H) based on the nucleotide ce ions in the genome. The geographical distribution of the genotypes is shown in Table 2 (KramVis et al., 2005, Vaccine 23(19):2409-2423; Magnius and Norder, 1995, Intervirology 38(1- 2):24-34; Sakamoto et al., 2006, J. Gen. Virol. 87:1873-1882; Lim et al., 2006,1nz‘. J. Med.
Sci. 3:14-20).
Table 2 HBV genotype Prevalent Geographical Distribution HBV/A Americas, Europe, Africa, ast Asia Asia (China, Japan, Southeast Asia), HBV/B United States Asia (China, Japan, Southeast Asia), HBV/C United States United States, Mediterranean, Middle HBV/D East and IndIa HBV/E Sub-Saharan and West Africa HBV/F Central and South America HBV/G France, Germany, United States Central America, United States HBV/H California The nucleic acid and amino acid sequence for HBV genes and the proteins encoded thereby are known in the art for each of the known genotypes. Table 3 provides reference to sequence identifiers for ary sentative) amino acid sequences of all of the HBV structural and non-structural proteins in each of the eight known genotypes of HBV, and further indicates the position of certain structural domains. It is noted that small variations may occur in the amino acid sequence between different viral isolates of the same protein or domain from the same HBV genotype. However, as sed above, strains and serotypes of HBV and genotypes of HBV display high amino acid identity even between serotypes and genotypes (e.g., see Table 4). Therefore, using the guidance provided herein and the reference to the exemplary HBV sequences, one of skill in the art will readily be able to produce a variety of HBV-based proteins, including fusion proteins, from any HBV strain (isolate), pe, or pe, for use in the compositions and methods of the present invention, and as such, the invention is not limited to the c sequences disclosed herein. Reference to an HBV protein or HBV antigen anywhere in this disclosure, or to any functional, structural, or immunogenic domain thereof, can accordingly be made by reference to a particular sequence from one or more of the sequences ted in this disclosure, or by reference to the same, similar or corresponding sequence from a ent HBV isolate (strain).
Table 3 sm, Genotype,m ce Identifier Gene Database Accession No. ion No. AAX83988.1 HBV, Genotype A, C POSItIons 30133-312 of SEQ ID . Core (HBcAg) *Positions 20-178 of SEQ ID NO:1 AcceSSIon No. BAI81985 HBV, Genotype A, P ' reverse *Positions 383-602 of SEQ ID N012 transcriptase SEQ ID NC).3 eurraceHBsAg _ Accession No BAD91280 1 HBV pe A S Surface HBsA- *Positions 0 of SEQ ID NO: 3 Surface HBsA- *Positions 175-400 of SEQ ID NC): 3 SEQ ID NC):4 HBV’ Genotype A’ X X (HBX) Accession No.AAK97189.1 .V V . . .V V siEQ-IDNo.5.
Precore Accession No. BAD90067 HBV, Genotype B, C ions 30133-212 of SEQ ID . Core (HBcAg) 0 e-antioen HBer *Positions 20-178 of SEQ ID NO:5 9566900681 HBV, Genotype B, P I I *Positions 381-600 of SEQ ID NO:6 transcriptase SEQ ID NC).7 _ Accession No BAJ06634 1 HBV GenOtype B S Surface HBsAo *Positions 120-400 of SEQ ID NO: 7 Surface HBsA- *Positions 175-400 of SEQ ID NO: 7 SEQ ID NO:8 HBV’ Genotype 8’ X X (HBX) Accession No. BAD90066.1 SEQ ID NQ:9 Precore Accession No. YP_355335 HBV, Genotype C, C *Positions 30:33:12 of SEQ ID . Core (HBcAg) 0 e-antioen HBer *Positions 20-178 of SEQ ID NO:9 SEQ ID NO:10 Pol merasey Accession No. ACH57822 HBV Genet 9 c P ’ yp ’ 0 reverse *Positions 381-600 of SEQ ID transcriptase NO:10 SEQ ID NO:11 surface HBSAQ (L) Accession No. BAJ06646.1 ons 13954120 0f SEQ 'D HBV, Genotype c, 3 Surface HBsAg (M) POSItIons "50-41010 of SEQ ID Surface HBsAg (S) SEQ ID NO: 12 HBV’ Genotype C’ X X (HBX) Accession No. BAJ06639. 1 SEQ ID NC): 13 Precore ion No. ADF29260.1 HBV Genot e D C I yp 1 *Positions 212 of SEQ ID 0 Core (HBcAg) NO'13 Organism, Genotype, Sequence Identifier Gene Database Accession No. —. e-antioen HBeA *Positions 20-178 ofSEQ ID NO:13 SEQ ID NO:14 Pol y Accession No. ADD12642.1 HBV Genet 6 D P ’ yp ’ 0 reverse *Positions 370-589 of SEQ ID transcriotase NO:14 SEQ ID No:15 surface HBSAQ (L) Accession No. 63.1 Pos't'ons 18353129 0f SEQ 'D HBV, Genotype D, 8 Surface HBsAg (M) POSItIons 1345—3129 of SEQ ID Surface HBsAg (S) SEQ ID NO: 16 Accession No. ACU25047. 1 HBV, Genotype E, C Positions 3011133712712 of SEQ ID . Core (HBcAg) 0 e-antioen HBer *Positions 20-178 of SEQ ID NO:17 SEQ ID NO:18 Pol y Accession No. AC089764.1 HBV Genet ’ yp6 E P’ *Positions 380-599 of SEQ ID transcriptase NO:18 SEQ ID NO:19 surface HBSAQ (L) Accession No. BAD91274.1 Pos't'ons HBV, Genotype E, S Surface HBsAg (M) 531? 0f SEQ 'D POSItIons 15:13-31? of SEQ ID Surface HBsAg (S) SEQ ID NO:20 1' I I I I» I SEQID NQ:21 Precore Accession No. BAB17946. 1 HBV, Genotype F, C Positions BONE-22112 of SEQ ID . Core (HBcAg) *Positions 20-178 of SEQ ID NO:21 SEQ ID NO:22 Pol merasey Accession No. ACD03788.2 HBV Geno ypet F P ’ ’ 0 reverse *Positions 381-600 of SEQ ID transcriptase NO:22 SEQ ID NO:23 surface HBSAg (L) Accession No. 33.1 POSItIons 135-42330 of SEQ ID HBV, Genotype F, 8 Surface HBsAg (M) Surface HBsAg (S) 1550-42030 of SEQ ID SEQ ID NO:24 HBV pe F X X (HBX) AcceSSIon No. AAM090541 1' 1' H ' SEQID NQ:25 Accession No. ADD62622. 1 HBV’ Genome G’ C Core HBcAo *Positions 14-194 of SEQ ID NO:25 o e-antien HBer *Positions 4-161 of SEQ ID NO:25 SEQ ID NO:26 HBV’ Gemtype G’ P Accession No. ADD62619.1 Organism, Genotype,m Sequence Identifier Gene se Accession No. 0 e *Positions 380-599 of SEQ ID riptase NO:26 SEQ ID N012? surface (HBSAQ) (L) Accession No. ADD62620.1 POSItIons 1&2? Of SEQ ID HBV, Genotype G, 8 Surface HBsAg (M) POSItIons K‘s-32979 of SEQ ID Surface HBsAg (S) SEQ ID NO:28 HBV’ Gemtype G X X(HEX) ion No BABBZ4OO_1 SEQ ID NO:29 Precore Accession No. BAD91265. 1 HBV, Genotype H, C Positions 32331-525? of SEQ ID . Core (HBcAg) 0 e-anti-en HBer *Positions 20—178 of SEQ ID NO:29 SEQ ID NO:30 Accession No. BAF49208.1 HBV Genot e H P ’ yp ’ 0 reverse *Positions 381-600 of SEQ ID transcriptase NO:3O SEQ ID NO:31 surface HBSAQ (L) Accession No. BAE20065.1 ons 13%?) 0f SEQ 'D HBV, Genotype H, 3 Surface HBsAg (M) Posmons K5620? of SEQ ID Surface HBsAg (S) SEQ ID NO:32 HBV’ Gemtype H’ X X (HBX) Accession No. 06.1 *Position numbering is approximate and may include additional amino acids flanking either side of the indicated position Hepatitis B Virus Antigens and Constructs. One embodiment of the invention relates to novel HBV antigens and fusion proteins and recombinant c acid molecules encoding these antigens and proteins. Described herein are several ent novel HBV antigens for use in a yeast-based immunotherapeutic composition or other ition (e.g., other immunotherapeutic or diagnostic ition) that provide one or multiple (two, three, four, five, six, seven, eight, nine or more) antigens and/or immunogenic domains from one or more proteins, all contained within the same fusion protein and encoded by the same recombinant nucleic acid construct (recombinant nucleic acid molecule). The antigens used in the compositions of the ion include at least one HBV protein or immunogenic domain thereof for immunizing an animal (prophylactically or therapeutically). The composition can include one, two, three, four, a few, several or a plurality of HBV antigens, including one, two, three, four, five, six, seven, eight, nine, ten, or more immunogenic domains of one, two, three, four or more HBV proteins. In some ments, the antigen is a fusion protein. In one aspect of the ion, fusion protein can include two or more proteins. In one aspect, the fusion n can include two or more immunogenic domains and/or two or more epitopes of one or more proteins. An immunotherapeutic composition containing such antigens may provide antigen-specific immunization in a broad range of patients. For example, an antigen or fusion protein encompassed by the invention can include at least a portion of, or the full-length of, any one or more HBV proteins selected from: HBV surface protein (also called surface antigen or envelope protein or HBsAg), including the large (L), middle (M) and/or small (S) forms of surface protein and/or the pre-Sl and/or pre-SZ domains thereof; HBV precore protein; HBV core n (also called core antigen or HBcAg); HBV e-antigen (also called HBeAg); HBV polymerase (including one or both domains of the polymerase, called the RT domain and the TP domain); HBV X antigen (also called X, X antigen, or HBX); and/or any one or more genic domains of any one or more of these HBV proteins. In one embodiment, an antigen useful in an immunotherapeutic composition of the invention is from a single HBV protein (full-length, near ength, or portion f comprising at least, one, two, three, four or more immunogenic domains of a full-length protein). In one embodiment of the invention, an immunotherapeutic composition includes one, two, three, four, five or more individual yeast vehicles, each expressing or containing a different HBV antigen(s).
Combinations ofHBV ns useful in the present invention include, but are not limited to (in any order within the fusion n): (1) surface protein (L, M and/or S and/or any one or combination of functional and/or immunological domains thereof, including, but not limited to pre-Sl and/or pre-S2 and/or the hepatocyte receptor domain of pre-Sl) in combination with any one or more of: (a) precore/core/e (precore, core, e—antigen, and/or any one or combination of functional and/or logical domains thereof); (b) polymerase (full-length, RT domain, TP domain and/or any one or combination of functional and/or immunological domains thereof); and/or (c) X antigen (or any one or combination of functional and/or immunological s thereof); (2) precore/core/e (precore, core, e-antigen, and/or any one or combination of functional and/or immunological domains thereof) in combination with any one or more of: (a) surface protein (L, M and/or S and/or any one or ation of functional and/or immunological s thereof, including, but not limited to pre- Sl and/or pre—S2 and/or the cyte receptor domain of pre-S l); (b) polymerase (full—length, RT domain, TP domain and/or any one or combination of functional WO 09404 and/or logical domains thereof); and/or (0) X antigen (or any one or combination of functional and/or immunological domains thereof); (3) polymerase (full-length, RT domain, TP domain and/or any one or combination of functional and/or immunological domains thereof) in combination with any one or more of: (a) surface protein (L, M and/or S and/or any one or combination of functional and/or immunological s thereof, including, but not limited to pre- Sl and/or pre-SZ and/or the hepatocyte receptor domain of pre-Sl); (b) e/core/e (precore, core, e-antigen, and/or any one or combination of functional and/or immunological domains thereof); and/or (c) X antigen (or any one or combination of functional and/or immunological domains thereof); or (4) X antigen (or any one or combination of functional and/or logical domains thereof) in combination with any one or more of: (a) surface protein (L, M and/or S and/or any one or combination of functional and/or immunological domains thereof, including, but not limited to pre-Sl and/or pre-SZ and/or the hepatocyte receptor domain of pre-Sl); (b) polymerase (full—length, RT domain, TP domain and/or any one or combination of functional and/or immunological domains thereof); and/or (0) precore/core/e (precore, core, e-antigen, and/or any one or combination of fimctional and/or immunological domains thereof).
] Recombinant nucleic acid molecules and the proteins encoded thereby, including fusion proteins, as one embodiment of the ion, may be used in yeast-based immunotherapy compositions, or for any other le e for HBV n(s), including in an in vitro assay, for the production of antibodies, or in another immunotherapy composition, including another vaccine, that is not based on the yeast- based immunotherapy described herein. Expression of the proteins by yeast is one preferred ment, although other expression systems may be used to produce the proteins for applications other than a based immunotherapy composition.
According to the present invention, the general use herein of the term "antigen" refers: to any portion of a protein (peptide, partial protein, full-length n), wherein the protein is naturally occurring or tically derived, to a cellular composition (Whole cell, cell lysate or disrupted cells), to an organism (whole organism, lysate or disrupted cells) or to a carbohydrate, or other molecule, or a portion thereof. An antigen may elicit an antigen-specific immune response (e.g., a humoral and/or a cell- mediated immune response) against the same or similar antigens that are encountered by an element of the immune system (e.g., T cells, antibodies).
An antigen can be as small as a single epitope, a single immunogenic domain or larger, and can include multiple epitopes or immunogenic domains. As such, the size of an antigen can be as small as about 8-12 amino acids (z'.e., a peptide) and as large as: a fiJll length protein, a multimer, a filSlOIl protein, a chimeric protein, a whole cell, a whole microorganism, or any portions thereof (e. g., lysates of whole cells or extracts of microorganisms). In addition, antigens can include carbohydrates, which can be loaded into a yeast vehicle or into a composition of the invention. It will be appreciated that in some embodiments (e.g., when the n is expressed by the yeast vehicle from a recombinant nucleic acid molecule), the antigen is a protein, fusion protein, chimeric protein, or nt f, rather than an entire cell or microorganism.
When the antigen is to be expressed in yeast, an antigen is of a minimum size capable of being expressed recombinantly in yeast, and is typically at least or greater than amino acids in length, or at least or greater than 26, at least or greater than 27, at least or r than 28, at least or greater than 29, at least or greater than 30, at least or greater than 31, at least or r than 32, at least or greater than 33, at least or greater than 34, at least or r than 35, at least or greater than 36, at least or greater than 37, at least or greater than 38, at least or greater than 39, at least or r than 40, at least or r than 41 at least or greater than 42, at least or greater than 43, at least or greater than 44, at least or greater than 45, at least or greater than 46, at least or greater than 47, at least or greater than 48, at least or greater than 49, or at least or greater than 50 amino acids in length, or is at least 25—50 amino acids in length, at least 30-50 amino acids in length, or at least 35-50 amino acids in length, or at least 40-50 amino acids in length, or at least 45-50 amino acids in . Smaller proteins may be expressed, and considerably larger proteins (eg, hundreds of amino acids in length or even a few thousand amino acids in length) may be expressed. In one aspect, a full-length protein, or a structural or functional domain f, or an ogenic domain thereof, that is lacking one or more amino acids from the N— and/or the C-terminus may be expressed (e.g., lacking between about 1 and about 20 amino acids from the N- and/or the C-terminus). Fusion proteins and chimeric proteins are also antigens that may be expressed in the invention. A "target antigen" is an antigen that is specifically targeted by an immunotherapeutic ition of the invention (z'.e., an antigen against which elicitation of an immune response is desired). An "HBV antigen" is an antigen derived, designed, or produced from one or more HBV ns such that ing the antigen also targets the hepatitis B virus.
When referring to stimulation of an immune response, the term "immunogen" is a subset of the term "antigen", and therefore, in some instances, can be used interchangeably with the term "antigen". An immunogen, as used herein, describes an antigen which s a humoral and/or cell-mediated immune response (116., is immunogenic), such that administration of the immunogen to an individual mounts an antigen—specific immune response t the same or similar antigens that are encountered by the immune system of the individual. In one embodiment, an immunogen elicits a cell-mediated immune response, including a CD4+ T cell response (e.g., THl, TH2 and/or TH17) and/or a CD8+ T cell response (e.g, a CTL se).
] An "immunogenic domain" of a given antigen can be any portion, nt or epitope of an antigen (e.g., a peptide fragment or subunit or an antibody e or other conformational epitope) that contains at least one e that acts as an immunogen when administered to an animal. Therefore, an immunogenic domain is larger than a single amino acid and is at least of a size sufficient to contain at least one epitope that can act as an immunogen. For example, a single protein can contain le different immunogenic domains. Immunogenic domains need not be linear ces within a protein, such as in the case of a humoral immune response, where conformational domains are contemplated.
A "functional domain" of a given n is a portion or functional unit of the protein that es sequence or structure that is directly or indirectly sible for at least one biological or chemical function associated with, ascribed to, or performed by the protein. For example, a functional domain can include an active site for enzymatic activity, a ligand binding site, a receptor binding site, a binding site for a molecule or moiety such as m, a phosphorylation site, or a transactivation domain. Examples of HBV functional domains include, but are not limited to, the viral cyte receptor domain in pre-S 1 or the e transcriptase domain or RNase H domain of polymerase.
A "structural domain" of a given protein is a portion of the n or an element in the protein’s overall structure that has an fiable structure (e.g., it may be a primary or tertiary structure belonging to and indicative of several proteins within a class or family of proteins), is self-stabilizing and/or may fold independently of the rest of the protein. A structural domain is frequently associated with or features prominently in the biological function of the protein to which it belongs.
An epitope is defined herein as a single immunogenic site within a given antigen that is sufficient to elicit an immune response when provided to the immune system in the context of appropriate costimulatory signals and/or activated cells of the immune system. In other words, an e is the part of an n that is actually recognized by components of the immune system, and may also be referred to as an antigenic determinant. Those of skill in the art will recognize that T cell epitopes are different in size and composition from B cell or antibody epitopes, and that epitopes presented through the Class I MHC pathway differ in size and structural attributes from epitopes presented through the Class II MHC pathway. For example, T cell epitopes presented by Class I MHC molecules are typically between 8 and 11 amino acids in length, whereas epitopes presented by Class II MHC molecules are less restricted in length and may be from 8 amino acids up to 25 amino acids or longer. In addition, T cell es have predicted structural characteristics depending on the specific MHC molecules bound by the epitope. Multiple different T cell epitopes have been identified in various HBV strains and for many human HLA types, several of which are identified in Table 5. In addition, epitopes for certain murine MHC haplotypes have been newly ered herein and are also presented in Table 5 or in the Examples. Epitopes can be linear sequence es or conformational epitopes (conserved binding regions). Most antibodies ize conformational epitopes.
One exemplary embodiment of the invention relates to a fusion protein comprising an HBV antigen that is a multi-protein HBV antigen, and in this example, a fusion comprised of HBV large (L) surface antigen, including all of the hydrophobic transmembrane domains, and core antigen (HBcAg), described in detail below. Surface antigen and core are abundantly sed in infected cells, are required for viral replication, and contain multiple CD4+ and CD8+ T cell es. In addition, these antigens, particularly surface antigen, contain known mutation sites that can be induced by anti—viral therapy; these regions can therefore be modified, as , to provide additional immunotherapy compositions to target the e" mutations. An onal advantage of targeting these proteins, and particularly both proteins in a single immunotherapeutic composition, is the high degree of conservation at the amino acid level among different HBV genotypes. Both the core and surface (L) ns are highly conserved between HBV genotypes A and C or between A and H, for example (see Table 4), which are genotypes ent in the as and Asia (Table 2). The core protein ys a 95% amino acid identity between genotypes A and C and between genotypes A and H. The large (L) surface protein is also highly conserved among the different HBV genotypes; a 90% amino acid identity exists between genotypes A and C, and 82% amino acid identity exists between genotypes A and H.
Table 4 Therefore, one therapeutic composition ed using one HBV genotype can be ed to induce an ive immune se against a highly similar HBV genotype, either through direct ing of conserved epitopes or through epitope spreading as a result of initially targeting epitopes that are conserved n genotypes.
Alternatively, because of the ease of producing the yeast-based immunotherapy compositions of the invention, it is straightforward to modify a sequence to encode a n, domain, or epitope from a different genotype, or to include in the same construct different T cell epitopes or entire domains and/or proteins from two or more different HBV genotypes, in order to increase the wide applicability of the therapy.
Examples of such HBV antigens are described in detail and exemplified below. While one immunotherapeutic ition of the present invention was designed to target two HBV antigens, surface and core protein, in a single product, this approach can y be expanded to incorporate the protein sequences of other essential, conserved, and immunogenic HBV viral proteins to result in even broader cellular immune responses.
Such additional fusion proteins and immunotherapeutic compositions are described and exemplified herein.
In one embodiment of the invention, the HBV antigen(s) for use in a composition or method of the invention are selected from HBV antigens that have been designed to optimize or enhance their usefulness as clinical products, including in the context of a yeast-based immunotherapeutic composition. Such HBV antigens have been designed to produce an HBV yeast-based immunotherapeutic product that achieves one or more ofthe following goals: (1) compliance with the guidelines of the Recombinant DNA Advisory Committee (RAC) of the National Institutes of Health (NIH), wherein no more than two thirds (2/3) of the genome of an infectious agent may be used in a recombinant therapeutic or e; (2) inclusion of a maximized number of known T cell epitopes associated with immune responses to acute/self—limiting HBV infections and/or chronic HBV infections (with prioritization in one aspect based on the acute/self—limiting e oire, as discussed below); (3) maximizing or prioritizing the inclusion of immunogenic domains, and more ularly T cell epitopes (CD4+ and/or CD8+ epitopes, and dominant and/or subdominant epitopes), that are the most conserved among HBV genotypes and/or sub-genotypes, or that can be y modified to a consensus sequence or included in two or more forms to cover the most important sequence differences among target genotypes; and/or (4) minimizing the number of non-natural junctions within the ce of the HBV antigen in the product.
Accordingly, the invention includes, in some embodiments, modification of HBV antigens from their naturally ing or wild-type sequences in a given strain to meet one or more of criteria bed above, as well as to include design elements and/or antigen design criteria described elsewhere herein. Such criteria and antigen design ce is applicable to yeast-based immunotherapeutics comprising HBV antigens that are individual HBV proteins or domains, as well as HBV antigens that include combinations of HBV ns or domains and particularly, multi-protein ns/fitsion proteins (e.g., HBV antigens from two or more different HBV proteins and/or domains f, such as combinations of ns from HBV surface protein, polymerase, core, e- antigen, and/or X n). It will be appreciated that as the complexity of the HBV antigen increases, the utilization of more of these criteria are implemented in the construction of the antigen.
Therefore, in one embodiment of the invention, an HBV antigen useful in the present invention as a protein or fusion protein to be expressed by a yeast includes HBV sequences encoded by nucleotide sequences representing less than two thirds (2/3) of the HBV genome (i.e., the antigens are encoded by nucleic acid sequences that in total make up less than two thirds (2/3) of the HBV genome or meet the requirements of RAC for recombinant therapeutics and prophylactics). In one aspect, this embodiment can be achieved by ing HBV antigens for expression in a yeast-based immunotherapeutic that meet the RAC requirements in their full-length or near-full-length form (e.g., X antigen is small and when used alone would meet the RAC requirements). In another , this embodiment is achieved by modifying the structure of the protein(s) and/or domain(s) to be included in the HBV antigen, such as by deletion of sequence to truncate proteins or remove internal sequences from proteins, by including only selected fianctional, structural or immunogenic domains of a protein, or by ng to eliminate the inclusion of a particular protein in the antigen construct altogether. In addition, HBV yeast-based therapeutics may, in one embodiment, be produced as individual antigen constructs, and then used in combination in a manner that does not contravene any restrictions related to the viral genome.
In r embodiment of the invention, as discussed above, the ion of T cell epitopes in an HBV antigen construct (protein or fusion protein) is maximized, for e, if the HBV antigen ed in the immunotherapeutic has been modified to meet another design eration, such as the RAC requirement discussed above. In this embodiment, HBV antigens useful in a yeast-based immunotherapeutic are modified with the goal of maximizing the number of immunogenic domains, and in one aspect, the number of T cell epitopes, that are retained in the HBV antigen. In one aspect, the inclusion ofT cell epitopes in an HBV antigen is prioritized as follows: Epitopes identified in immune responses to both acute/self-limiting HBV infections flchronic HBV infections > Epitopes identified in immune responses to acute/self-limiting HBV infections > Epitopes fied in immune responses to chronic HBV infections.
In this embodiment, t being bound by theory, the inventors believe that immune responses from individuals who had acute or self-limiting HBV infections may be more productive in eliminating the viral infection than the immune responses from individuals who have chronic HBV infections. Therefore, the inclusion of T cell es that appear to be associated with clearance of virus in these acute or imiting infections (whether nt or minant) is prioritized as being more likely to elicit a beneficial immune response in an immunized individual. In addition, and again without being bound by theory, the inventors believe that the generation of an immune response against one or more HBV target antigens using yeast-based immunotherapy will result in an immune response in the zed individual against not only the epitopes included in the yeast— based therapeutic, but also against other HBV epitopes present in the individual.
This phenomenon, referred to as "epitope spreading" allows for the design of HBV antigens that are focused on epitopes that appear to be most relevant to therapeutic , and the mechanism of action of a based immunotherapeutic product then allows the immune system to expand the immune response to cover additional target epitopes, thereby enhancing a therapeutically productive or beneficial immune response against HBV.
Accordingly, an HBV antigen in one embodiment comprises one or more CTL epitopes (e.g, epitopes that are recognized by a T cell receptor of a cytotoxic T lymphocyte (CTL), when presented in the context of an appropriate Class I MHC WO 09404 molecule). In one aspect, the HBV antigen comprises one or more CD4+ T cell epitopes (e.g, epitopes that are recognized by a T cell receptor of a CD4+ T cell, in the context of an appropriate Class II MHC molecule). In one aspect, the HBV antigen ses one or more CTL epitopes and one or more CD4+ T cell epitopes. In one aspect, an HBV antigen useful in an immunotherapeutic composition of the invention comprises one or more of the exemplary HBV CTL epitopes described in Table 5. One of skill in the art will readily be able to identify the position of the corresponding sequence for each epitope in Table 5 in a given HBV sequence of any genotype, sub-genotype, or strain/isolate, given the guidance provided below, even though some amino acids may differ from those in Table 5.
Examples of such differences are rated in Table 5. The invention is not limited to antigens comprising these epitopes as others will be known in the art and are contemplated for use in the invention. In one embodiment, the epitope can be modified to correspond to the sequence of the epitope within a given genotype, sub-genotype or /isolate of HBV, since there may be one or more amino acid differences at these epitopes among genotypes, sub-genotypes or stain/isolates.
Table 5 Sequence Epitope Identifier HBV Anti - en HLA Preference Surface (e.g. positions 20-28 of S; 9.9. corresponding to positions 194-202 of SEQ ID NO:11, ons 201-209 of SEQ ID NO:34, or positions 51-59 of LTI1’2’3 SEQ ID NO 42 SEQ ID NO:36 A*0201 Surface (e.g. positions 185-194 of S; e.g. corresponding to positions 359-368 of SEQ ID NO:11, positions 366-375 of SEQ ID NO:34, or positions 5** GLSPTVWLSV5 SEQ ID NO 43 of SEQ ID NO:36 A*0201 Core (e.g. ons 47-56 of e; e.g. corresponding to positions 47-56 of SEQ ID NO:9, positions 424-433 of SEQ ID NO:34, or ons 621-630 FLPSDFFPSI2’3’4 SEQ ID NO:44 of SEQ ID NO:36 A*0201 Polymerase (e.g. positions 575-583 of Pol; e.g. corresponding to positions 1 of SEQ ID NO:10, or positions 486-494 FLLSLGIHL1 SEQ ID NO 45 of SEQ ID NO:36 A*0201 Surface (e.g. positions 172-180 of S; 6.9. corresponding to positions 346-354 of SEQ ID NO:11, positions 353-361T of SEQ ID NO:34, or positions 203-211 WLSLLVPFV1’3’5 SEQ ID NO 46 of SEQ ID NO:36 A*0201 Epitope Identifier HBV Anti - en HLA Preference Polymerase (e.g. positions 756-764 of Pol; e.g. corresponding to positions 756-764 of WLL SEQ ID NO:47 SEQ ID NO:1O A*2402 Core (e.g. positions 117-125 of e; e.g. corresponding to positions 117- 125 of SEQ ID NO:9, positions 494- 502 of SEQ ID NO:34, or positions WNVNMGLK4 SEQ ID NO:48 691-699 of SEQ ID NO:36 A*1101 Core (e.g. positions 146-154 of Precore; e.g. corresponding to positions 146- 154 of SEQ ID NO:9, positions 523- 531 of SEQ ID NO:34, or positions GVW SEQ ID NO:49 8 of SEQ ID NO:36 A*2402 Polymerase (e.g. positions 3 of Pol; e.g. corresponding to positions 453-461I of SEQ ID NO:10, or positions 366- GLSRYVARL3 SEQ ID NO:5O 374 of SEQ ID NO:36 A*0201 (e.g. positions 115-123 of X; e.g. corresponding to positions 1 15-123§ of SEQ ID NO:12, or positions 900- CLFKDWEEL5 SEQ ID NO:51 908§ of SEQ ID NO:36 A*02 Surface (e.g. positions 21-28 of Pre-S1; e.g. corresponding to positions 21-28 of SEQ ID NO:11, positions 28-35 of SEQ ID NO:34;, or positions 6-13 of PLGFFPDH5 SEQ ID NO:52 SEQ ID NO:36 A*11 Surface (e.g. positions 150-158 of S; e.g. corresponding to ons 324-332 of SEQ ID NO:11, positions 331-339 of SEQ ID NO:34, or positions 181-189 IPIPSSWAF5 SEQ ID NO:53 of SEQ ID NO:36 B*07 Core (e.g. ons 48-56 of Precore; e.g. corresponding to positions 48-56" of SEQ ID NO:9, positions 425-433" of SEQ ID NO:34, or positions 619—630" LPSDFFPSV5 SEQ ID NO:54 of SEQ ID NO:36 B*51 Surface (e.g. ons 1-15 of pre-SZ; e.g. ***corresponding to positions 120-134 MQWNSTALHQALQDP5 SEQ ID NO:55 of SEQ ID NO:3 >*3 Surface (e.g. positions 12-20 of pre-SZ; e.g. ***corresponding to positions 131-139 LLDPRVRGL5 SEQ ID NO:56 of SEQ ID NO:3 >*2 Surface (e. 9. positions 44-53 of a pre-SZ; e.g. ***corresponding to positions 163-172 SILSKTGDPV5 SEQ ID NO:57 of SEQ ID NO:3 A*2 Sequence Epitope ldenfifier HBV Anti - en HLA Preference Su rface (e.g. ons 14-22 of S; e.g. ***corresponding to positions 188-196 FLL5 SEQ ID NO:58 of SEQ ID NO:3 A*2 Surface (e.g. positions 20-28 of S; e.g. ***corresponding to positions 194-202 FLLTRILTI5 SEQ ID NO:59 of SEQ ID NO:3 A*2 Surface (e.g. positions 41-49 of S; e.g. ***corresponding to positions 215-223 FLGGTPVCL5 SEQ ID NO:60 of SEQ ID NO:3 A*2 Surface (e.g. ons 88-96 of S; e.g. ***corresponding to ons 262-270 LLCLIFLLV5 SEQ ID NO:61 of SEQ ID NO:3 A*2 Surface (e.g. positions 95-104 of S; e.g. ***corresponding to positions 269-278 LVLLDYQGML5 SEQ ID NO:62 of SEQ ID NO:3 A*2 Surface (e.g. positions 97-106 of S; e.g. ***corresponding to positions 271-280 LLDYQGMLPV5 SEQ ID NO:63 of SEQ ID NO:3 A*2 Surface (e.g. positions 207-216 of S; e.g. ***corresponding to positions 381-390 SIVSPFIPLL5 SEQ ID NO:64 of SEQ ID NO:3 A* Surface (e.g. positions 208-216 of S; e.g. ***corresponding to positions 382-390 ILSPFLPLL5 SEQ ID NO:65 of SEQ ID NO:3 A*2 Polymerase (e.g. positions 367-376 of Pol; e.g. ***corresponding to positions 367-376 TPARVTGGVF5 SEQ ID NO:66 of SEQ ID NO:2 03*7 Polymerase (e.g. positions 390-399 of Pol; e.g. ***corresponding to positions 390-399 FSR5 SEQ ID NO:67 of SEQ ID NO:2 A*3 Polymerase (e.g. positions 533-541 of Pol; e.g. ***corresponding to positions 533-541 SAICSWRR5 SEQ ID NO:68 of SEQ ID NO:2 A*3 Polymerase (e.g. positions 551-559 of Pol; e.g. ***corresponding to ons 551-559 YMDDWLGA5 SEQ ID NO:69 of SEQ ID NO:2 A*N Polymerase (e.g. positions 655-663 of Pol; e.g. ***corresponding to ons 655-663 ALMPLYACI5 SEQ ID NO:7O of SEQ ID NO:2 A*2 Polymerase (e.g. ons 667—676 of Pol; e.g. ***corresponding to positions 667-676 QAFTFSPTYK5 SEQ ID NO:71 of SEQ ID NO:2 A*3 Epitope Identifier HBV Anti - en HLA Preference Core (e.g. positions 40-56 of Precore; e.g. ***corresponding to positions 40-56 of ATVELLSFLPSDFFPSV5 SEQ ID NO:72 SEQ ID NO:1 A*2 Core (e.g. positions 48-56 of Precore; e.g. ***corresponding to ons 48-56 of LPSDFFPSV5 SEQ ID NO:73 SEQ ID NO:1 B*51 Core (e.g. positions 4 of Precore; e.g. ***corresponding to positions CLTFGRETV5 SEQ ID NO:74 136-144 of SEQ ID NO:1 A*2 Core (e.g. ons 144-153 of Precore; e.g. ***corresponding to positions VLEYLVSFGV5 SEQ ID NO:75 144-153 of SEQ ID NO:1 A*2 Core (e.g. positions 168-177 of Precore; e.g. ***corresponding to positions ILSTLPETTV5 SEQ ID NO:76 168-177 of SEQ ID NO:1 A*2 Core (e.g. positions 170-180 of Precore; e.g. ***corresponding to positions STLPETTWRR5 SEQ ID NO:77 170-180 of SEQ ID NO:1 A*3 (e.g. positions 52-60 of X; e.g. responding to positions 52-60 of LFV5 SEQ ID NO:78 SEQ ID NO:4 A* (e.g. positions 92-100 of X; e.g. ***corresponding to ons 92-100 VLHKRTLGL5 SEQ ID NO:79 of SEQ ID NO:4 A*2 (e.g. positions 99—108 of X; e.g. ***corresponding to positions 99-108 TTDL5 SEQ ID NO:80 of SEQ ID NO:4 A*2 (e.g. positions 133-141 of X; e.g. ***corresponding to positions of 133- HKL5 SEQ ID NO:81 141 SEQ ID NO:4 A*2 Polymerase (e.g. positions 49-57 of Pol; e.g. ***corresponding to positions 49-57 of NVSIWTHK5 SEQ ID NO:82 SEQ ID NO:2 A*3 Polymerase (e.g. positions 57-65 of Pol; e.g. ***corresponding to positions 57-65 of KVGNFTGLY5 SEQ ID NO:83 SEQ ID N022 A*00 Polymerase (e.g. positions 63-71 of Pol; e.g. ***corresponding to positions 63-71 of GLYSSTVPV5 SEQ ID NO:84 SEQ ID NO:2 A*2 Polymerase (e.g. positions 152-161 of Pol; e.g. ***corresponding to positions of SEQ TLWKAGILYK5 SEQ ID NO:85 A*3 WO 09404 2012/024409 e Identifier HBV Anti - en HLA Preference Polymerase (e.g. positions 4 of Pol; e.g. ***corresponding to positions 6 KYTSFPWLL5 SEQ ID NO:86 of SEQ ID NO:2 A*24 Polymerase (e.g. positions 773-782 of Pol; e.g. ***corresponding to positions 773-782 ILRGTSFVW5 SEQ ID N018? of SEQ ID N022 A*2 rase (e.g. positions 4 of Pol; e.g. ***corresponding to positions 816-824 SLYADSPSV5 SEQ ID NO:88 of SEQ ID NO:2 A*2 Polymerase (e.g., positions 502-510 of Pol; e.g., ***corresponding to positions 502-510 KLHLYSHPI6 SEQ ID NO:135 of SEQ ID NO:2 A*2 Surface (e.g., positions 349-358 of S; e.g., ***corresponding to positions 349—358 LLVPFVQWFV6’7 SEQ ID NO:136 of SEQ ID NO:3 A*2 Polymerase (e.g., positions 504-512 of Pol; e.g., ***corresponding to positions 504-512 HLYSHPIIL8 SEQ ID NO:137 of SEQ ID NO:2 A*2 Surface (e.g., positions 77-84 of S; e.g., responding to positions 77-84 of WSPQAQGIL9 SEQ ID NO:138 SEQ ID NO:3 H-2Db Surface (e.g., positions 270-277 of S; e.g., ***corresponding to positions 270-277 VLLDYQGM1o SEQ ID NO:139 of SEQ ID NO:3 H-2Kb Su rface (e.g., positions 340—347 of S; e.g., ***corresponding to positions 7 ASVRFSWL1o SEQ ID NO:140 of SEQ ID NO:3 H-2Kb **Substitution of an Ala for Val at position 9 of SEQ ID NO:43; at position 225 in SEQ ID NO:36.
TSubstitution of Gin-Ala for Leu-Val at positions 5 and 6 of SEQ ID NO:46; at positions 357 and 358 in SEQ ID NO:34. iSubstitution of Pro for Ser at on 3 of SEQ ID NO:50; at position 455 in SEQ ID NO:10.
§Substitution of Val for Leu at position 2 ofSEQ ID NO:51; at position 116 in SEQ ID NO:12 and position 901 in SEQ ID NO:36.
||Substitution of lie for Val at position 9 of SEQ ID NO:54; at position 56 in SEQ ID NO:9, position 433 of SEQ ID NO:34, and position 630 ofSEQ ID NO:36.
***One or more amino acid differences between the epitope sequence and the actual sequence of the corresponding larger protein or domain may exist due to genotype, sub-genotype or strain differences, although position of the epitope within the larger n or domain can readily be ined. 1Zhang et al., Journal of Hepatology 50:1163-1173 (2009) 2Lopes et al., J. Clin. Invest. 118:1835—1845 (2008) 3Boettler et al., J Virol 80(7):3532—3540 (2006) 4Peng et al., Mol. Immunol. 45:963-970 (2008) nd 2008; www.allelefrequencies.net or Desmond et al., Antiviral Ther. 13:16-175 (2008) 6Webster et al., 2004, J. Virol. 78(11)5707-5719 7Vitie|lo, 1997, Eur. J. Immunol. 27(3): 671-678 8Sette et al., 1994, J. Immunol. 153(12): 5586-5592 9Murine H-2Db epitope, not previously reported 1OMurine H-2Kb epitope, not previously reported In one embodiment of the invention, useful HBV antigens can include in one or more yeast-based immunotherapeutic itions an antigen comprising one or more T cell epitopes that has been described as or determined to be a "dominant" epitope (i.e., a T cell epitope that contributes to the development of a T cell se against the whole protein, and/or that is among the relatively small number of T cell epitopes within the large group of possible epitopes that most likely or most readily elicit CD4+ and CD8+ T cell responses, also referred to as an odominant epitope"). In another embodiment, HBV antigens useful in the invention can include in the same or a different or additional yeast-based compositions, an HBV antigen sing one or more T cell epitopes that has been described as or determined to be a "subdorninant" epitope (i.e., a T cell epitope that is immunogenic, but to a lesser extent than an immunodominant epitope; the immune response generated by a minant epitope may be suppressed or outcompeted by the immune se to an immunodominant epitope). For an example of this effect with CTL responses to HBV T cell epitopes in mice, see Schirmbeck R., et al. J. immunology 168: 6253-6262, 2010; or Sette et al. Jlmmunology 89-1397, 2001. In one aspect of the invention, different compositions comprising immunodominant or minant epitopes could be administered at the same site in an individual, or in one ment, at different sites in an individual (i.e., the composition comprising dominant epitopes being administered to one site and the composition comprising sub—dominant epitopes being administered to a different site). In some cases, a sub-dominant epitope may elicit a more therapeutically beneficial immune response than a dominant e. Therefore, if administered to separate sites, it may decrease the chance that an immune response to a dominant epitope would suppress or outcompete an immune se to a sub-dominant epitope, thereby maximizing the immune response as a whole and maximizing the protective or therapeutic benefit in an individual. This approach of providing different antigens in different compositions administered to different sites in the individual can also be utilized even if all epitopes are dominant or sub—dominant. dominant es and sub—dominant epitopes have been ized to play a role in HBV infection and immune responses (see, e.g., Sette et al., 2001, supra. and Schirmbeck et al., 2002, supra).
In one embodiment of the invention, an HBV antigen useful in a based immunotherapeutic maximizes the inclusion of immunogenic domains, and particularly, T cell epitopes, that are conserved among genotypes and/or sub-genotypes, and/or includes immunogenic domains from several different genotypes and/or sub-genotypes and/or includes immunogenic domains that can readily be modified to produce multiple yeast- based immunotherapeutic products that differ in some minor respects, but are tailored to treat different individuals or tions of individuals based on the HBV genotype(s) or sub-genotype(s) that infect such individuals or populations of duals. For example, the HBV antigen can be produced based on a genotype or sub-genotype that is most prevalent among duals or populations of individuals to be protected or d, and the HBV n includes the most conserved immunogenic domains from those genotypes. Alternatively or in addition, immunogenic domains can be d to correspond to a consensus sequence for that domain or epitope, or more than one version of the epitope can be included in the construct.
In any embodiment of the invention related to the design of an HBV antigen for a yeast-based immunotherapeutic composition, in one aspect, artificial junctions between segments of a fusion protein comprising HBV antigens is minimized (i.e., the inclusion of tural sequences is limited or minimized to the extent possible).
Without being bound by theory, it is ed that natural evolution has resulted in: i) contiguous sequences in the virus that most likely to be expressed well in another cell, such as a yeast; and ii) an proteasome in antigen presenting cells that can properly digest and present those sequences to the immune system. The yeast-based immunotherapeutic product of the invention allows the host immune system to process and present target antigens; accordingly, a fusion n with many unnatural junctions may be less useful in a yeast-based immunotherapeutic as compared to one that retains more ofthe l HBV protein ces.
In any of the HBV ns described herein, including any of the fusion proteins, the following additional embodiments can apply. First, the N-terminal expression sequence and the C-terminal tag included in some of the fusion proteins are optional, and if used, may be selected from several different sequences described ere herein to improve expression, stability, and/or allow for identification and/or purification of the protein. Alternatively, one or both of the N- or C-terminal sequences are omitted altogether. In addition, many different promoters suitable for use in yeast are known in the art and are encompassed for use to express HBV antigens according to the present invention. Furthermore, short intervening linker sequences (e.g., l, 2, 3, 4, or 5, or larger, amino acid es) may be introduced between portions of the fusion protein for a variety of s, including the introduction of restriction enzyme sites to facilitate cloning and future manipulation of the constructs. Finally, as discussed in detail elsewhere herein, the sequences described herein are exemplary, and may be modified as WO 09404 2012/024409 described in detail elsewhere herein to substitute, add, or delete ces in order to accommodate preferences for HBV genotype, HBV subgenotype, HBV strain or e, or consensus sequences and inclusion of preferred T cell epitopes, including dominant and/or subdominant T cell epitopes. A description of several different ary HBV antigens useful in the invention is provided below.
In any of the embodiments of the invention described herein, including any embodiment related to an immunotherapeutic composition, HBV n, fusion protein or use of such composition, HBV antigen or fusion protein, in one aspect, an amino acid of an HBV surface antigen useful as an HBV antigen or in a fusion protein or an immunotherapeutic composition ofthe invention can include, but is not limited to, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:11, positions 21-47 of SEQ ID NO:11, positions 176- 400 of SEQ ID NO:ll, SEQ ID NO:15, SEQ ID NO:l9, SEQ ID NO:23, SEQ ID NO:27, SEQ ID NO:31, positions 9-407 of SEQ ID NO:34, positions 6-257 of SEQ ID NO:36, ons 6-257 of SEQ ID NO:41, positions 92-343 of SEQ ID NO:92, positions 90-488 of SEQ ID NO:93, SEQ ID NO:97, positions 90-338 of SEQ ID NO:101, positions 7-254 of SEQ ID NO:102, positions 1-249 of SEQ ID NO:lO7, ons 1-249 of SEQ ID NO:108, positions 1-249 of SEQ ID NO:109, positions 1—249 of SEQ ID NO:110, positions 1-399 of SEQ ID NO:112, positions 1-399 of SEQ ID NO:114, or positions 1- 399 of SEQ ID NO:116, positions 1-399 of SEQ ID , positions 1-399 of SEQ ID NO:120, positions 1-399 of SEQ ID NO:122, positions 1-399 of SEQ ID NO:124, positions 1-399 of SEQ ID NO:126, positions 231-629 of SEQ ID NO:128, positions 63- 461 of SEQ ID NO:130, positions 289-687 of SEQ ID NO:132, positions 289-687 of SEQ ID NO: 134, or a corresponding sequence from a ent HBV strain.
In any of the embodiments of the invention described herein, including any embodiment d to an immunotherapeutic composition, HBV antigen, fusion protein or use of such composition, HBV n or fusion protein, in one aspect, an amino acid of an HBV polymerase antigen useful as an HBV antigen or in a fusion protein or an immunotherapeutic composition of the invention can include, but is not limited to, positions 383-602 of SEQ ID NO:2, ons 381-600 of SEQ ID N026, positions 381- 600 of SEQ ID NO:lO, positions 453 to 680 of SEQ ID NO:lO, positions 370-589 of SEQ ID NO:14, positions 380-599 of SEQ ID NO:18, positions 381-600 of SEQ ID N0222, positions 380-599 of SEQ ID NO:26, positions 381-600 of SEQ ID NO:30, positions 260 to 604 of SEQ ID NO:36, positions 7-351 of SEQ ID NO:38, positions 7-351 of SEQ ID NO:40, 260 to 604 of SEQ ID NO:41, positions 346 to 690 of SEQ ID NO:92, positions 90-434 of SEQ ID NO:94, SEQ ID NO:98, positions 339 to 566 of SEQ ID NO:101, positions 255 to 482 of SEQ ID NO:102, positions 250-477 of SEQ ID NO:107, positions 250—477 of SEQ ID NO:108, positions 250—477 of SEQ ID NO:109, positions 250—477 of SEQ ID NO:110, positions 582 to 809 of SEQ ID NO:120, positions 582 to 809 of SEQ ID NO:124, positions 642 to 869 of SEQ ID NO:126, positions 1 to 228 of SEQ ID NO:128, positions 1 to 228 of SEQ ID NO:132, positions 61 to 288 of SEQ ID NO:134, or a ponding sequence from a different HBV strain.
In any of the embodiments of the invention described herein, including any embodiment related to an immunotherapeutic composition, HBV antigen, fiision protein or use of such composition, HBV n or fusion protein, in one aspect, an amino acid of an HBV core antigen useful as an HBV antigen or in a fusion protein or an immunotherapeutic composition of the invention can include, but is not limited to, ons 31-212 of SEQ ID NO:1, positions 31-212 of SEQ ID NO:5, positions 31-212 of SEQ ID N029, positions 37 to 188 of SEQ ID NO:9, positions 31-212 of SEQ ID NO:13, positions 31-212 of SEQ ID NO:l7, positions 31—212 of SEQ ID NO:21, positions 14-194 of SEQ ID NO:25, positions 31-212 of SEQ ID NO:29, positions 9 of SEQ ID NO:34, positions 605 to 786 of SEQ ID NO:36, positions 352-533 of SEQ ID NO:38, positions 160-341 of SEQ ID NO:39, positions 605-786 of SEQ ID NO:41, positions 691- 872 of SEQ ID NO:92, positions 90-271 of SEQ ID NO:95, SEQ ID NO:99, positions 567 to 718 of SEQ ID NO:101, positions 483 to 634 of SEQ ID NO:102, positions 2-183 of SEQ ID NO:105, positions 184-395 of SEQ ID , positions 396-578 of SEQ ID NO:105, positions 1 of SEQ ID NO:105, positions 2-183 of SEQ ID NO:106, 338- 520 of SEQ ID , ons 478-629 of SEQ ID , positions 478-629 of SEQ ID NO:108, positions 478-629 of SEQ ID NO: 109, positions 478-629 of SEQ ID NO:110, positions 1 of SEQ ID , positions 400-581 of SEQ ID NO:114, positions 400-581 of SEQ ID NO:116, positions 400—581 of SEQ ID NO:118, positions 400 to 581 of SEQ ID , positions 400 to 581 of SEQ ID NO:122, positions 400 to 581 of SEQ ID NO:124, positions 400 to 581 of SEQ ID NO:126, positions 630 to 811 of SEQ ID NO:128, positions 462 to 643 of SEQ ID NO:130, positions 688 to 869 of SEQ ID NO:132, ons 688 to 869 of SEQ ID , or a corresponding sequence from a different HBV strain.
In any of the embodiments of the invention described herein, including any embodiment related to an immunotherapeutic ition, HBV antigen, fusion protein or use of such composition, HBV antigen or fusion protein, in one aspect, an amino acid of W0 2012/109404 2012/024409 an HBV X antigen usefiil as an HBV antigen or in a fiision protein or an immunotherapeutic composition ofthe invention can include, but is not limited to, SEQ ID NO:4, SEQ ID N028, SEQ ID NO:12, ons 2 to 154 of SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:32, positions 52-68 followed by positions 84—126 of SEQ ID NO:4, positions 52-68 followed by positions 84— 126 of SEQ ID NO:8, positions 52-68 followed by positions 84-126 of SEQ ID NO:12, ons 52-68 ed by positions 84-126 of SEQ ID NO:16, positions 52-68 followed by positions 84—126 of SEQ ID NO:20, positions 52-68 followed by positions 84-126 of SEQ ID NO:24, ons 52-68 followed by positions 84-126 of SEQ ID NO:28, positions 52—68 followed by positions 84—126 of SEQ ID N0232, positions 787 to 939 of SEQ ID NO:36, positions 7-159 of SEQ ID NO:39, positions 873-1025 of SEQ ID NO:92, positions 90-242 of SEQ ID NO:96, SEQ ID NO:100, positions 8 of SEQ ID NO:101, positions 635-694 of SEQ ID NO:102, positions 184-337 of SEQ ID NO:106, positions 521-674 of SEQ ID NO:106, positions 630-689 of SEQ ID NO:107, positions 630-689 of SEQ ID NO:108, positions 630-689 of SEQ ID , positions 630-689 of SEQ ID NO:110, positions 582-641 of SEQ ID NO:122, positions 810-869 of SEQ ID NO:124, positions 582-641 of SEQ ID NO:126, positions 1-60 of SEQ ID NO:130, positions 229 to 288 of SEQ ID NO:132, positions 1 to 60 of SEQ ID NO:134, or a corresponding sequence from a different HBV strain.
HBV Antigens Comprising Surface Antigen and Core Protein. In one embodiment of the invention, the HBV n(s) for use in a composition or method of the invention is a fusion protein comprising HBV antigens, wherein the HBV antigens comprise or consist of HBV large (L) e antigen or at least one immunogenic domain thereof and HBV core protein (HBcAg) or at least one immunogenic domain thereof In one aspect, the HBV large (L) surface antigen and/or the HBV core protein is fiill-length or near full-length. According to any embodiment of the t ion, nce to a "full-length" protein (or a ength functional domain or full-length immunological domain) includes the full-length amino acid sequence of the protein or functional domain or immunological domain, as described herein or as otherwise known or described in a publicly available sequence. A protein or domain that is "near full-length", which is also a type of homologue of a protein, differs from a full-length protein or domain, by the addition or deletion or omission of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids from the N- and/or C-terminus of such a full-length protein or full-length domain. General reference to a protein or domain can include both fiill-length and near fiill—length proteins, as well as other homologues thereof.
In one aspect, the HBV large (L) surface antigen or the HBV core protein ses at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the linear sequence of a full-length HBV large (L) surface n or HBV core protein, respectively, or of the linear sequence of a portion of HBV large surface antigen that comprises the hepatocyte receptor binding portion of pre-Sl and all or a portion of HBV small (S) surface antigen, of the linear amino acid sequences represented by SEQ ID NO:97 (optimized HBV surface antigen, described below), SEQ ID NO:99 (optimized core protein, described below), or a corresponding sequence from another HBV strain, as applicable. A y of other sequences for le HBV surface antigens and HBV core antigens useful in the invention are described herein. In one aspect, the HBV large (L) surface antigen or the HBV core protein is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a full-length HBV large (L) surface antigen or HBV core protein, respectively, or to r HBV surface antigen or HBV core antigen described , including the amino acid sequence represented by SEQ ID NO:97 (optimized HBV surface antigen, described below), SEQ ID NO:99 (optimized core protein, described below), or a corresponding sequence from another HBV strain, as applicable.
Such a fusion protein is schematically represented in Fig. 2. One example of a composition comprising such a fusion protein is described in Example 1. In this ment, yeast (e.g., Saccharomyces cerevisiae) were engineered to express various HBV surface-core fusion ns as shown in Fig. 2 under the control of the copper- inducible er, CUP] or the TEF2 promoter. In each case, the HBV fusion protein was a single polypeptide with the ing sequence elements fused in frame from N- to C-terminus, represented by SEQ ID NO:34: (1) an N-terminal peptide to impart resistance to proteasomal degradation and stabilize expression (e.g., positions 1 to 6 of SEQ ID NO:34); 2) a two amino acid spacer to introduce a Spel ction enzyme site; 3) the amino acid sequence of a near full-length (minus position 1) HBV genotype C large (L) surface antigen (e.g., positions 9 to 407 of SEQ ID NO:34 or positions 2-400 of SEQ ID NO:ll (which differs from SEQ ID NO:34 at positions 350-351 of SEQ ID NO:11, where a Leu—Val ce in SEQ ID NO:11 is replaced with a Gln-Ala sequence at positions 357-358 of SEQ ID ); 4) the amino acid sequence of an HBV core antigen (e.g, positions 31-212 of SEQ ID NO:9 or positions 408 to 589 of SEQ ID N034); and 5) a hexahistidine tag (e.g., positions 590-595 of SEQ ID . Positions 28-54 of SEQ ID NO:34 comprise the hepatocyte receptor portion of large (L) surface protein. SEQ ID NO:34 ns multiple epitopes or domains that are believed to enhance the immunogenicity of the fusion n. For example, positions 209-220, positions 389-397, positions 360-367, and positions 499—506, with respect to SEQ ID NO:34, comprise known MHC Class 1 binding and/or CTL epitopes. Positions 305-328 of SEQ ID NO:34 comprise an antibody epitope. A nucleic acid sequence encoding the fusion protein of SEQ ID NO:34 (codon optimized for yeast expression) is represented herein by SEQ ID NOz33. A based immunotherapy composition expressing this fusion protein is also referred to herein as GI—l3002.
The amino acid segments used in any of the fusion proteins bed herein can be modified by the use of additional amino acids flanking either end of any domain; the descriptions provided herein are exemplary. For example, a fusion protein according to this embodiment can include 1) the amino acid ce of a near full-length (minus on 1) HBV genotype C large (L) surface n (6g, positions 2-400 of SEQ ID NO:11 or positions 9 to 407 of SEQ ID NO:34); and 2) the amino acid sequence of an HBV core antigen (e.g, positions 31-212 of SEQ ID N09 or positions 408 to 589 of SEQ ID N034), and utilize no N— or C-terminal sequences, or utilize different N— or C-terminal sequences and/or linkers or no linkers between HBV sequences. In one embodiment, instead of the N-terminal peptide represented by ons 1—6 of SEQ DI NO:34, an N- terminal peptide represented by SEQ ID NO:89 or SEQ ID NO:90 is utilized, followed by the remainder of the fiision protein, including or not including the hexahistidine C- terminal tag. The fusion n may also include one, two, three, four, five, six, or more linker (spacer) amino acids between HBV proteins or domains. The same ate embodiments apply to any fusion protein or HBV antigen construct used in the invention as described herein.
The HBV sequences used to design this fusion protein and many of the others described and/or exemplified herein are based on isolates of a particular HBV genotype (e.g, pe A, B, C, or D). However, it is an embodiment of the invention to add to or substitute into any portion of an HBV antigen described herein that is based on or derived from one ular genotype, sub-genotype, or strain, a corresponding sequence, or even a single or small amino acid tution, insertion or deletion that occurs in a corresponding sequence, from any other HBV pe(s), sub-genotype(s), or strain(s). In one embodiment, an HBV antigen can be produced by substituting an entire sequence(s) of an HBV antigen described herein with the corresponding sequence(s) from one or more different HBV genotypes, sub-genotypes or strain/isolates. Adding to or substituting a sequence from one HBV genotype or notype for another, for example, allows for the customization of the immunotherapeutic composition for a particular individual or population of individuals (e.g, a population of individuals within a given country or region of a country, in order to target the HBV genotype(s) that is most prevalent in that country or region of the country). Similarly, it is also an embodiment of the ion to use all or a portion of a consensus sequence derived from, determined from, or published for, a given HBV strain, genotype or subtype to make s in the sequence of a given HBV n to more closely or exactly correspond to the sus sequence. ing to the present invention and as generally understood in the art, a "consensus sequence" is typically a sequence based on the most common nucleotide or amino acid at a particular position of a given sequence after multiple sequences are aligned.
As a particular example of the above-mentioned types of modifications, an HBV n can be modified to change a T cell epitope in a given ce from one isolate to correspond more closely or exactly with a T cell epitope from a different isolate, or to correspond more closely or exactly with a consensus sequence for the T cell epitope.
Such T cell epitopes can include dominant epitopes and/or sub-dominant epitopes. Indeed, according to the invention, HBV antigens can be designed that incorporate consensus ces from a variety of HBV genotypes and/or subtypes, or mixtures of ces from different HBV genotypes and/or subtypes. Alignments of major HBV proteins across exemplary sequences from each of the major known genotypes can be readily generated using publicly available software, which will inform the generation of consensus ces, for example. Furthermore, consensus ces for many HBV proteins have been published. Since there is a high degree of conservation at the amino acid level among different HBV genotypes, sub-genotypes and strains, it is straightforward to use the corresponding portions of HBV proteins from genotypes, sub-genotypes or s other than those exemplified herein to create HBV antigens having a r or the same overall structure as those described . Examples of such ations are illustrated and exemplified herein.
By way of example, there can be minor ences among sequences of the same protein even within the same serotype and genotype (i.e., due to strain or isolate variations), although such differences in sequence identity will typically be less than 20% across the full length of the sequences being compared (z'.e., the sequences will be at least W0 2012/109404 80% identical), and more typically, the sequences will be at least 85% identical, 90% identical, 91% identical, 92% identical, 93% identical, 94% identical, 95% identical, 96% identical, 97% identical, 98% cal, 99% identical, or 100% identical, over the full length of the ed sequences. For example, in the fusion protein described above (SEQ ID N0234), the sequence for the large (L) surface antigen used in the fusion (positions 9-407 of SEQ ID NO:34) is from an HBV genotype C isolate, and is about 99% identical to positions 2-400 of SEQ ID NO:11, which is also from large (L) surface antigen from an HBV genotype C isolate (i.e., there are two different amino acids, at positions 350-351 of SEQ ID NO:11 (Gln-Ala) as compared to positions 357-358 of SEQ ID NO:34 (Leu-Val). However, either sequence is suitable for use in a fusion protein described herein, as are sequences from other HBV strains. Accordingly, in one embodiment, the sequences ed in any of the HBV antigens described herein, including any of the fusion proteins described herein, can e the corresponding sequences from one or more ent HBV genotypes, notypes, or strains.
The above-described utilization of consensus sequences and individual HBV pes has been applied to various HBV antigens described herein. For example, consensus sequence design has been applied to the fusion protein bed above with reference to SEQ ID NO:34, which contains HBV surface proteins and HBV core proteins.
Example 7 describes additional fusion ns that are similar in design to the fusion protein ented by SEQ ID N0234, but that are based on a consensus sequence for HBV genotypes A, B, C and D, respectively. A fusion protein sing HBV surface and core proteins that is based on a consensus sequence for HBV genotype A, which is also schematically illustrated in Fig. 2, is a single polypeptide with the following sequence elements fused in frame from N— to C-terminus, represented by SEQ ID NO:112 nal ces that are not HBV sequences are not included in the base sequence of SEQ ID NO:112, but may be added to this sequence as in the construct bed in Example 7): (1) optionally, an N-terminal peptide that is a synthetic N-terminal peptide designed to impart resistance to proteasomal degradation and stabilize expression represented by SEQ ID NO:37, which may be substituted by an N-terminal peptide represented by SEQ ID NO:89, SEQ ID NO:90, or another inal peptide suitable for use with a based immunotherapeutic as described herein; (2) optionally, a linker peptide of from one to three or more amino acids linker sequences of one, two, three or more amino acids, such as the two amino acid linker of Thr-Ser; (3) the amino acid sequence of a near full-length (minus position 1) a consensus sequence for HBV genotype A large (L) surface antigen represented by positions 1 to 399 of SEQ ID NO:112; (4) the amino acid sequence of a consensus sequence for HBV genotype A core antigen ented by positions 400 to 581 of SEQ ID NO:112; and (5) optionally, a stidine tag. A nucleic acid sequence encoding the fiision protein of SEQ ID NO:112 (codon optimized for yeast expression) is ented herein by SEQ ID . A yeast-based immunotherapy composition sing this fusion n is also referred to herein as GI-13006.
Example 7 also describes a fusion protein that is similar in design to the fusion protein ented by SEQ ID NO:34, but that is based on a consensus sequence for HBV genotype B. This fusion protein, which is also schematically illustrated in Fig. 2, is a single polypeptide with the following sequence elements fused in frame from N— to C— terminus, represented by SEQ ID NO:114 (optional sequences that are not HBV sequences are not included in the base sequence of SEQ ID NO:ll4, but may be added to this sequence as in the construct described in Example 7): (1) optionally, an N-terminal peptide that is a synthetic N—terminal peptide designed to impart resistance to proteasomal degradation and stabilize expression represented by SEQ ID N037, which may be substituted by an N—terminal e represented by SEQ ID NO:89, SEQ ID NO:90, or another N—terminal peptide suitable for use with a yeast-based immunotherapeutic as described herein; (2) optionally, a linker peptide of from one to three or more amino acids linker sequences of one, two, three or more amino acids, such as the two amino acid linker of Thr-Ser; (3) the amino acid sequence of a near full-length (minus position 1) a consensus sequence for HBV genotype B large (L) surface antigen represented by positions 1 to 399 of SEQ ID NO:ll4, (4) the amino acid sequence of a consensus sequence for HBV genotype B core antigen ented by positions 400 to 581 of SEQ ID NO:1 l4; and (5) optionally, a hexahistidine tag. A nucleic acid ce encoding the fusion n of SEQ ID NO:114 (codon optimized for yeast expression) is represented herein by SEQ ID NO:1 13. A yeast-based immunotherapy composition expressing this fusion protein is also referred to herein as GI—13007.
Example 7 describes a fusion protein that is similar in design to the fusion protein represented by SEQ ID NO:34, but that is based on a sus sequence for HBV pe C. This fusion protein, which is also schematically illustrated in Fig. 2, is a single polypeptide with the following sequence elements fused in frame from N— to C— terrninus, ented by SEQ ID NO:116 (optional sequences that are not HBV sequences are not included in the base sequence of SEQ ID NO:ll6, but may be added to this sequence as in the construct described in Example 7): (1) optionally, an N-terminal WO 09404 peptide that is a synthetic N—terminal peptide designed to impart resistance to proteasomal degradation and stabilize expression represented by SEQ ID NO:37, which may be substituted by an N—terminal peptide represented by SEQ ID N0289, SEQ ID N0290, or another N—terminal peptide suitable for use with a yeast-based immunotherapeutic as described herein; (2) optionally, a linker peptide of from one to three or more amino acids linker sequences of one, two, three or more amino acids, such as the two amino acid linker of Thr-Ser; (3) the amino acid sequence of a near full-length (minus position 1) a consensus ce for HBV genotype C large (L) surface antigen represented by positions 1 to 399 of SEQ ID NO:116; (4) the amino acid sequence of a consensus sequence for HBV genotype C core antigen represented by positions 400 to 581 of SEQ ID NO:116; and (5) optionally, a hexahistidine tag. A nucleic acid sequence encoding the fusion protein of SEQ ID NO:116 (codon optimized for yeast expression) is ented herein by SEQ ID NO:115. A yeast-based immunotherapy composition expressing this fusion protein is also referred to herein as GI-l3008.
Example 7 describes a fusion protein that is similar in design to the fusion protein represented by SEQ ID NO:34, but that is based on a consensus sequence for HBV genotype D. This fusion n, which is also schematically illustrated in Fig. 2, is a single ptide with the ing sequence elements fused in frame from N— to C- terminus, represented by SEQ ID NO:118 (optional sequences that are not HBV sequences are not included in the base sequence of SEQ ID , but may be added to this sequence as in the construct described in Example 7): (1) optionally, an N-terminal peptide that is a synthetic N-terminal e designed to impart resistance to proteasomal degradation and stabilize expression represented by SEQ ID NO:37, which may be tuted by an N—terminal peptide represented by SEQ ID N0289, SEQ ID N0290, or another N—terminal peptide suitable for use with a yeast-based immunotherapeutic as described herein; (2) optionally, a linker e of from one to three or more amino acids linker sequences of one, two, three or more amino acids, such as the two amino acid linker of Thr-Ser; (3) the amino acid sequence of a near full-length (minus position 1) a consensus sequence for HBV genotype D large (L) surface antigen represented by positions 1 to 399 of SEQ ID NO:118; (4) the amino acid sequence of a consensus sequence for HBV genotype D core n represented by positions 400 to 581 of SEQ ID NO:118; and (5) ally, a hexahistidine tag. The amino acid ce of a complete fusion protein described in e 7 comprising SEQ ID NO:118 and including the N- and C-terminal peptides and linkers is represented herein by SEQ ID NO:151. A nucleic acid sequence encoding the fusion protein of SEQ ID NO:118 or SEQ ID NO:151 (codon optimized for yeast sion) is represented herein by SEQ ID NO:117. A yeast-based immunotherapy composition expressing this fusion protein is also referred to herein as GI-13009.
HBVAntigens Comprising Surface n, Core Protein, Polymerase and X Antigen. In one embodiment of the invention, the HBV antigen(s) for use in a composition or method of the ion is a fusion protein comprising HBV antigens, wherein the HBV antigens comprise or t of: the HBV surface antigen (large (L), medium (M) or small (8)) or at least one structural, functional or immunogenic domain thereof), HBV polymerase or at least one structural, functional or immunogenic domain f, the HBV core protein (HBcAg) or HBV e-antigen (HBeAg) or at least one structural, onal or immunogenic domain f, and the HBV X antigen (HBx) or at least one structural, functional or immunogenic domain thereof. In one aspect, any one or more of the HBV surface antigen, HBV polymerase, HBV core protein, HBV e-antigen, HBV X antigen, or domain thereof, is fiJll-length or near full-length. In one aspect, any one or more of the HBV surface antigen, HBV polymerase, HBV core protein, HBV e- antigen, HBV X antigen, or domain thereof ses at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the linear sequence of a ength HBV surface antigen, HBV polymerase, HBV core protein, HBV e-antigen, HBV X antigen, or domain thereof, respectively, or of the linear amino acid sequences represented by SEQ ID NO:97 (optimized HBV surface antigen, described below), SEQ ID NO:98 (optimized HBV polymerase, described below), SEQ ID NO:99 (optimized core protein, described below), SEQ ID NO:100 (optimized X n, described below), or a ponding sequence from r HBV strain, as applicable. In one aspect, any one or more of the HBV surface antigen, HBV polymerase, HBV core protein, HBV e-antigen, HBV X antigen, or domain thereof is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a full-length HBV surface antigen, HBV rase, HBV core protein, HBV e-antigen, HBV X antigen, or domain thereof, respectively, or to the amino acid ces represented by SEQ ID NO:97 (optimized HBV surface antigen, described below), SEQ ID NO:98 (optimized HBV polymerase, described below), SEQ ID NO:99 (optimized core protein, bed below), or SEQ ID NO:100 (optimized X antigen, described below), or a corresponding sequence from another HBV strain, as applicable. A variety of le and exemplary sequences for HBV surface antigens, HBV polymerase antigens, HBV core antigens, and HBV X antigens are described herein.
W0 2012/109404 In one embodiment of the ion, the HBV antigen(s) for use in a composition or method of the invention is a fusion protein sing HBV antigens, n the HBV antigens comprise or consist of: the hepatocyte receptor portion of Pre— S1 of the HBV large (L) surface antigen or at least one immunogenic domain f, an HBV small (S) surface antigen (HBsAg) or at least one immunogenic domain thereof, the reverse transcriptase (RT) domain of HBV polymerase or at least one immunogenic domain f, the HBV core protein (HBcAg) or at least one immunogenic domain thereof, and the HBV X antigen (HBx) or at least one immunogenic domain thereof. In one aspect, any one or more of the hepatocyte receptor portion of Pre-Sl of the HBV large (L) surface antigen, the HBV small (S) surface n, the RT domain of HBV polymerase, the HBV core protein, X antigen, or domain thereof, is full-length or near full-length. In one aspect, any one or more of the hepatocyte receptor portion of Pre-Sl of the HBV large (L) surface n, the HBV small (S) surface antigen, the RT domain of HBV polymerase, the HBV core protein, X antigen, or domain thereof, comprises at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the linear ce of a ength Pre—Sl of the HBV large (L) surface antigen, the HBV small (S) surface antigen, the RT domain of HBV polymerase, the HBV core protein, X antigen, or domain thereof, respectively. In one aspect, any one or more of the hepatocyte receptor portion of Pre-Sl of the HBV large (L) surface antigen, the HBV small (S) surface antigen, the RT domain of HBV polymerase, the HBV core protein, X antigen, or domain thereof is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a full-length hepatocyte receptor portion of Pre-Sl of the HBV large (L) surface antigen, the HBV small (S) surface n, the RT domain of HBV polymerase, the HBV core protein, X antigen, or domain thereof, respectively.
Such a fusion n is schematically represented in Fig. 3. An example of a composition comprising this fusion protein is described in Example 2. In this embodiment, yeast (e.g, Saccharomyces cerevisiae) were engineered to express various HBV fusion proteins as schematically shown in Fig. 3 under the control of the copper-inducible promoter, CUP], or the TEF2 promoter. In one case, the fusion protein is a single polypeptide with the following sequence elements fused in frame from N— to C-terminus, represented by SEQ ID N0236: (1) an N-terminal peptide to impart resistance to proteasomal degradation and stabilize sion (e.g., positions 1 to 5 of SEQ ID N036); 2) the amino acid sequence of an HBV pe C hepatocyte receptor domain of the pre-Sl portion of HBV large (L) surface protein (unique to L) (e.g, positions 21—47 of W0 2012/109404 SEQ ID NO:11 or positions 6 to 32 of SEQ ID NO:36); 3) the amino acid sequence of a ength HBV genotype C small (S) surface antigen (e.g., positions 176 to 400 of SEQ ID NO:ll or positions 33 to 257 of SEQ ID NO:36); 4) a two amino acid /linker to facilitate cloning and manipulation of the sequences (e.g., positions 258 and 259 of SEQ ID NO:36); 5) the amino acid sequence of a portion of the HBV genotype C polymerase including the reverse transcriptase domain (e.g., positions 247 to 691 of SEQ ID NO:10 or positions 260 to 604 of SEQ ID NO:36); 6) an HBV genotype C core n (e.g., positions 31-212 of SEQ ID NO:9 or ons 605 to 786 of SEQ ID NO:36); 7) the amino acid sequence of an HBV genotype C X antigen (e.g., positions 2 to 154 of SEQ ID NO:12 or positions 787 to 939 of SEQ ID N036); and 8) a hexahistidine tag (e.g., positions 940 to 945 of SEQ ID NO:36). A nucleic acid sequence encoding the fusion protein of SEQ ID NO:36 (codon optimized for yeast expression) is represented herein by SEQ ID NO:35. A yeast-based immunotherapy composition expressing this fusion protein is referred to herein as GI-13005.
In one alternate example of this embodiment, a fusion n according to the embodiment described above or that below can include 1) the amino acid sequence of an HBV genotype C cyte receptor domain of the pre-Sl portion of HBV large (L) surface protein (unique to L) (e.g., positions 21-47 of SEQ ID NO:ll or positions 6 to 32 of SEQ ID NO:36); 2) the amino acid sequence of a full-length HBV genotype C small (S) surface antigen (e.g., positions 176 to 400 of SEQ ID NO:11 or positions 33 to 257 of SEQ ID NO:36); 3) the amino acid sequence of a portion of the HBV genotype C polymerase ing the e transcriptase domain (e.g, positions 247 to 691 of SEQ ID NO:10 or positions 260 to 604 of SEQ ID NO:36); 4) an HBV genotype C core protein (e.g., positions 31-212 of SEQ ID N029 or positions 605 to 786 of SEQ ID N036); and 5) the amino acid sequence of an HBV genotype C X n (e.g, positions 2 to 154 of SEQ ID NO:12 or positions 787 to 939 of SEQ ID N036), and utilize no N— or C-terminal sequences, or utilize different N— or C-terminal sequences, and/or use linkers or no linkers between HBV sequences.
] In one embodiment, instead of the N-terminal peptide represented by ons 1-5 of SEQ DI NO:36, an N—terminal peptide represented by SEQ ID NO:89 or SEQ ID NO:90 is utilized (or a homologue thereof), followed by the remainder of the fusion protein as described. Example 2 describes such a fusion n, which is also illustrated by the schematic depiction of the construct in Fig. 3. In this ment, yeast (e.g, Saccharomyces cerevisiae) were again engineered to express various HBV fusion proteins W0 2012/109404 as schematically shown in Fig. 3 under the control of the copper-inducible promoter, CUP], or the TEF2 promoter. In this second case, the fusion protein is a single polypeptide with the following ce elements fused in frame from N— to C-terrninus, represented by SEQ ID NO:92: (1) an N—terminal peptide to impart resistance to proteasomal degradation and stabilize or enhance expression (SEQ ID NO:89, positions 1 to 89 of SEQ ID ; 2) a two amino acid /linker (Thr-Ser) to facilitate cloning and manipulation of the sequences (positions 90 to 91 of SEQ ID NO:92); 3) the amino acid sequence of an HBV genotype C hepatocyte receptor domain of the pre-Sl portion of HBV large (L) surface protein (unique to L) (e. g., positions 21-47 of SEQ ID NO:11 or positions 92 to 118 of SEQ ID NO:92); 4) the amino acid sequence of a full—length HBV genotype C small (S) surface antigen (e.g., ons 176 to 400 of SEQ ID NO:11 or positions 119 to 343 of SEQ ID NO:92); 5) a two amino acid spacer/linker (Leu-Glu) to facilitate g and manipulation of the sequences (e.g., ons 344 to 345 of SEQ ID NO:92); 6) the amino acid sequence of a portion of the HBV genotype C polymerase including the reverse transcriptase domain (e.g, positions 247 to 691 of SEQ ID NO:10 or positions 346 to 690 of SEQ ID NO:92); 7) an HBV genotype C core protein (e.g., positions 31—212 of SEQ ID N029 or positions 691 to 872 of SEQ ID NO:92); 8) the amino acid sequence of an HBV genotype C X antigen (e.g., positions 2 to 154 of SEQ ID NO:12 or positions 873 to 1025 of SEQ ID NO:92); and 9) a hexahistidine tag (e.g., positions 1026 to 1031 of SEQ ID NO:92). A nucleic acid sequence encoding the fusion protein of SEQ ID NO:92 (codon-optimized for expression in yeast) is represented herein by SEQ ID NO:91. A yeast-based therapy composition expressing this fusion protein is ed to herein as GI-13004.
SEQ ID NO:36 and SEQ ID NO:92 contain multiple epitopes or domains that are believed to e the immunogenicity of the fusion protein, including several described above for SEQ ID NO:34. In addition, the reverse transcriptase domain used in this fusion protein contains l amino acid ons that are known to become mutated as a drug-resistance response to treatment with various anti-Viral drugs, and therefore, any one or more of these may be d in this fusion protein in order to provide a therapeutic or prophylactic immunotherapeutic that targets specific drug resistance (escape) mutations. These amino acid positions are, with respect to SEQ ID NO:36, at amino acid position: 432 (Val, known to mutate to a Leu after lamivudine therapy); position 439 (Leu, known to mutate to a Met after dine therapy); position 453 (Ala, known to mutate to a Thr after tenofovir therapy); position 463 (Met, known to mutate to an Ile or Val after lamivudine therapy); and position 495 (Asn, known to mutate to Thr after adefovir therapy). These amino acid positions are, with respect to SEQ ID NO:92, at amino acid position: 518 (Val, known to mutate to a Leu after lamivudine therapy); position 525 (Leu, known to mutate to a Met after lamivudine therapy); position 539 (Ala, known to mutate to a Thr after tenofovir therapy); position 549 (Met, known to mutate to an Ile or Val after lamivudine therapy); and position 581 (Asn, known to mutate to Thr after adefovir therapy). onal drug resistance mutations that are identified or that have been identified can be added, as desired, to create additional immunotherapeutics targeting such mutations, using the guidance provided .
In one embodiment of the invention, the valine at position 901 in SEQ ID NO:36 or the valine at position 987 of SEQ ID NO:92 (or the valine at on 116 of SEQ ID NO:12 or in any X antigen or domain thereof containing this corresponding position) is substituted with a leucine, to create the T cell epitope identified as SEQ ID NO:51 (see Table 5).
As discussed above, the invention includes the modification of HBV antigens from their lly occurring or wild-type sequences for inclusion in a yeast-based immunotherapeutic that improve the clinical y or meet required ia for therapeutics or prophylactics related to infectious agents. By way of example, the ing discussion and Examples 5-8 describe the design and construction of yeast- based immunotherapeutics that takes into consideration one or more criteria of RAC requirements, zation of immunogenic domains associated with the most beneficial immune responses, maximization of conserved T cell es, utilization of consensus sequences for a particular HBV pe, and/or zation of artificial junctions within the HBV antigen. For example, the following yeast-based immunotherapeutic composition exemplifies an HBV fusion protein meeting the requirements of the goals specified above, and comprising portions of each of the HBV major proteins: HBV surface antigen, polymerase, core and X antigen. To design this fusion protein, individual HBV antigens within the fusion were optimized or modified to reduce the size of the segments in the protein (e.g, to ensure that the protein represented less than 2/3 of the HBV genome), as well as to maximize the inclusion of T cell epitopes that have been associated with an immune response in acute/self—limiting HBV ion and/or chronic HBV infection, to maximize conserved epitopes, and to minimize non-natural ces.
One of skill in the art using this guidance can produce alternate optimized HBV proteins for use in an HBV antigen of the invention.
As described in more detail in Example 5, to uct an HBV e antigen segment, a full-length large (L) surface n protein from HBV genotype C was reduced in size by tion of the N- and C-terminal sequences, while maximizing the inclusion of known MHC T cell epitopes, using the prioritization for inclusion of T cell epitopes associated with acute/self-limiting infections. The resulting e antigen segment is represented by SEQ ID NO:97.
To construct the segment of the fusion protein comprising HBV polymerase (see Example 5), substantial portions of a fiJll—length polymerase from HBV genotype C were eliminated by focusing on inclusion of the active site domain (from the RT domain), which is the most conserved region of the protein among HBV pes and isolates, and which includes several sites where drug resistance ons have been known to occur.
The HBV polymerase segment was designed to maximize known T cell es, using the prioritization strategy discussed above, and to modify one of the T cell epitopes to correspond exactly to a known T cell epitope that differed by a single amino acid. The ing HBV polymerase antigen segment is represented by SEQ ID NO:98.
] To construct the t of the fusion protein comprising HBV Core antigen (see Example 5), a full—length Core protein from HBV genotype C was modified to reduce the size of the protein while maximizing the number of T cell epitopes by inclusion and by ation of sequence to created perfect matches to certain known T cell epitopes. In addition, sequence was removed that contained exceptionally positively charged C- terminus which may be toxic to yeast by competitive interference with natural yeast RNA binding proteins which often are ne rich (positively charged). The resulting HBV Core antigen segment is represented by SEQ ID NO:99.
To construct the segment of the fusion protein comprising HBV X antigen (see Example 5), a full-length X antigen from HBV genotype C was truncated to reduce the size of the protein, while maximizing the retention of most of the known T cell epitopes.
Single amino acid changes were also introduced to correspond to the published T cell epitope sequences, and sequence flanking the T cell es at the ends of the segment was retained to facilitate efficient processing and presentation of the t epitopes by an antigen presenting cell. The resulting HBV X antigen segment is represented by SEQ ID NO:100.
Finally, as described in Example 5, a complete fusion protein was constructed by linking the four HBV segments described above to form a single protein optimized for clinical use. Two ent exemplary fusion proteins were d, each with a different N-terminal peptide added to enhance and/or stabilize expression of the fusion protein in yeast. As described previously herein with respect to all of the other ns used in a yeast-based immunotherapeutic compositions described herein, the N—terminal peptide can be replaced with a different synthetic or natural N—terminal peptide or with a homologue thereof, or the N-terminal peptide can be omitted altogether and a methionine included at position one. In addition, linker sequences of one, two, three or more amino acids may be added between segments of the fusion protein, if desired. For example, a two amino acid linker sequence such as r may be inserted between the N-terminal peptide and the first HBV antigen in the fusion protein, and/or between two HBV antigens in the fusion n. Also, while these constructs were designed using HBV proteins from genotype C as the ne, any other HBV genotype, sub-genotype, or HBV proteins from different strains or isolates can be used to design the protein segments. In one aspect, consensus sequences from a given HBV genotype can be used to design or form the protein segments, as described in additional fusion ns below. Finally, if one or more segments are excluded from the fusion protein as described herein, then the sequence from the remaining segments can be expanded in length, if desired, to include additional T cell epitopes and/or flanking regions of the remaining ns.
Example 5 bes an HBV fusion protein, which is also illustrated by the schematic depiction of the construct in Fig. 3, that is a single ptide with the following sequence ts fused in frame from N— to C-terminus, represented by SEQ ID NO:101: (1) an N—terminal peptide that is an alpha factor prepro ce, to impart resistance to proteasomal degradation and stabilize expression represented by SEQ ID NO:89 ions 1-89 of SEQ ID ); (2) an optimized portion of an HBV large (L) surface antigen represented by SEQ ID NO:97 (positions 90 to 338 of SEQ ID NO:101, e.g., corresponding to positions 120 to 368 of SEQ ID NO:11 plus optimization of epitopes); (3) an optimized portion of the reverse transcriptase (RT) domain of HBV polymerase represented by SEQ ID NO:98 (positions 339 to 566 of SEQ ID NO:101, e.g, corresponding to positions 453 to 680 of SEQ ID NO:10 plus optimization of es); (4) an optimized portion of HBV Core protein represented by SEQ ID NO:99 (positions 567 to 718 of SEQ ID NO:101 e.g., corresponding to positions 37 to 188 of SEQ ID NO:9 plus optimization of epitopes); (5) an optimized n of HBV X n represented by SEQ ID NO:100 (positions 719 to 778 of SEQ ID NO:101, e.g, corresponding to positions 52 to 127 of SEQ ID NO:12 plus optimization of epitopes); and (6) a hexahistidine tag (e.g., positions 779 to 784 of SEQ ID NO: 101). In one embodiment, the linker ce of threonine (Thr or T)—serine (Ser or S) is used between the N-terminal peptide of SEQ ID NO:89 and the first HBV n (optimized portion of HBV large surface antigen), thereby extending the total length of SEQ ID NO:101 by two amino acids.
Example 5 also describes a fusion n, which is also illustrated by the schematic depiction of the construct in Fig. 3, that is a single polypeptide with the following sequence elements fiJsed in frame from N— to C-terminus, represented by SEQ ID NO:102: (1) an N—terminal peptide that is a synthetic N-terminal peptide designed to impart resistance to proteasomal degradation and stabilize expression represented by SEQ ID NO:37 ions l-6 of SEQ ID NO:102); (2) an optimized portion of an HBV large (L) surface antigen represented by ons 2 to 248 of SEQ ID NO:97 (positions 7 to 254 of SEQ ID , e.g., corresponding to positions 120 to 368 of SEQ ID NO:1l plus zation of epitopes); (3) an optimized portion of the reverse riptase (RT) domain of HBV polymerase represented by SEQ ID NO:98 (positions 255 to 482 of SEQ ID NO:102, e. g., corresponding to positions 453 to 680 of SEQ ID NO:10 plus optimization of epitopes); (4) an optimized portion of HBV Core protein represented by SEQ ID NO:99 (positions 483 to 634 of SEQ ID NO:102, e.g, corresponding to positions 37 to 188 of SEQ ID N029 plus optimization of epitopes); (5) an zed portion of HBV X antigen represented by SEQ ID NO:100 (positions 635 to 694 of SEQ ID NO:102, e.g., corresponding to positions 52 to 127 of SEQ ID NO:12 plus optimization of epitopes); and (6) a hexahistidine tag (e.g., positions 695 to 700 of SEQ ID ). In one embodiment, the linker sequence of threonine (Thr or ine (Ser or S) is used between the N-terminal e of SEQ ID N037 and the first HBV protein (optimized portion of HBV large surface antigen), thereby extending the total length of SEQ ID NO:102 by two amino acids. In one embodiment, an optimized portion of an HBV large (L) surface antigen used in the fusion protein described above is represented by ons 1 to 248 of SEQ ID NO:97 (thereby extending the total length of SEQ ID NO:102 by one amino acid). In one embodiment both the T-S linker and positions 1-248 of SEQ ID NO:97 are used in SEQ ID NO:102.
As sed above, the invention es the modification of HBV antigens from their naturally occurring or wild-type sequences for inclusion in a yeast-based immunotherapeutic that improve the clinical utility or meet required criteria for therapeutics or prophylactics related to infectious agents, utilizing consensus sequences from a given HBV genotype to design or form the protein segments. By way of example, additional HBV antigens for use in a yeast-based immunotherapeutic of the invention were designed to illustrate this type of modification. As in the design of the HBV fusion ns represented bed above, to produce these additional fusion ns, individual HBV ns within the fusion were optimized or modified to reduce the size of the segments in the protein (e.g., to ensure that the protein ented less than 2/3 of the HBV genome), as well as to maximize the inclusion of T cell epitopes that have been associated with an immune response in acute/self—limiting HBV infection and/or chronic HBV infection, to maximize conserved epitopes, to minimize non-natural sequences, and also to utilize sus sequences for each of genotype A-D that were built from multiple sources of HBV sequences (e.g, Yu and Yuan et al, 2010, for S, Core and X, where consensus sequences were ted from 322 HBV sequences, or for Pol (RT), from the Stanford University HIV Drug Resistance Database, HBVseq and HBV Site Release . In designing the following four exemplary fusion proteins comprising HBV antigens, the consensus sequence for the given HBV genotype was used unless using the sus sequence altered one of the known acute self-limiting T cells epitopes or one of the known polymerase escape mutation sites, in which case, these positions followed the published sequence for these epitopes or mutation sites. Additional antigens could be constructed based solely on consensus sequences or using other published epitopes as they become known.
] Example 7 describes a fusion protein that is r in design to the fusion protein represented by SEQ ID NO:101 or SEQ ID NO:102 (illustrated schematically by Fig. 3), but that is based on a consensus ce for HBV genotype A. This fusion protein is a single polypeptide with the following sequence elements fused in frame from N— to C-terminus, represented by SEQ ID NO:107 (optional sequences that are not HBV sequences are not included in the base sequence of SEQ ID NO: 107, but may be added to this sequence as in the construct described in Example 7): (1) optionally, an N—terminal peptide that is a synthetic N—terminal peptide designed to impart resistance to proteasomal degradation and stabilize expression represented by SEQ ID NO:37, which may be tuted by an N—terminal peptide represented by SEQ ID N0289, SEQ ID NO:90, or another N-terminal peptide suitable for use with a yeast-based immunotherapeutic as described ; (2) optionally, a linker peptide of from one to three or more amino acids linker sequences of one, two, three or more amino acids, such as the two amino acid linker of r; (3) an optimized portion of an HBV large (L) surface antigen represented by positions 1 to 249 of SEQ ID NO:107, which is a consensus sequence for HBV genotype A utilizing the design strategy discussed above; (4) an optimized portion of the reverse transcriptase (RT) domain of HBV polymerase represented by positions 250 to 477 of SEQ ID NO: 107, which is a consensus sequence for HBV genotype A utilizing the design strategy sed above; (5) an zed portion of HBV Core protein represented by positions 478 to 629 of SEQ ID NO:107, which is a consensus sequence for HBV genotype A utilizing the design strategy discussed above; (6) an optimized portion of HBV X antigen represented by positions 630 to 689 of SEQ ID NO:107, which is a consensus sequence for HBV genotype A utilizing the design strategy discussed above; and (7) optionally, a hexahistidine tag. A yeast-based immunotherapy composition expressing this fusion n is also referred to herein as GI-l3010.
Example 7 also describes a fusion protein that is r in design to the fusion protein represented by SEQ ID NO:101 or SEQ ID NO:102 (illustrated schematically by Fig. 3), but that is based on a consensus sequence for HBV genotype B. This fusion protein is a single polypeptide with the following sequence elements fused in frame from N— to C-terminus, represented by SEQ ID NO:108 (optional sequences that are not HBV ces are not included in the base sequence of SEQ ID NO: 108, but may be added to this sequence as in the construct described in e 7): (l) optionally, an N-terminal peptide that is a synthetic N—terminal e designed to impart resistance to proteasomal ation and stabilize expression represented by SEQ ID NO:37, which may be substituted by an N—terminal peptide represented by SEQ ID NO:89, SEQ ID NO:90, or another N-terminal peptide suitable for use with a yeast-based therapeutic as described herein; (2) optionally, a linker peptide of from one to three or more amino acids linker sequences of one, two, three or more amino acids, such as the two amino acid linker of Thr-Ser; (3) an optimized portion of an HBV large (L) surface antigen represented by positions 1 to 249 of SEQ ID NO:108, which is a consensus sequence for HBV genotype B utilizing the design strategy discussed above; (4) an optimized portion of the reverse transcriptase (RT) domain of HBV rase represented by positions 250 to 477 of SEQ ID NO:108, which is a consensus sequence for HBV genotype B utilizing the design strategy discussed above; (5) an optimized portion of HBV Core protein represented by positions 478 to 629 of SEQ ID NO:108, which is a consensus ce for HBV genotype B utilizing the design strategy discussed above; (6) an optimized portion of HBV X antigen represented by positions 630 to 689 of SEQ ID , which is a consensus sequence for HBV genotype B utilizing the design strategy discussed above; and (7) optionally, a hexahistidine tag. A yeast-based therapy composition sing this fusion protein is also referred to herein as GI-l301 l.
Example 7 also describes a fusion n that is similar in design to the fusion protein represented by SEQ ID NO:lOl or SEQ ID NO:102 (illustrated schematically by Fig. 3), but that is based on a consensus sequence for HBV pe C. This fusion protein is a single polypeptide with the following sequence elements fused in frame from N— to C-terminus, represented by SEQ ID NO:109 (optional sequences that are not HBV sequences are not included in the base sequence of SEQ ID NO: 109, but may be added to this sequence as in the construct described in Example 7): (1) optionally, an N-terminal e that is a synthetic N—terminal peptide designed to impart ance to proteasomal degradation and stabilize expression represented by SEQ ID NO:37, which may be substituted by an N—terminal peptide represented by SEQ ID N0289, SEQ ID N0290, or another inal peptide suitable for use with a yeast-based immunotherapeutic as described herein; (2) optionally, a linker peptide of from one to three or more amino acids linker sequences of one, two, three or more amino acids, such as the two amino acid linker of Thr-Ser; (3) an optimized n of an HBV large (L) surface antigen represented by ons I to 249 of SEQ ID NO:lO9, which is a consensus sequence for HBV genotype C ing the design strategy discussed above; (4) an optimized n of the reverse transcriptase (RT) domain of HBV rase represented by positions 250 to 477 of SEQ ID , which is a consensus sequence for HBV genotype C utilizing the design strategy discussed above; (5) an optimized portion of HBV Core protein represented by positions 478 to 629 of SEQ ID NO:lO9, which is a consensus sequence for HBV genotype C utilizing the design strategy discussed above; (6) an optimized n of HBV X antigen represented by positions 630 to 689 of SEQ ID NO:lO9, which is a consensus sequence for HBV genotype C utilizing the design strategy discussed above; and (7) optionally, a hexahistidine tag. A yeast-based immunotherapy composition expressing this fusion protein is also referred to herein as GI-l3012.
Example 7 also bes a fusion protein that is similar in design to the fusion protein represented by SEQ ID NO:lOl or SEQ ID NO:lO2 (illustrated schematically by Fig. 3), but that is based on a consensus sequence for HBV genotype D. This fusion protein is a single polypeptide with the following sequence elements fused in frame from N— to C-terminus, represented by SEQ ID NO:llO (optional sequences that are not HBV sequences are not included in the base sequence of SEQ ID N021 10, but may be added to this sequence as in the construct described in Example 7): (l) optionally, an N—terminal e that is a synthetic N—terminal peptide ed to impart resistance to somal degradation and stabilize expression represented by SEQ ID NO:37, which may be substituted by an N—terminal peptide represented by SEQ ID N0289, SEQ ID NO:90, or another N—terminal peptide suitable for use with a yeast-based immunotherapeutic as described herein; (2) optionally, a linker peptide of from one to three or more amino acids linker sequences of one, two, three or more amino acids, such as the two amino acid linker of Thr-Ser; (3) an optimized portion of an HBV large (L) surface antigen ented by ons 1 to 249 of SEQ ID NO:llO, which is a consensus sequence for HBV genotype D utilizing the design strategy discussed above; (4) an optimized portion of the e transcriptase (RT) domain of HBV polymerase represented by positions 250 to 477 of SEQ ID NO: 1 10, which is a sus sequence for HBV genotype D utilizing the design strategy discussed above; (5) an optimized portion of HBV Core protein represented by positions 478 to 629 of SEQ ID , which is a consensus sequence for HBV genotype D utilizing the design strategy discussed above; (6) an optimized portion of HBV X antigen represented by positions 630 to 689 of SEQ ID NO:llO, which is a consensus sequence for HBV genotype D utilizing the design gy sed above; and (7) ally, a stidine tag. A yeast-based immunotherapy composition expressing this fusion protein which comprises an N—terminal sequence represented by SEQ ID NO:37 is referred to herein as GI-l3013. A yeast-based immunotherapy composition expressing this fusion n which comprises an N—terminal ce represented by SEQ ID N0289 is referred to herein as GI—l3014.
As discussed above, it is one embodiment of the invention to change the order of HBV n segments within a fusion protein described herein. Accordingly, although the constructs utilizing four HBV proteins as bed above are ed in the order of a surface antigen fused to a polymerase antigen fused to a Core antigen fused to an X antigen, the invention is not limited to this particular order of proteins within the construct, and indeed, other arrangements of fusion segments may be used and in some aspects, may improve the resulting immunotherapeutic compositions. For example, rearrangement of segments within a fusion protein may improve or modify expression of the HBV antigen in yeast, or may improve or modify the immunogenicity or other functional attribute of the HBV antigen. In one aspect of this embodiment, the invention plates beginning with one HBV antigen that expresses well in yeast and/or provides positive functional data (e.g., is immunogenic), and adding additional HBV proteins or domains to that HBV antigen in order to expand the potential antigens or epitopes that are contained within the HBV antigen. Example 8 provides an example of additional ements of the four HBV proteins described above.
W0 2012/109404 2012/024409 Example 8 describes a fusion protein that contains sequences from HBV surface antigen, core protein, polymerase and X antigen, where the sequences were derived from segments of the fusion ns represented by SEQ ID NO:110 and SEQ ID NO:118, and where the fusion protein utilizes a different order of fusion segments as compared to SEQ ID NO:110. This antigen is based on a consensus sequence for HBV genotype D; however, it would be straightforward to produce a fusion protein having a similar overall structure using the corresponding fusion segments from the fusion proteins represented by SEQ ID NO:107 or SEQ ID NO:112 (genotype A), SEQ ID NO:108 or SEQ ID NO:114 (genotype B), SEQ ID NO:109 or SEQ ID NO:116 (genotype C), or using the corresponding sequences from a different HBV genotype, sub-genotype, consensus sequence or strain. In this example, yeast (e.g., Saccharomyces cerevisiae) were engineered to express this fusion protein under the control of the copper-inducible promoter, CUPI, and the resulting HBV immunotherapy composition can be referred to herein as GI-13017, schematically illustrated in Fig. 10. The fusion protein represented by SEQ ID NO:124 comprises, in order, surface antigen, core, polymerase and X antigen ces, as a single polypeptide with the following sequence elements fused in frame from N— to C-terminus, represented by SEQ ID NO:124 (optional sequences that are not HBV sequences are not included in the base ce of SEQ ID NO:124, but may be added to this ce as in the construct described in Example 8): (1) ally, an N-terminal peptide that is a synthetic inal peptide designed to impart resistance to proteasomal degradation and stabilize expression represented by SEQ ID NO:37 (in the construct bed in Example 8), which may be substituted by an N-terminal peptide represented by SEQ ID NO:89, SEQ ID NO:90, or another N—terminal peptide suitable for use with a yeast-based immunotherapeutic as described herein; (2) optionally, a linker peptide of from one to three or more amino acids, such as the two amino acid linker of Thr-Ser (in the construct described in Example 8); (3) the amino acid sequence of a near full-length (minus position 1) consensus ce for HBV genotype D large (L) surface antigen represented by ons 1 to 399 of SEQ ID NO: 124 (corresponding to positions 1 to 399 of SEQ ID NO:118); 4) the amino acid sequence of a consensus sequence for HBV genotype D core antigen represented by ons 400 to 581 of SEQ ID NO:124 (corresponding to positions 400 to 581 of SEQ ID NO:118); (5) an optimized portion of the reverse transcriptase (RT) domain of HBV polymerase using a consensus sequence for HBV genotype D, represented by positions 582 to 809 of SEQ ID NO:124 (corresponding to ons to 250 to 477 of SEQ ID NO:110); (6) an zed portion of HBV X 2012/024409 antigen using a consensus sequence for HBV genotype D, ented by ons 810 to 869 of SEQ ID NO:124 (corresponding to positions 630 to 689 of SEQ ID NO:110); and (7) optionally, a hexahistidine tag (in the construct bed in Example 8). SEQ ID NO:I24 contains le T cell epitopes (human and murine), which can be found in Table 5. A nucleic acid sequence encoding the fusion protein of SEQ ID NO: 124 (codon— optimized for expression in yeast) is represented herein by SEQ ID NO: 123.
Example 8 also describes another fusion protein that contains sequences from HBV surface antigen, core protein, X n, and polymerase, where the sequences were derived from segments of the fusion proteins ented by SEQ ID NO:110 and SEQ ID NO:118, but where the fusion protein utilizes a different order of fusion segments as compared to SEQ ID NO:110. This antigen is also based on a consensus sequence for HBV genotype D; however, it would be straightforward to produce a fusion protein having a similar overall structure using the corresponding fusion segments from the fusion proteins represented by SEQ ID NO:107 or SEQ ID NO:112 (genotype A), SEQ ID NO:lO8 or SEQ ID NO:ll4 ype B), SEQ ID NOle9 or SEQ ID NO:ll6 (genotype C), or using the corresponding sequences from a different HBV genotype, sub-genotype, consensus sequence or strain. In this example, yeast (e.g., Saccharomyces cerevisiae) were ered to express this filSIOIl protein under the control of the copper-inducible promoter, CUPI, and the resulting yeast-HBV immunotherapy composition can be referred to herein as 18, schematically illustrated in Fig. 11. The fusion protein represented by SEQ ID NO:126 comprises, in order, surface antigen, core, X antigen, and polymerase sequences, as a single polypeptide with the following sequence elements fused in frame from N— to C-terminus, represented by SEQ ID NO:126 (optional sequences that are not HBV sequences are not included in the base sequence of SEQ ID NO: 126, but may be added to this sequence as in the construct described in Example 8): (1) optionally, an N—terminal peptide that is a synthetic N—terminal peptide designed to impart resistance to proteasomal degradation and stabilize expression represented by SEQ ID NO:37 (in the uct bed in Example 8), which may be substituted by an N-terminal peptide represented by SEQ ID NO:89, SEQ ID NO:90, or another N-terminal peptide suitable for use with a yeast-based therapeutic as bed herein; (2) optionally, a linker peptide of from one to three or more amino acids, such as the two amino acid linker of Thr-Ser (in the construct described in Example 8); (3) the amino acid sequence of a near full-length (minus position 1) consensus sequence for HBV genotype D large (L) surface antigen represented by positions 1 to 399 of SEQ ID NO:126 sponding to positions 1 to 399 of SEQ ID NO:118); 4) the amino acid sequence of a consensus sequence for HBV genotype D core antigen represented by ons 400 to 581 of SEQ ID NO:126 (corresponding to ons 400 to 581 of SEQ ID NO:118); (5) an optimized portion of HBV X antigen using a consensus sequence for HBV genotype D, represented by positions 582 to 641 of SEQ ID NO:126 (corresponding to positions 630 to 689 of SEQ ID NO:110); (5) an optimized portion of the reverse transcriptase (RT) domain of HBV polymerase using a sus sequence for HBV genotype D, ented by positions 642 to 869 of SEQ ID NO:126 (corresponding to positions to 250 to 477 of SEQ ID NO:1 10); and (7) optionally, a stidine tag (in the construct described in Example 8).
SEQ ID NO:126 contains multiple T cell epitopes (human and murine), which can be found in Table 5. A nucleic acid sequence encoding the fusion protein of SEQ ID NO: 126 (codon-optimized for expression in yeast) is represented herein by SEQ ID NO:125.
] Example 8 describes another fusion protein that contains sequences from HBV polymerase, X antigen, surface antigen, core protein, where the sequences were derived from segments of the fusion proteins represented by SEQ ID NO:110 and SEQ ID , but where the fusion protein es a different order of fiision segments as compared to SEQ ID NO:110. This antigen is based on a consensus sequence for HBV genotype D; however, it would be straightforward to produce a fiJsion protein having a similar overall structure using the corresponding fusion segments from the fusion proteins represented by SEQ ID NO:107 or SEQ ID NO:112 (genotype A), SEQ ID NO:108 or SEQ ID NO:114 (genotype B), SEQ ID NO:109 or SEQ ID NO:116 (genotype C), or using the corresponding sequences from a different HBV genotype, sub-genotype, sus sequence or strain. In this example, yeast (e.g., Saccharomyces cerevisiae) were engineered to express this fusion protein under the control of the -inducible promoter, CUP], and the resulting HBV immunotherapy composition can be referred to herein as GI-1302l, schematically illustrated in Fig. 14. The fusion protein represented by SEQ ID NO:132 comprises, in order, polymerase, X antigen, surface antigen, and core, as a single ptide with the following sequence elements fused in frame from N- to C-terminus, ented by SEQ ID NO: 132 nal sequences that are not HBV sequences are not included in the base sequence of SEQ ID NO:132, but may be added to this sequence as in the construct described in Example 8): (1) optionally, an N— terminal peptide that is a synthetic N—terminal peptide designed to impart resistance to proteasomal degradation and stabilize expression represented by SEQ ID NO:37 (in the uct bed in Example 8), which may be substituted by an N-terminal peptide represented by SEQ ID NO:89, SEQ ID NO:90, or another N—terminal peptide suitable for use with a yeast-based immunotherapeutic as described herein; (2) optionally, a linker peptide of from one to three or more amino acids, such as the two amino acid linker of Thr-Ser (in the construct described in Example 8); (3) an optimized portion of the e riptase (RT) domain of HBV polymerase using a consensus sequence for HBV pe D, represented by positions 1 to 228 of SEQ ID NO:l32 (corresponding to ons to 250 to 477 of SEQ ID NO:1 10); (4) an optimized portion of HBV X antigen using a consensus sequence for HBV genotype D, represented by positions 229 to 288 of SEQ ID NO:l32 (corresponding to positions 630 to 689 of SEQ ID NOleO); (5) the amino acid sequence of a near full-length (minus position 1) consensus sequence for HBV genotype D large (L) surface antigen represented by positions 289 to 687 of SEQ ID NOzl32 (corresponding to positions 1 to 399 of SEQ ID NOzll8); (6) the amino acid sequence of a consensus sequence for HBV pe D core antigen represented by positions 688 to 869 of SEQ ID NO:l32 (corresponding to positions 400 to 581 of SEQ ID NO:1 l8); and (7) optionally, a hexahistidine tag (in the construct described in Example 8).
SEQ ID NO:l32 contains le T cell epitopes (human and murine), which can be found in Table 5. A nucleic acid sequence encoding the fusion protein of SEQ ID NO: 132 (codon-optimized for expression in yeast) is represented herein by SEQ ID NO:13 1.
Example 8 also describes a fusion n that contains sequences from HBV X n, polymerase, surface antigen, and core n, where the sequences were derived from segments of the fusion proteins represented by SEQ ID NO] 10 and SEQ ID , but where the fiision protein es a different order of fusion segments as compared to SEQ ID NO:1 10. This antigen is based on a consensus sequence for HBV genotype D; however, it would be straightforward to produce a fusion protein having a similar overall ure using the corresponding fusion segments from the fusion proteins ented by SEQ ID NO:lO7 or SEQ ID NO:ll2 (genotype A), SEQ ID N02108 or SEQ ID NO:ll4 (genotype B), SEQ ID NOle9 or SEQ ID NO:ll6 (genotype C), or using the corresponding sequences from a different HBV genotype, sub-genotype, consensus sequence or strain. In this example, yeast (e.g., Saccharomyces cerevisiae) were engineered to express this fusion protein under the control of the copper-inducible promoter, CUP], and the resulting yeast-HBV therapy composition can be referred to herein as GI-l3022, tically illustrated in Fig. 15. The fusion protein represented by SEQ ID NO:134 comprises, in order, X antigen, polymerase, surface antigen, and core protein, as a single polypeptide with the following sequence elements fused in frame from N- to C-terminus, represented by SEQ ID NO: 134 (optional sequences that are not HBV sequences are not included in the base sequence of SEQ ID NO:134, but may be added to this sequence as in the construct bed in Example 8): (l) optionally, an N—terminal peptide that is a tic N—terminal peptide designed to impart resistance to proteasomal degradation and stabilize expression represented by SEQ ID NO:37 (in the construct described in Example 8), which may be substituted by an N- terminal peptide represented by SEQ ID NO:89, SEQ ID NO:90, or another N-terminal peptide suitable for use with a yeast-based immunotherapeutic as described herein; (2) optionally, a linker peptide of from one to three or more amino acids, such as the two amino acid linker of Thr—Ser (in the construct described in Example 8); (3) an optimized portion of HBV X antigen using a consensus sequence for HBV genotype D, represented by positions 1 to 60 of SEQ ID NO:134 (corresponding to positions 630 to 689 of SEQ ID NO:110); (4) an optimized portion of the e transcriptase (RT) domain of HBV polymerase using a consensus sequence for HBV genotype D, ented by positions 61 to 288 of SEQ ID NO:134 sponding to positions to 250 to 477 of SEQ ID NO:1 10); (5) the amino acid sequence of a near full-length (minus position 1) consensus sequence for HBV genotype D large (L) surface antigen represented by positions 289 to 687 of SEQ ID NO:134 (corresponding to positions 1 to 399 of SEQ ID ); (6) the amino acid sequence of a consensus sequence for HBV genotype D core antigen ented by positions 688 to 869 of SEQ ID NO:134 (corresponding to positions 400 to 581 of SEQ ID NO:118); and (7) optionally, a hexahistidine tag (in the construct described in Example 8).
SEQ ID NO:134 contains multiple T cell epitopes (human and murine), which can be found in Table 5. A nucleic acid sequence ng the fusion protein of SEQ ID NO:134 -optimized for expression in yeast) is represented herein by SEQ ID NO:133.
HBVAntigens Comprising Surface Antigen, Core n and X Antigen. In one embodiment of the invention, the HBV n(s) for use in a composition or method of the invention is a fusion protein comprising HBV antigens, n the HBV antigens comprise or consist of: the HBV surface antigen (large (L), medium (M) or small (8)) or at least one ural, functional or immunogenic domain thereof), the HBV core protein (HBcAg) or HBV e-antigen (HBeAg) or at least one structural, functional or immunogenic domain thereof, and the HBV X antigen (HBx) or at least one structural, functional or immunogenic domain thereof. In one aspect, any one or more of the HBV surface n, HBV core protein, HBV e-antigen, HBV X antigen, or domain thereof, is full-length or near full-length. In one aspect, any one or more of the HBV surface antigen, HBV core protein, HBV e-antigen, HBV X antigen, or domain thereof ses at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the linear sequence of a full-length HBV surface antigen, HBV core protein, HBV gen, HBV X antigen, or domain thereof, respectively, or of the amino acid sequences represented by SEQ ID NO:97 (optimized HBV surface antigen), SEQ ID NO:99 (optimized core protein), SEQ ID NO: 100 (optimized X antigen), or a ponding sequence from another HBV strain, as applicable. In one aspect, any one or more of the HBV surface antigen, HBV core protein, HBV e—antigen, HBV X antigen, or domain thereof is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a ength HBV surface antigen, HBV core protein, HBV e—antigen, HBV X antigen, or domain thereof, respectively, or to the amino acid sequences represented by SEQ ID NO:97 (optimized HBV surface antigen), SEQ ID NO:99 (optimized core protein), SEQ ID NO:100 (optimized X n), or a corresponding sequence from another HBV strain, as applicable. A variety of suitable and ary sequences for additional HBV surface antigens, HBV core antigens, and HBV X antigens useful in this construct are described herein.
] Example 8 describes a fusion protein that contains sequences from HBV surface antigen, core protein, and X antigen, where the sequences were d from segments of the fusion proteins represented by SEQ ID NO:110 and SEQ ID .
This antigen is based on a sus sequence for HBV genotype D; however, it would be straightforward to produce a fusion protein having a similar overall structure using the corresponding fusion segments from the fusion ns represented by SEQ ID NO:107 or SEQ ID NO:112 (genotype A), SEQ ID NO: 108 or SEQ ID NO:114 (genotype B), SEQ ID NO:109 or SEQ ID NO:116 (genotype C), or using the corresponding sequences from a different HBV genotype, sub-genotype, consensus ce or strain. In this example, yeast (e.g., Saccharomyces cerevisiae) were ered to express this fusion protein under the control of the copper—inducible promoter, CUP], and the resulting yeast-HBV immunotherapy composition can be referred to herein as GI-13016, schematically illustrated in Fig. 9. The fusion protein represented by SEQ ID NO:122 comprises, in order, surface antigen, core, and X antigen sequences, as a single polypeptide with the following sequence elements fused in frame from N— to C-terminus, represented by SEQ ID NO:122 nal sequences that are not HBV sequences are not ed in the base sequence of SEQ ID NO:122, but may be added to this sequence as in the construct described in Example 8): (1) optionally, an N—tenninal peptide that is a synthetic N- terminal peptide designed to impart resistance to proteasomal degradation and stabilize sion represented by SEQ ID NO:37 (in the construct described in Example 8), which may be tuted by an N—terminal peptide represented by SEQ ID NO:89, SEQ ID NO:90, or another N—terminal peptide suitable for use with a yeast-based immunotherapeutic as described herein; (2) optionally, a linker peptide of from one to three or more amino acids, such as the two amino acid linker of Thr-Ser (in the construct bed in Example 8); (3) the amino acid sequence of a near fiill-length (minus position 1) consensus sequence for HBV genotype D large (L) surface antigen represented by positions 1 to 399 of SEQ ID NO:122 (corresponding to positions 1 to 399 of SEQ ID NO:118); 4) the amino acid sequence of a consensus sequence for HBV genotype D core antigen represented by ons 400 to 581 of SEQ ID NO:122 (corresponding to positions 400 to 581 of SEQ ID NO:118); (5) an optimized portion of HBV X n using a sus sequence for HBV genotype D, represented by positions 582 to 641 of SEQ ID NO:122 (corresponding to positions 630 to 689 of SEQ ID NO:110); and (6) optionally, a hexahistidine tag (in the construct described in Example 8). SEQ ID NO: 122 contains multiple T cell epitopes (human and murine), which can be found in Table 5. A nucleic acid sequence encoding the fusion protein of SEQ ID NO:122 (codon-optimized for expression in yeast) is represented herein by SEQ ID NO: 121.
Example 8 also describes a fusion protein that contains sequences from HBV surface antigen, core protein, and X antigen, where, as in the fusion n comprising SEQ ID NO:122, the sequences were derived from segments of the fusion ns ented by SEQ ID NO:110 and SEQ ID NO:118. This fusion protein differs from the fusion protein comprising SEQ ID NO:122, however, in the arrangement of the fusion segments within the fusion protein. This antigen is based on a consensus sequence for HBV genotype D; however, it would be straightforward to produce a fusion protein having a similar overall structure using the corresponding fusion segments from the fusion proteins ented by SEQ ID NO:107 or SEQ ID NO:112 (genotype A), SEQ ID NO:108 or SEQ ID NO:114 (genotype B), SEQ ID NO:109 or SEQ ID NO:116 ype C), or using the corresponding ces from a different HBV genotype, sub-genotype, consensus sequence or strain. In this example, yeast (e.g., Saccharomyces cerevisiae) were engineered to express this fusion protein under the l of the copper-inducible promoter, CUPI , and the resulting yeast-HBV immunotherapy composition can be referred to herein as GI-13020, schematically rated in Fig. 13. The fusion protein represented by SEQ ID NO:130 ses, in order, X antigen, surface antigen, and core WO 09404 antigen sequences, as a single polypeptide with the following sequence elements fused in frame from N— to C-terminus, represented by SEQ ID NO:130 nal sequences that are not HBV sequences are not included in the base sequence of SEQ ID NO:130, with the exception of the Leu-Glu linker between the X antigen segment and the surface antigen segment in the construct exemplified here, but may be added to this sequence as in the construct described in Example 8): (l) optionally, an N—terminal peptide that is a synthetic N—terminal peptide designed to impart resistance to proteasomal degradation and stabilize sion represented by SEQ ID NOZ37 (in the construct described in Example 8), which may be tuted by an N-terminal peptide represented by SEQ ID NO:89, SEQ ID NOZ90, or another N—terminal peptide suitable for use with a yeast-based immunotherapeutic as described herein; (2) optionally, a linker peptide of from one to three or more amino acids, such as the two amino acid linker of Thr—Ser (in the construct described in Example 8); (3) an optimized portion of HBV X n using a consensus sequence for HBV genotype D, represented by positions 1 to 60 of SEQ ID NO:130 (corresponding to positions 630 to 689 of SEQ ID ); (4) optionally, a linker peptide of from one to three or more amino acids, such as the two amino acid linker of Leu—Glu (in the construct described in Example 8), represented by positions 61 to 62 of SEQ ID ; (5) the amino acid sequence of a near full-length (minus position 1) consensus sequence for HBV genotype D large (L) surface antigen represented by positions 63 to 461 of SEQ ID NO:130 (corresponding to positions 1 to 399 of SEQ ID NO:118); (6) the amino acid sequence of a consensus sequence for HBV genotype D core antigen represented by positions 462 to 643 of SEQ ID NO:130 (corresponding to positions 400 to 581 of SEQ ID ); and (7) optionally, a hexahistidine tag (in the construct described in Example 8). SEQ ID NO:130 contains multiple T cell epitopes (human and murine), which can be found in Table 5. The amino acid sequence of the complete fusion protein described in e 8 sing SEQ ID NO:130 and ing the N— and inal peptides and all linkers is represented herein by SEQ ID NO:150. A nucleic acid ce encoding the fusion protein of SEQ ID NO: 130 or SEQ ID NO:150 (codon-optimized for expression in yeast) is represented herein by SEQ ID NO:129.
HBV Antigens sing Surface Antigen, Core Protein and Polymerase.
In one embodiment of the ion, the HBV antigen(s) for use in a composition or method of the invention is a fusion protein comprising HBV antigens, wherein the HBV antigens comprise or consist of: the HBV surface antigen (large (L), medium (M) or small (S)) or at least one structural, functional or immunogenic domain f), the HBV core protein (HBcAg) or HBV e-antigen (HBeAg) or at least one structural, functional or immunogenic domain thereof, and the HBV polymerase or at least one structural, functional or immunogenic domain thereof (e.g., the reverse riptase (RT) domain).
In one aspect, any one or more of the HBV surface antigen, HBV core protein, HBV e— antigen, HBV polymerase, or domain thereof, is full-length or near full—length. In one aspect, any one or more of the HBV surface antigen, HBV core protein, HBV e-antigen, HBV polymerase, or domain thereof comprises at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the linear sequence of a full-length HBV surface antigen, HBV core protein, HBV e—antigen, HBV polymerase, or domain thereof, respectively, or of the amino acid sequences represented by SEQ ID NO:97 (optimized HBV surface antigen), SEQ ID NO:99 (optimized core protein), SEQ ID NO:98 (optimized polymerase), or a corresponding ce from another HBV strain, as applicable. In one aspect, any one or more of the HBV surface antigen, HBV core protein, HBV e—antigen, HBV polymerase, or domain thereof is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a full-length HBV surface n, HBV core protein, HBV e—antigen, HBV polymerase, or domain thereof, respectively, or to the amino acid ces represented by SEQ ID NO:97 (optimized HBV e antigen), SEQ ID NO:99 (optimized core n), SEQ ID NO:98 (optimized polymerase), or a corresponding sequence from another HBV strain, as applicable. A variety of suitable and exemplary sequences for HBV surface antigens, HBV polymerase antigens, and HBV core antigens are described herein.
One example of such a filSlOI‘l protein is schematically ented in Fig. 7.
An example of a composition comprising this fusion protein is bed in Example 3. In this embodiment, yeast (e.g., Saccharomyces cerevisiae) are engineered to express s HBV e-polymerase-core fusion proteins under the control of the copper-inducible promoter, CUP], or the TEF2 promoter. In each case, the fusion protein is a single polypeptide with the following ce elements fused in frame from N— to inus, represented by SEQ ID NO:41: (1) an N-terminal peptide to impart resistance to proteasomal degradation and stabilize expression (e.g., positions 1 to 5 of SEQ ID NO:41); 2) an amino acid sequence of the amino HBV hepatocyte receptor domain of the pre—Sl portion of HBV large (L) surface protein (unique to L) (e.g., positions 21-47 of SEQ ID NO:11 or positions 6 to 32 of SEQ ID N0241); 3) the amino acid sequence of an HBV small (S) surface protein (e.g., positions 176 to 400 of SEQ ID NO:11 or positions 33 to 257 of SEQ ID NO:41); 4) a two amino acid spacer/linker to facilitate cloning and manipulation of the sequences (e.g., positions 258 and 259 of SEQ ID NO:41); 5) the amino acid sequence of an HBV polymerase comprising the reverse riptase domain (e.g., positions 247 to 691 of SEQ ID NO:10 or positions 260 to 604 of SEQ ID NO:41); 6) the amino acid sequence of an HBV core protein (e.g., positions 31-212 of SEQ ID NO:9 or positions 605 to 786 of SEQ ID NO:41); and 7) a hexahistidine tag (e.g., positions 787 to 792 of SEQ ID NO:41). The sequence also contains epitopes or domains that are believed to enhance the immunogenicity of the fiision protein. In addition, in one embodiment, the sequence of this construct can be modified to introduce one or more or all of the ing anti-viral resistance mutations: rtM204l, rtLl80M, rtM204V, rtVl73L, rtN236T, rtAl94T (positions given with respect to the fiill-length amino acid ce for HBV rase). In one embodiment, six different therapy compositions are d, each one containing one of these ons. In other embodiments, all or some of the mutations are ed in a single fusion protein. In one embodiment, this construct also contains one or more anti-viral resistance mutations in the surface antigen. The amino acid segments used in any of the fusion proteins described herein can be modified by the use of additional amino acids flanking either end of any domain; the examples provided herein are exemplary. For example, a fiision protein according to this embodiment can include 1) an amino acid sequence of the amino HBV hepatocyte receptor domain of the pre-Sl portion of HBV large (L) surface protein (unique to L) (e.g, positions 21—47 of SEQ ID NO:ll or positions 6 to 32 of SEQ ID NO:41); 2) the amino acid sequence of an HBV small (S) surface protein (e.g., positions 176 to 400 of SEQ ID NO:11 or ons 33 to 257 of SEQ ID ; 3) the amino acid sequence of an HBV polymerase comprising the reverse riptase domain (e.g., positions 247 to 691 of SEQ ID NO:10 or positions 260 to 604 of SEQ ID NO:41); and 4) the amino acid sequence of an HBV core protein (e.g, positions 31-212 of SEQ ID NO:9 or positions 605 to 786 of SEQ ID , and utilize no N— or C-terminal sequences, or utilize different N- or C- terminal sequences, and/or use linkers or no linkers between HBV sequences. In one embodiment, instead of the N-terminal peptide represented by positions l-5 of SEQ ID NO:41, an N—terminal peptide represented by SEQ ID NO:89 or SEQ ID NO:90 is utilized, ed by the remainder of the fusion protein as described.
Another example of such a fusion protein is described in Example 8. e 8 exemplifies a fusion protein that contains ces from HBV surface antigen, core protein, and polymerase where the sequences were derived from segments of the fusion WO 09404 2012/024409 proteins represented by SEQ ID NO:110 and SEQ ID N011 18. This antigen is based on a consensus sequence for HBV genotype D; however, it would be straightforward to produce a fusion protein having a similar overall structure using the corresponding fusion segments from the fusion proteins represented by SEQ ID NO:107 or SEQ ID NO:112 (genotype A), SEQ ID NO:108 or SEQ ID NO:114 (genotype B), SEQ ID NO:109 or SEQ ID NO:116 (genotype C), or using the corresponding sequences from a different HBV genotype, sub-genotype, consensus sequence or strain. In this example, yeast (e.g, Saccharomyces siae) were engineered to express this fusion n under the control of the copper-inducible promoter, CUP], and the resulting HBV immunotherapy composition can be referred to herein as GI-l3015, schematically illustrated in Fig. 8. The fusion protein represented by SEQ ID NO:120 comprises, in order, surface antigen, core protein, and polymerase sequences, as a single polypeptide with the following sequence elements fused in frame from N— to inus, represented by SEQ ID NO:120 (optional sequences that are not HBV sequences are not included in the base sequence of SEQ ID NO:120, but may be added to this ce as in the construct described in Example 8): (l) optionally, an N—terminal peptide that is a synthetic N—terminal peptide designed to impart resistance to proteasomal degradation and stabilize sion ented by SEQ ID NO:37 (in the construct described in Example 8), which may be tuted by an N-terminal peptide represented by SEQ ID NO:89, SEQ ID NO:90, or another N-terminal peptide suitable for use with a yeast-based immunotherapeutic as described herein; (2) optionally, a linker peptide of from one to three or more amino acids, such as the two amino acid linker of Thr-Ser (in the uct described in Example 8); (3) the amino acid sequence of a near fiill-length (minus position 1) consensus sequence for HBV pe D large (L) surface antigen represented by positions 1 to 399 of SEQ ID NO:120 (corresponding to positions 1 to 399 of SEQ ID NO:118); (4) the amino acid sequence of a sus sequence for HBV genotype D core antigen represented by positions 400 to 581 of SEQ ID NO:120 sponding to positions 400 to 581 of SEQ ID NO:ll8); (5) an optimized portion of the reverse transcriptase (RT) domain of HBV polymerase using a consensus ce for HBV genotype D, represented by positions 582 to 809 of SEQ ID NO:120 (corresponding to positions to 250 to 477 of SEQ ID NO:110); and (6) optionally, a hexahistidine tag (in the construct described in Example 8). SEQ ID NO:120 contains multiple T cell epitopes (human and murine), which can be found in Table 5. A nucleic acid sequence encoding the fusion protein of SEQ ID NO:120 (codon-optimized for expression in yeast) is represented herein by SEQ ID NO:119.
Yet another example of such a fusion protein is described in Example 8.
Example 8 ifies a fusion protein that contains sequences from HBV polymerase, surface antigen, and core protein, where the sequences were derived from segments of the fusion proteins represented by SEQ ID NO:110 and SEQ ID NO:118. This fusion protein differs from the fusion protein comprising SEQ ID NO:120 in the arrangement of the fusion segments within the fusion protein. This antigen is based on a consensus sequence for HBV genotype D; however, it would be htforward to produce a fusion protein having a similar overall structure using the corresponding fusion segments from the fusion ns represented by SEQ ID NO:107 or SEQ ID NO:112 (genotype A), SEQ ID NO:108 or SEQ ID NO:114 (genotype B), SEQ ID NO:109 or SEQ ID NO:116 (genotype C), or using the corresponding sequences from a different HBV genotype, sub-genotype, sus sequence or . In this example, yeast (e.g., Saccharomyces siae) were engineered to express this fusion protein under the control of the copper—inducible promoter, CUP], and the resulting yeast-HBV immunotherapy composition can be referred to herein as GI-l3019, schematically illustrated in Fig. 12. The fusion protein represented by SEQ ID NO: 128 comprises, in order, polymerase, surface antigen, and core sequences, as a single polypeptide with the following ce elements fused in frame from N— to C-terminus, represented by SEQ ID NO:128 nal sequences that are not HBV sequences are not included in the base sequence of SEQ ID NO:128, with the exception of the Leu-Glu linker between the polymerase segment and the e antigen t in the construct exemplified here, but may be added to this sequence as in the construct described in Example 8): (1) optionally, an inal peptide that is a tic N—terminal peptide designed to impart resistance to proteasomal degradation and stabilize expression ented by SEQ ID N0237 (in the construct described in Example 8), which may be substituted by an N—terminal e represented by SEQ ID NO:89, SEQ ID NO:90, or another N—terminal peptide suitable for use with a yeast-based immunotherapeutic as described herein; (2) optionally, a linker peptide of from one to three or more amino acids, such as the two amino acid linker of Thr-Ser (in the construct described in Example 8); (3) an optimized portion of the reverse transcriptase (RT) domain ofHBV polymerase using a consensus sequence for HBV pe D, represented by positions 1 to 228 of SEQ ID NO:128 (corresponding to ons to 250 to 477 of SEQ ID NO:110); (4) optionally, a linker peptide of from one to three or more amino acids, 2012/024409 such as the two amino acid linker of Leu-Glu (in the construct described in Example 8), represented by positions 229 to 230 of SEQ ID ; (5) the amino acid sequence of a near full-length (minus on 1) consensus sequence for HBV genotype D large (L) e antigen represented by ons 231 to 629 of SEQ ID NO:128 (corresponding to ons 1 to 399 of SEQ ID NO:118); (6) the amino acid ce of a sus sequence for HBV genotype D core antigen represented by positions 630 to 811 of SEQ ID NO: 128 (corresponding to positions 400 to 581 of SEQ ID NO:118); and (7) optionally, a hexahistidine tag (in the construct described in Example 8). SEQ ID NO:128 contains multiple T cell es (human and murine), which can be found in Table 5. A nucleic acid sequence encoding the fusion protein of SEQ ID NO:128 (codon-optimized for expression in yeast) is represented herein by SEQ ID NO: 127.
HBVAntigens Comprising Polymerase and Core Protein. In one embodiment of the invention, the HBV antigen(s) for use in a composition or method of the invention is a fusion protein comprising HBV antigens, wherein the HBV antigens comprise or consist of HBV polymerase (the RT domain) or at least one immunogenic domain thereof and an HBV core protein (HBcAg) or at least one immunogenic domain thereof. In one aspect, one or both of the RT domain of HBV polymerase or the HBV core protein is full—length or near fiJll-length. In one aspect, one or both of the RT domain of HBV polymerase or the HBV core protein or a domain thereof comprises at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the linear sequence of a ength the RT domain of HBV polymerase or the HBV core protein or a domain thereof, respectively, or to the amino acid sequences represented by SEQ ID NO:98 (optimized HBV polymerase), SEQ ID NO:99 (optimized core protein), or a corresponding sequence from another HBV strain, as applicable. In one aspect, one or both of the RT domain of HBV polymerase or the HBV core protein or a domain thereof is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a full—length RT domain of HBV polymerase or the HBV core n or a domain thereof, respectively, or to the amino acid sequences represented by SEQ ID NO:98 (optimized HBV polymerase), SEQ ID NO:99 ized core protein), or a corresponding sequence from another HBV strain, as applicable. A variety of suitable and exemplary ces for HBV polymerase antigens and HBV core antigens are described herein.
One example of this antigen is schematically represented in Fig. 4. One example of a composition comprising this fusion protein is described in e 3. In this embodiment, yeast (e.g., Saccharomyces cerevisiae) are engineered to express various HBV polymerase—core fusion proteins as shown schematically in Fig. 4 under the l of the copper-inducible promoter, CUPI or the TEFZ promoter. In each case, the fusion protein is a single ptide with the following sequence elements fused in frame from N- to C-terminus, represented by SEQ ID N0:38: (1) an N-terminal e to impart resistance to somal degradation and stabilize expression (e.g., SEQ ID NO:37 or positions 1 to 6 of SEQ ID NO:38), 2) the amino acid sequence of a portion of the HBV pe C polymerase including the reverse transcriptase domain (e.g, positions 347 to 691 of SEQ ID NO:10 or positions 7 to 351 of SEQ ID NO:38), 3) an HBV genotype C core protein (e.g, positions 31 to 212 of SEQ ID NO:9 or positions 352 to 533 of SEQ ID NO:38), and 4) a hexahistidine tag (e.g., positions 534 to 539 of SEQ ID NO:38). The ce also contains epitopes or domains that are believed to enhance the immunogenicity of the fusion protein. The amino acid segments used in any of the fusion proteins described herein can be modified by the use of additional amino acids flanking either end of any domain; the examples provided herein are exemplary. For example, a fusion protein according to this embodiment can include 1) the amino acid sequence of a portion of the HBV genotype C polymerase including the reverse riptase domain (e.g., positions 347 to 691 of SEQ ID N0210 or positions 7 to 351 of SEQ ID NO:38), and 2) an HBV genotype C core protein (e.g, positions 31 to 212 of SEQ ID N09 or positions 352 to 533 of SEQ ID NO:38), and utilize no N- or C-terminal sequences, or utilize different N- or C-terminal sequences, and/or use linkers or no linkers between HBV sequences. In one embodiment, instead of the N-terminal peptide represented by SEQ ID NO:37, an N-terminal peptide represented by SEQ ID N0289 or SEQ ID N0290 is utilized, followed by the der of the fusion n.
HBVAntz'gens Comprising X Antigen and Core Protein. In one embodiment of the invention, the HBV antigen(s) for use in a composition or method of the invention is a fusion protein comprising HBV antigens, wherein the HBV antigens comprise or consist of HBV X antigen or at least one immunogenic domain f and HBV core protein ) or at least one immunogenic domain thereof. In one aspect, one or both of the HBV X antigen or the HBV core protein is ength or near full-length. In one aspect, one or both of the HBV X n or the HBV core protein or a domain thereof comprises at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the linear sequence of a full-length HBV X n or HBV core protein or domain thereof, respectively, or to the amino acid sequences represented by SEQ ID NO:99 (optimized core protein), SEQ ID N0:100 (optimized X antigen), or a corresponding sequence from W0 2012/109404 another HBV strain, as applicable. In one aspect, one or both of the HBV X antigen or the HBV core protein or a domain thereof is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a full-length HBV X antigen or HBV core protein or domain thereof, respectively, or to the amino acid sequences represented by SEQ ID NO:99 (optimized core protein), SEQ ID NO:100 (optimized X antigen), or a corresponding ce from another HBV strain, as applicable. A variety of suitable and exemplary sequences for HBV core antigens and HBV X antigens are described herein.
] This fusion protein is tically represented in Fig. 5. An example of a composition comprising this fusion protein is described in Example 3. In this embodiment, yeast (e.g, Saccharomyces cerevisiae) are engineered to express various HBV X—core fusion proteins as shown schematically in Fig. 5 under the control of the copper—inducible promoter, CUP], or the TEF2 er. In each case, the fusion protein is a single polypeptide with the following sequence elements fused in frame from N- to C—terminus, represented by SEQ ID NO:39 (1) an N-terminal peptide to impart resistance to proteasomal degradation and stabilize expression (e.g. SEQ ID NO:37 or positions 1 to 6 of SEQ ID NO:39); 2) the amino acid sequence of a near full-length (minus on 1) HBV genotype C X antigen (e.g., positions 2 to 154 of SEQ ID NO:12 or positions 7 to 159 of SEQ ID NO:39); 3) an HBV genotype C core n (e.g, positions 31 to 212 of SEQ ID N029 or positions 160 to 341 of SEQ ID NO:39); and 4) a hexahistidine tag (e.g., positions 342 to 347 of SEQ ID NO:39). The sequence also ns epitopes or domains that are believed to enhance the immunogenicity of the fusion n. The amino acid segments used in any of the fusion proteins described herein can be modified by the use of additional amino acids flanking either end of any domain; the examples provided herein are exemplary. For example, a fusion protein according to this embodiment can e 1) the amino acid sequence of a near ength (minus position 1) HBV genotype C X antigen (e.g., positions 2 to 154 of SEQ ID NO:12 or positions 7 to 159 of SEQ ID N039); and 2) an HBV genotype C core protein (e.g, positions 31 to 212 of SEQ ID N09 or positions 160 to 341 of SEQ ID , and utilize no N- or C-terminal sequences, or utilize different N- or C-terminal sequences, and/or use linkers or no linkers between HBV sequences. In one embodiment, instead of the inal peptide represented by SEQ ID NO:37, an N-terminal peptide represented by SEQ ID NO:89 or SEQ ID NO:90 is utilized, ed by the der of the fusion protein as described.
HBV Antigens Comprising Single HBV ns. In one embodiment of the invention, an HBV antigen is comprised of a single HBV protein (e.g., one HBV protein selected from surface, core, e-antigen, polymerase, or X antigen) or one or more domains (structural, functional, and/or immunological) from a single HBV protein. This embodiment of the invention is ularly useful for creating a yeast-based immunotherapeutic composition that can be used, for example, in combination with one or more other yeast-based immunotherapeutic compositions for the treatment or laxis of HBV, or in sequence with one or more other yeast-based immunotherapeutic compositions for the treatment or prophylaxis of HBV, or to follow a prophylactic approach with a therapeutic approach if the patient becomes infected. For e, the yeast-based immunotherapeutic composition including an HBV surface antigen of this embodiment can be ed with a second yeast-based immunotherapeutic composition ing a different HBV protein/antigen, such as an HBV X n (described below), and further, with additional "single HBV protein" yeast-based immunotherapeutics, as desired (e.g, a yeast-based immunotherapeutic ition including an HBV Precore, Core or gen and/or a yeast-based immunotherapeutic composition including an HBV polymerase n or domain thereof). These "single HBV n yeast immunotherapeutics" can be used in combination or sequence with each other and/or in combination or sequence with other multi-HBV protein yeast-based immunotherapeutics, such as those described in the Examples or elsewhere herein. Alternatively, or in addition, a "single HBV protein yeast immunotherapeutic" such as this HBV surface antigen yeast— based immunotherapeutic can be produced using the HBV sequence for any given genotype or sub-genotype, and additional HBV e antigen yeast-based immunotherapeutics can be produced using the HBV sequences for any one or more additional genotype or sub-genotype. This strategy effectively creates a "spice rack" of different HBV ns and genotypes and/or sub—genotypes to each of which is provided in the context of a yeast-based immunotherapeutic of the invention, or in a strategy that includes at least one yeast—based immunotherapeutic of the invention. Accordingly, any combination of one, two, three, four, five, six, seven, eight, nine, ten or more of these yeast-based therapeutics can be selected for use to treat a particular patient or population of patients who are infected with HBV, illustrating the flexibility of the present invention to be customized or tailored to meet the needs of a particular patient, population of patients, demographic, or other patient ng.
In one embodiment of the ion, the HBV antigen(s) for use in a composition or method of the invention is an HBV antigen comprising or consisting of: (a) an HBV surface antigen n and/or one or more domains (structural, functional or immunogenic) thereof, which can include the hepatocyte receptor portion of Pre—Sl of the HBV large (L) surface antigen, the HBV large (L) surface antigen, the HBV middle (M) surface n, the HBV small (S) surface antigen (HBsAg), or any domain or combination thereof; (b) an HBV polymerase antigen, which can include one or more domains (structural, functional, or immunogenic) of HBV polymerase, such as the reverse riptase (RT) domain (a functional domain) of HBV polymerase; (c) an HBV e n, an HBV core antigen and/or HBV e-antigen, or one or more domains thereof (structural, functional or immunogenic), which can include one or more domains or portions of HBV Precore containing ces from both HBV core and HBV e-antigen, or one or the other of these proteins; or (d) an HBV X antigen, which can include one or more domains (structural, fianctional or immunogenic) of HBV X antigen. In one aspect, any one or more of these proteins or domains is full-length or near full-length. In one aspect, one or more of these proteins or s comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more immunogenic domains. In one aspect, any one or more of these proteins or domains comprises at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the linear ce of the corresponding lull-length sequence or a domain thereof. In one aspect, any one or more of these proteins or domains is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% cal to the corresponding full-length sequence or a domain thereof. A variety of suitable and exemplary sequences for HBV surface antigens, HBV polymerase antigens, HBV core antigens, and HBV X antigens are described herein.
An example of a composition comprising a surface antigen protein is described in Example 5. In this embodiment, yeast (e.g, Saccharomyces cerevisiae) are engineered to express HBV surface proteins under the control of a suitable promoter, such as the copper—inducible promoter, CUP], or the TEF2 promoter. The protein is a single polypeptide comprising HBV near-full-length HBV large (L) surface antigen (to accommodate the ce of an N-terminal ce selected to enhance or stabilize expression of the antigen), represented by SEQ ID NO:93: (1) an N—terminal e of SEQ ID N0289 (positions 1-89 of SEQ ID NO:93); 2) the amino acid ce of a near full-length (minus position 1) HBV pe C large (L) surface antigen (e.g, positions 2- 400 of SEQ ID NO:11 or positions 90 to 488 of SEQ ID NO:93); and 3) a hexahistidine tag (e.g., positions 489 to 494 of SEQ ID NO:93). Alternatively, the N—terminal peptide can be replaced with SEQ ID NO:37 or a homologue thereof or another N-terminal peptide bed herein. In one ment, this construct also contains one or more anti-viral resistance mutations in the surface antigen. While this example utilizes large (L) surface antigen as an HBV antigen that may ze the exposure of immunogenic epitopes generated by the immune , small portions of e antigen, including any domains or combinations of s of surface antigen, can be produced using the guidance provided herein. In addition, while the exemplary immunotherapeutic is shown using a genotype C sequence, sequences from other genotypes, sub-genotypes, and/or strains or isolates of HBV can be used instead.
An example of a composition comprising an HBV polymerase antigen is described in Example 3 and also in Example 5. The HBV antigen described in Example 5 is schematically represented in Fig. 6. In this embodiment, yeast (e.g., Saccharomyces cerevisiae) are engineered to express various HBV polymerase proteins under the control of the copper-inducible promoter, CUPI or the TEF2 promoter. In each case, the fusion protein is a single polypeptide with the following sequence elements fused in frame from N— to C-terminus, represented by SEQ ID NO:40 (1) an inal peptide to impart resistance to proteasomal degradation and ize expression (SEQ ID NO:37, or ons 1 to 6 of SEQ ID NO:40; 2) the amino acid sequence of a portion of the HBV genotype C polymerase ing the reverse transcriptase domain (e.g, positions 347 to 691 of SEQ ID NO:10 or positions 7 to 351 of SEQ ID NO:40), and 3) a hexahistidine tag (e.g., positions 352 to 357 of SEQ ID NO:40). The sequence also contains epitopes or domains that are believed to enhance the immunogenicity of the fusion protein. In addition, in one embodiment, the sequence of this construct can be modified to introduce one or more or all of the following anti-viral resistance mutations: rtM204l, rtL180M, rtM204V, rtV173L, rtN236T, rtA194T (positions given with respect to the full-length amino acid sequence for HBV polymerase). In one embodiment, six different therapy compositions are created, each one containing one of these ons. In other embodiments, all or some of the mutations are included in a single fusion n.
The amino acid segments used in any of the fusion proteins described herein can be modified by the use of onal amino acids flanking either end of any domain; the examples provided herein are exemplary. For example, a fiision protein ing to this embodiment can include the amino acid sequence of a portion of the HBV genotype C polymerase including the reverse transcriptase domain (e.g., positions 347 to 691 of SEQ ID NO:10 or ons 7 to 351 of SEQ ID NO:40), and utilize no N- or C-terminal sequences, or e different N— or C-terminal sequences, and/or use linkers or no linkers between HBV ces. In one embodiment, instead of the N—terminal peptide W0 2012/109404 represented by SEQ ID NO:37, an N-terminal peptide represented by SEQ ID NO:89 or SEQ ID NO:90 is utilized, followed by the remainder of the fusion protein as described.
In the ment shown in Example 5, yeast (e.g, Saccharomyces cerevisiae) are engineered to express HBV polymerase proteins under the control of a suitable promoter, such as the copper-inducible promoter, CUP], or the TEF2 promoter.
The n is a single polypeptide comprising HBV reverse transcriptase (RT) domain of polymerase (Pol), represented by SEQ ID NO:94: (1) an N—terminal peptide of SEQ ID NO:89 (positions 1-89 of SEQ ID ; 2) the amino acid sequence of a portion of the HBV genotype C polymerase including the reverse transcriptase domain (e.g., positions 347 to 691 of SEQ ID NO:10 or positions 90 to 434 of SEQ ID N094); and 3) a hexahistidine tag (e.g., positions 435 to 440 of SEQ ID NO:94). The sequence also contains epitopes or domains that are believed to enhance the immunogenicity of the fusion protein. In addition, in one embodiment, the sequence of this construct can be ed to introduce one or more or all of the following anti-viral resistance mutations: rtM204l, rtL180M, rtM204V, rtV173L, rtN236T, rtAl94T (positions given with respect to the full-length amino acid sequence for HBV polymerase). atively, the N—terminal peptide can be replaced with SEQ ID NO:37 or a gue thereof or another N— terminal peptide described herein.
An example of a composition comprising an HBV Precore, Core or e-antigen is described in Example 5. Yeast (e.g, Saccharomyces siae) are engineered to express HBV Core proteins under the control of a suitable promoter, such as the copper- ble promoter, CUP], or the TEFZ promoter. The protein is a single polypeptide comprising near full-length HBV Core protein, represented by SEQ ID NO:95: (1) an N- terminal peptide of SEQ ID NO:89 (positions 1—89 of SEQ ID ; 2) the amino acid sequence of a n of the HBV genotype C Core protein (e.g., ons 31 to 212 of SEQ ID N029 or positions 90 to 271 of SEQ ID NO:95); and 3) a hexahistidine tag (e.g., positions 272 to 277 of SEQ ID NO:95). The sequence also contains epitopes or domains that are believed to enhance the immunogenicity of the fusion protein. Alternatively, the N-terminal peptide can be ed with SEQ ID NO:37 or a homologue thereof or r N-terminal e described herein.
An example of a yeast—based immunotherapeutic composition comprising an HBV X antigen is described in Example 5. Yeast (e.g., Saccharomyces cerevisiae) are engineered to express HBV X antigens under the control of a suitable promoter, such as the copper-inducible promoter, CUP], or the TEFZ promoter. The protein is a single polypeptide comprising near full-length HBV X antigen, represented by SEQ ID NO:96: (1) an inal peptide of SEQ ID NO:89 (positions 1-89 of SEQ ID NO:96); 2) the amino acid sequence of a portion of the HBV genotype C X antigen (e.g, positions 2 to 154 of SEQ ID NO:12 or positions 90 to 242 of SEQ ID NO:96); and 3) a hexahistidine tag (e.g., ons 243 to 248 of SEQ ID N0296). The ce also contains epitopes or s that are believed to enhance the immunogenicity of the fusion protein.
Alternatively, the N—terminal peptide can be replaced with SEQ ID NO:37 or a homologue thereof or another N-terminal peptide described herein.
HBV Antigens Comprising HBV Proteins from Two or More pes. r ment of the invention relates to HBV antigens for use in an immunotherapeutic composition of the invention that maximizes the targeting of HBV genotypes and/or sub-genotypes in order to provide itions with the potential to treat a large number of individuals or populations of individuals using one composition.
Such compositions are generally more efficient to produce (i. e., have a production advantage by ing multiple ns and/or a consensus approach to targeting genotypes) and are more efficient to utilize in a wide variety of clinical settings (e.g., one composition may serve many different types of patient populations in many ent geographical settings). As discussed above, to produce such HBV antigens, conserved antigens and/or conserved domains (among HBV genotypes) can be selected, and the antigens can be designed to maximize the inclusion of conserved immunological domains.
In one aspect of this embodiment, an HBV antigen is provided that includes in a single yeast-based immunotherapeutic a single HBV protein or domain thereof (e.g, e, polymerase, core/e or X) that is repeated two, three, four, five or more times within the antigen construct, each time using a sequence from a different HBV genotype or subgenotype. In this aspect, multiple nt or prevalent genotypes can be targeted in one yeast-based immunotherapeutic, increasing clinical and manufacturing efficacy.
These antigens can be modified, if desired, to maximize the ion of consensus sequences, including consensus T cell epitopes within the antigens, which may otherwise n subtle differences due to sub-genotype, strain or isolate ences.
Accordingly, in one embodiment of the invention, the HBV antigen(s) for use in a composition or method of the invention is an HBV antigen comprising or consisting of two or more repeated HBV antigens of the same protein or , but of different HBV genotypes (e.g., two or more HBV Core or e-antigens, which can include one or more domains (structural, functional or immunogenic) of HBV Core or e-antigen, wherein W0 2012/109404 the antigens include the same or similar n from each of HBV genotype C and HBV pe D, to form a Core-Core fusion where each Core protein is a different genotype).
In one aspect, the HBV protein used in such constructs is full-length or near full—length protein or domain. In one aspect, the HBV n comprises or consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more immunogenic domains. In one aspect, any one or more of these proteins or domains comprises at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the linear sequence of the corresponding ength sequence. In one aspect, any one or more of these proteins or domains is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of the corresponding full-length sequence.
Such an antigen is exemplified in e 6. In this embodiment, yeast (e.g, Saccharomyces cerevisiae) are engineered to express an HBV fusion protein under the control of a suitable promoter, such as the copper-inducible promoter, CUP] or the TEF2 promoter. The protein is a single ptide comprising four Core antigens, each one from a different genotype (HBV genotypes A, B, C and D), ented by SEQ ID NO:105: 1) an N-terminal methionine at position 1 of SEQ ID NO:105; 2) the amino acid sequence of a near ength Core protein from HBV genotype A (e.g., positions 31 to 212 of SEQ ID NO:1 or positions 2 to 183 of SEQ ID NO: 105); 3) the amino acid ce of a near full-length Core protein from HBV pe B (e.g., positions 30 to 212 of SEQ ID N025 or positions 184 to 395 of SEQ ID NO: 105); 4) the amino acid ce of a near fiJll-length Core protein from HBV genotype C (e.g., positions 30 to 212 of SEQ ID N029 or positions 396 to 578 of SEQ ID NO: 105); 5) the amino acid sequence of a near full-length Core protein from HBV genotype D (e.g., positions 30 to 212 of SEQ ID NO: 13 or positions 579 to 761 of SEQ ID NO: 105); and 5) a stidine tag (e.g., positions 762 to 767 of SEQ ID NO: 105). The sequence also contains epitopes or domains that are believed to enhance the immunogenicity of the fusion protein. The N- terminal nine at position 1 can be substituted with SEQ ID NO:37 or a homologue thereof, or with an alpha prepro sequence of SEQ ID NO:89 or SEQ ID NO:90, or a homologue thereof, or any other suitable N-terminal sequence if desired. In on, linker sequences can be inserted between HBV proteins to facilitate cloning and manipulation of the construct, if desired. This is an exemplary construct, as any other combination of HBV genotypes and/or sub-genotypes can be substituted into this design as desired to construct a single antigen yeast-based HBV immunotherapeutic product with broad clinical applicability and efficient design for manufacturing. The amino acid sequence of SEQ ID NOle5 also contains several known T cell epitopes, and certain epitopes have been modified to correspond to the published sequence for the given epitope (see Table 5).
In another aspect of this embodiment, more than one protein or domain from a single HBV pe is included in an HBV antigen useful in the invention, which may be selected to maximize the most ved n ces encoded by the HBV genome or to maximize the inclusion of therapeutically or prophylactically useful immunogenic domains within the antigen. These antigens are then repeated within the same fusion protein, but using the same or similar sequences from a ent HBV pe or subgenotype. In this , multiple nt or prevalent genotypes can also be targeted in one yeast-based immunotherapeutic, again increasing clinical and manufacturing eff1cacy. These antigens can also be modified, if desired, to maximize the inclusion of consensus T cell es within the antigens, which may otherwise contain subtle differences due to sub-genotype, strain or isolate differences.
Accordingly, in one embodiment of the invention, the HBV antigen(s) for use in a composition or method of the invention is an HBV n comprising or consisting of at least two different HBV proteins or domains thereof, each of which is repeated two or more times, but wherein the repeated sequences are from ent HBV genotypes (e.g., two or more HBV Core and two or more X antigens, or domains thereof, wherein the antigens e the same or similar antigen from each of HBV genotype C and HBV genotype D, to form a Core-X-Core-X fusion (or any other order of segments within the fusion) where each Core protein is a different genotype and each X antigen is a different pe). In one aspect, the HBV protein used in such constructs is lull-length or near full-length protein or domain. In one aspect, the HBV antigen comprises or consists of l, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more immunogenic s. In one , any one or more of these proteins or domains comprises at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the linear sequence of the corresponding full-length sequence. In one aspect, any one or more of these proteins or domains is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% to the sequence of the corresponding full-length sequence.
Such an antigen is exemplified in Example 6. In this embodiment, yeast (e.g, Saccharomyces cerevisiae) are engineered to express an HBV fusion protein under the control of a suitable promoter, such as the copper-inducible promoter, CUP], or the TEF2 promoter. The protein is a single polypeptide comprising two Core antigens and two X antigens, each one of the pair from a different genotype (HBV pes A and C), represented by SEQ ID NO:106: 1) an N—terminal methionine at position 1 of SEQ ID NO:106; 2) the amino acid ce of a near full—length Core protein from HBV genotype A (e.g., positions 31 to 212 of SEQ ID NO] or positions 2 to 183 of SEQ ID NO:106); 3) the amino acid sequence of a ength X antigen from HBV genotype A (e.g., positions SEQ ID NO :4 or positions 184 to 337 of SEQ ID NO:106); 4) the amino acid sequence of a near full-length Core protein from HBV genotype C (e.g., positions 30 to 212 of SEQ ID N029 or positions 338 to 520 of SEQ ID NO:106); 5) the amino acid sequence of a full-length X antigen from HBV genotype C (e. g., SEQ ID NO:8 or positions 521 to 674 of SEQ ID NO:106); and 5) a hexahistidine tag (e.g., positions 675 to 680 of SEQ ID NO:106). The sequence also contains epitopes or domains that are believed to enhance the immunogenicity of the fusion protein. The N—terminal methionine at position 1 can be substituted with SEQ ID NO:37 or a homologue thereof, or with an alpha prepro sequence of SEQ ID NO:89 or SEQ ID NO:90, or a homologue thereof The amino acid sequence of SEQ ID NO:106 also contains l known T cell epitopes, and certain epitopes have been modified to correspond to the published sequence for the given epitope (see Table 5).
Additional Embodiments Regarding HBV Antigens. In some aspects of the ion, amino acid insertions, deletions, and/or substitutions can be made for one, two, three, four, five, six, seven, eight, nine, ten, or more amino acids of a wild-type or reference HBV protein, provided that the ing HBV protein, when used as an antigen in a yeast-HBV immunotherapeutic composition of the invention, elicits an immune response against the target or wild-type or reference HBV protein, which may include an enhanced immune response, a diminished immune response, or a substantially similar immune response. For example, the invention includes the use of HBV agonist antigens, which may include one or more T cell epitopes that have been d to enhance the T cell response against the HBV t, such as by improving the avidity or affinity of the epitope for an MHC molecule or for the T cell receptor that izes the epitope in the t of MHC presentation. HBV protein agonists may therefore improve the y or efficiency of a T cell response t native HBV proteins that infect a host.
Referring to any of the above-described HBV antigens, including the fusion proteins that have amino acid sequences including or represented by SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:92, SEQ ID , SEQ ID NO:102, SEQ ID NO:107, SEQ ID NO:108, SEQ ID , SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132, SEQ ID NO:134, SEQ ID NO:150 or SEQ ID NO:151, it is an aspect of the invention to use one or more of the HBV antigens from individual HBV proteins within the fusion protein (e.g., from HBV surface antigen, HBV polymerase, HBV core/e-antigen, and/or HBV X antigen) to uct "single protein" antigens (e.g., ns from only one of these HBV proteins), or to construct fusion proteins using only two or three of the HBV protein segments, if applicable to the given nce fusion protein. It is also an aspect of the invention to change the order of HBV n segments within the fusion protein. As another alternate design, HBV genotypes and/or consensus sequences can be combined, where two, three, four or more genotypes and/or sus sequences are used to uct the fusion n.
The invention also includes homologues of any of the above-described fusion proteins, as well as the use of homologues, variants, or mutants of the individual HBV proteins or portions thereof (including any fiinctional and/or immunogenic s) that are part of such filSIOIl proteins or otherwise described herein. In one aspect, the invention includes the use of fusion proteins or individual (single) HBV proteins or HBV antigens, having amino acid sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of any one of the fiasion proteins or individual HBV proteins or HBV antigens, respectively, described herein, including any of the HBV proteins, HBV antigens and fusion proteins referenced by a specific sequence identifier herein, over the fill length of the fusion protein, or with respect to a d segment in the fusion n or a defined protein or domain thereof (immunogenic domain or functional domain (z'.e., a domain with at least one ical ty)) that forms part of the fusion protein. Many CTL epitopes (epitopes that are recognized by cytotoxic T lymphocytes from patients infected with HBV) and escape mutations (mutations that arise in an HBV protein due to selective pressure from an anti-viral drug) are known in the art, and this information can also be used to make substitutions or create variants or homologues of the HBV antigens described herein in order to provide a c sequence in the HBV antigen of the invention.
Yeast-Based Immunotherapy Compositions. In various embodiments of the invention, the invention es the use of at least one "yeast-based immunotherapeutic composition" (which phrase may be used interchangeably with "yeast-based therapy product", "yeast-based immunotherapy composition", "yeast—based composition", -based immunotherapeutic", "yeast-based vaccine", or derivatives of these phrases). An "immunotherapeutic composition" is a composition that elicits an immune response sufficient to achieve at least one therapeutic benefit in a subject. As used herein, yeast-based therapeutic composition refers to a composition that includes a yeast vehicle component and that elicits an immune response sufficient to achieve at least one therapeutic benefit in a subject. More particularly, a yeast-based therapeutic composition is a composition that includes a yeast vehicle component and can elicit or induce an immune response, such as a cellular immune response, including without limitation a T cell-mediated cellular immune response. In one aspect, a yeast-based immunotherapeutic composition useful in the invention is capable of inducing a CD8+ and/or a CD4+ T cell-mediated immune response and in one aspect, a CD8+ and a CD4+ T cell-mediated immune response. Optionally, a yeast—based immunotherapeutic composition is capable of eliciting a l immune response. A yeast-based immunotherapeutic composition useful in the present ion can, for example, elicit an immune response in an individual such that the individual is protected from HBV infection and/or is treated for HBV infection or for symptoms resulting from HBV infection.
Yeast-based immunotherapy itions of the invention may be either "prophylactic" or "therapeutic". When ed prophylactically, the compositions of the present invention are provided in e of any symptom of HBV infection. Such a composition could be administered at birth, in early ood, or to . The prophylactic administration of the immunotherapy itions serves to prevent subsequent HBV infection, to resolve an infection more quickly or more completely if HBV infection subsequently ensues, and/or to ameliorate the symptoms of HBV ion if infection subsequently ensues. When provided therapeutically, the immunotherapy compositions are provided at or after the onset of HBV infection, with the goal of ameliorating at least one symptom of the infection and preferably, with a goal of eliminating the infection, providing a long lasting remission of infection, and/or providing long term immunity against subsequent infections or vations of the virus. In one aspect, a goal of ent is loss of detectable HBV viral load or reduction of HBV viral load (e.g., below detectable levels by PCR or <2000 IU/ml). In one aspect, a goal of treatment is sustained viral clearance for at least 6 months after the completion of therapy.
In one , a goal of treatment is the loss of detectable serum HBeAg and/or HBsAg proteins. In one aspect, a goal of ent is the development of antibodies against the hepatitis B surface antigen (anti-HBs) and/or antibodies against HBeAg. In one aspect, the goal of treatment is seroconversion, which may be defined by: (a) 10 or more sample ratio units (SRU) as ined by radioirnrnunoassay; (b) a positive result as determined by enzyme immunoassay; or (c) detection of an antibody concentration of 310 mIU/ml (10 SRU is comparable to 10 mIU/mL of antibody). In one aspect, a goal of treatment is normalization of serum alanine ransferase (ALT) levels, improvement in liver inflammation and/or improvement in liver fibrosis.
Typically, a yeast-based immunotherapy composition includes a yeast vehicle and at least one antigen or immunogenic domain f expressed by, attached to, or mixed with the yeast vehicle, wherein the antigen is heterologous to the yeast, and wherein the antigen comprises one or more HBV antigens or immunogenic domains thereof. In some embodiments, the antigen or immunogenic domain thereof is provided as a fusion protein. l HBV fusion proteins suitable for use in the compositions and methods of the invention have been described above. In one aspect of the invention, fusion protein can include two or more antigens. In one , the fusion protein can e two or more genic domains of one or more antigens, or two or more epitopes of one or more antigens.
In any of the yeast-based immunotherapy itions used in the present invention, the following s related to the yeast vehicle are included in the invention. ing to the present invention, a yeast vehicle is any yeast cell (e. g., a whole or intact cell) or a tive thereof (see below) that can be used in conjunction with one or more antigens, immunogenic domains thereof or epitopes thereof in a therapeutic composition of the invention, or in one aspect, the yeast vehicle can be used alone or as an adjuvant.
The yeast vehicle can therefore include, but is not limited to, a live intact ) yeast rganism (z'.e., a yeast cell having all its components including a cell wall), a killed (dead) or inactivated intact yeast microorganism, or derivatives of intact/whole yeast including: a yeast spheroplast (z‘.e., a yeast cell lacking a cell wall), a yeast ast (z'.e., a yeast cell lacking a cell wall and nucleus), a yeast ghost (i.e., a yeast cell lacking a cell wall, nucleus and cytoplasm), a subcellular yeast membrane extract or fraction thereof (also referred to as a yeast membrane particle and previously as a subcellular yeast particle), any other yeast particle, or a yeast cell wall preparation.
] Yeast spheroplasts are typically produced by enzymatic digestion of the yeast cell wall. Such a method is described, for example, in Franzusoff et al., 1991, Meth.
Enzymol. 194, 662-674., incorporated herein by reference in its entirety.
Yeast cytoplasts are typically produced by enucleation of yeast cells. Such a method is described, for example, in Coon, 1978, Natl. Cancer Inst. Monogr. 48, 45-55 orated herein by reference in its ty.
Yeast ghosts are typically produced by ing a permeabilized or lysed cell and can, but need not, contain at least some of the organelles of that cell. Such a method is described, for example, in Franzusoff et al., 1983, J. Biol. Chem. 258, 3608-3614 and Bussey et al., 1979, Biochim. Biophys. Acta 553, 185-196, each of which is incorporated herein by reference in its entirety.
A yeast membrane particle (subcellular yeast membrane extract or on thereof) refers to a yeast membrane that lacks a natural nucleus or cytoplasm. The particle can be of any size, including sizes ranging from the size of a natural yeast membrane to microparticles produced by sonication or other membrane disruption methods known to those skilled in the art, followed by resealing. A method for producing lular yeast membrane extracts is described, for example, in Franzusoff et al., 1991, Meth. Enzymol. 194, 662-674. One may also use ons of yeast membrane particles that contain yeast membrane portions and, when the antigen or other protein was expressed recombinantly by the yeast prior to ation of the yeast membrane particles, the antigen or other n of st. Antigens or other proteins of interest can be carried inside the membrane, on either surface of the membrane, or combinations thereof (1.6., the protein can be both inside and outside the membrane and/or spanning the ne of the yeast membrane particle). In one embodiment, a yeast membrane particle is a recombinant yeast membrane particle that can be an intact, disrupted, or disrupted and resealed yeast membrane that includes at least one desired antigen or other protein of interest on the surface of the membrane or at least partially embedded within the membrane.
] An example of a yeast cell wall preparation is a preparation of isolated yeast cell walls carrying an antigen on its surface or at least partially embedded within the cell wall such that the yeast cell wall preparation, when administered to an animal, stimulates a desired immune response against a disease target.
Any yeast strain can be used to produce a yeast vehicle of the present ion. Yeast are unicellular microorganisms that belong to one of three classes: Ascomycetes, Basidiomycetes and Fungi Imperfecti. One consideration for the selection of a type of yeast for use as an immune modulator is the pathogenicity of the yeast. In one embodiment, the yeast is a thogenic strain such as Saccharomyces cerevisiae. The selection of a thogenic yeast strain minimizes any e effects to the individual to whom the yeast vehicle is administered. However, pathogenic yeast may be used if the pathogenicity of the yeast can be d by any means known to one of skill in the art (e.g, mutant strains).
Genera of yeast strains that may be used in the ion include but are not d to Saccharomyces, Candida, Cryptococcas, Hansenula, romyces, Pichia, Rhodotorala, Schizosaccharomyces and Yarrowia. In one aspect, yeast genera are selected from Saccharomyces, Candida, Hansenala, Pichia or Schizosaccharomyces, and in one aspect, yeast genera are selected from Saccharomyces, Hansenula, and Pichia, and in one aspect, Saccharomyces is used. s of yeast strains that may be used in the invention include but are not limited to Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Candida albicans, Candida kefi/r, Candida tropicalis, Cryptococcas tii, Cryptococcus mans, Hansenula anomala, Hansenula polymorpha, Kluyveromyces fragilis, Kluyveromyces lactis, Klayvemmyces marxianus var. lactis, Pichia pastoris, Rhodotorala rabra, Schizosaccharomyces pombe, and Yarrowia lipolytica.
It is to be appreciated that a number of these species include a variety of subspecies, types, subtypes, etc. that are intended to be included within the aforementioned s. In one aspect, yeast species used in the ion include S. cerevisiae, C. ns, H. polymorpha, P. pastoris and S. pombe. S. siae is useful as it is relatively easy to manipulate and being "Generally Recognized As Safe" or "GRAS" for use as food additives (GRAS, FDA proposed Rule 62FR18938, April 17, 1997). One embodiment of the present invention is a yeast strain that is capable of replicating plasmids to a ularly high copy number, such as a S. cerevisiae cir° strain. The S. cerevisiae strain is one such strain that is capable of supporting expression vectors that allow one or more target antigen(s) and/or antigen fusion protein(s) and/or other proteins to be expressed at high levels. In addition, any mutant yeast strains can be used in the present invention, including those that exhibit reduced post-translational modifications of expressed target antigens or other proteins, such as mutations in the enzymes that extend N-linked glycosylation.
In most embodiments of the invention, the yeast-based immunotherapy composition includes at least one antigen, genic domain thereof, or epitope thereof.
The antigens contemplated for use in this invention include any HBV antigen or immunogenic domain thereof, including mutants, variants and ts of HBV proteins or domains thereof, t which it is desired to elicit an immune response for the purpose of prophylactically or therapeutically immunizing a host against HBV infection. HBV antigens that are useful in various embodiments of the invention have been described in detail above.
Optionally, proteins, including fusion ns, which are used as a component of the yeast-based immunotherapeutic composition of the invention are produced using constructs that are particularly useful for improving or enhancing the expression, or the stability of expression, of recombinant antigens in yeast. Typically, the desired antigenic protein(s) or peptide(s) are fused at their amino-terminal end to: (a) a specific synthetic peptide that stabilizes the expression of the fusion n in the yeast vehicle or prevents posttranslational modification of the expressed filSlOIl protein (such es are described in detail, for e, in US. Patent Publication No. 2004—0156858 Al, published August 12, 2004, incorporated herein by reference in its ty); (b) at least a portion of an endogenous yeast protein, including but not limited to alpha factor, wherein either fusion partner provides improved stability of sion of the protein in the yeast and/or a prevents post-translational modification of the proteins by the yeast cells (such proteins are also described in , for example, in US. Patent Publication No. 2004-0156858 Al, supra); and/or (0) at least a portion of a yeast n that causes the fusion protein to be expressed on the surface of the yeast (e.g, an Aga protein, described in more detail herein).
In addition, the present invention ally includes the use of peptides that are fused to the C-terminus of the antigen-encoding construct, particularly for use in the selection and identification of the protein. Such peptides include, but are not d to, any synthetic or natural peptide, such as a peptide tag (e.g., hexahistidine) or any other short epitope tag.
Peptides attached to the C-terminus of an n according to the invention can be used with or without the addition of the N—terminal peptides discussed above.
In one ment, a synthetic peptide useful in a fusion protein is linked to the inus of the antigen, the peptide consisting of at least two amino acid residues that are heterologous to the antigen, wherein the peptide stabilizes the expression of the fusion protein in the yeast vehicle or prevents posttranslational modification of the expressed fusion protein. The synthetic peptide and N—terminal portion of the n together form a fiision n that has the following requirements: (1) the amino acid residue at position one of the fusion protein is a methionine (i.e., the first amino acid in the tic peptide is a methionine); (2) the amino acid residue at position two of the fusion protein is not a glycine or a proline (i.e., the second amino acid in the synthetic peptide is not a glycine or a proline); (3) none of the amino acid residues at positions 2-6 of the fusion protein is a methionine (i.e., the amino acids at positions 2—6, whether part of the tic peptide or the protein, if the synthetic peptide is shorter than 6 amino acids, do not include a methionine); and (4) none of the amino acids at positions 2-6 of the fusion protein is a lysine or an arginine (i.e., the amino acids at positions 2-6, whether part of the synthetic peptide or the n, if the synthetic peptide is shorter than 5 amino acids, do not include a lysine or an arginine). The synthetic peptide can be as short as two amino acids, but in one aspect, is 2-6 amino acids (including 3, 4, 5 amino acids), and can be longer than 6 amino acids, in whole integers, up to about 200 amino acids, 300 amino acids, 400 amino acids, 500 amino acids, or more.
In one embodiment, a fusion protein comprises an amino acid sequence of M- X2-X3-X4-X5—X6, wherein M is nine; wherein X2 is any amino acid except glycine, proline, lysine or ne; wherein X3 is any amino acid except methionine, lysine or arginine; wherein X4 is any amino acid except methionine, lysine or arginine; wherein X5 is any amino acid except methionine, lysine or arginine; and wherein X6 is any amino acid except methionine, lysine or arginine. In one embodiment, the X6 residue is a e. An exemplary synthetic sequence that enhances the stability of expression of an antigen in a yeast cell and/or prevents post-translational modification of the protein in the yeast includes the sequence M-A-D—E—A-P (SEQ ID NO:37). Another exemplary synthetic sequence with the same properties is M-V. In addition to the enhanced ity of the expression product, these fusion partners do not appear to negatively impact the immune response against the immunizing antigen in the construct. In addition, the synthetic fusion es can be designed to provide an epitope that can be recognized by a selection agent, such as an antibody.
In one embodiment, the HBV antigen is linked at the N—terminus to a yeast protein, such as an alpha factor prepro sequence (also referred to as the alpha factor signal leader sequence, the amino acid sequence of which is exemplified herein by SEQ ID NO:89 or SEQ ID NO:90. Other sequences for yeast alpha factor prepro ce are known in the art and are encompassed for use in the present invention.
] In one aspect of the invention, the yeast vehicle is manipulated such that the antigen is sed or provided by delivery or translocation of an sed protein product, partially or wholly, on the surface of the yeast e (extracellular expression).
One method for lishing this aspect of the invention is to use a spacer arm for positioning one or more protein(s) on the surface of the yeast vehicle. For example, one can use a spacer arm to create a fusion protein of the antigen(s) or other protein of interest with a protein that targets the antigen(s) or other protein of interest to the yeast cell wall.
For example, one such protein that can be used to target other proteins is a yeast protein (e.g, cell wall protein 2 (cwp2), Aga2, Pir4 or Flol protein) that enables the antigen(s) or other protein to be targeted to the yeast cell wall such that the antigen or other protein is located on the surface of the yeast. Proteins other than yeast proteins may be used for the spacer arm; however, for any spacer arm protein, it is most desirable to have the immunogenic response be directed against the target antigen rather than the spacer arm protein. As such, if other proteins are used for the spacer arm, then the spacer arm protein that is used should not generate such a large immune response to the spacer arm protein itself such that the immune response to the target n(s) is overwhelmed. One of skill in the art should aim for a small immune response to the spacer arm protein relative to the immune response for the target antigen(s). Spacer arms can be constructed to have cleavage sites (e.g., protease cleavage sites) that allow the antigen to be readily removed or processed away from the yeast, if desired. Any known method of determining the magnitude of immune responses can be used (e.g, antibody production, lytic assays, etc.) and are readily known to one of skill in the art. r method for positioning the target antigen(s) or other ns to be exposed on the yeast e is to use signal ces such as glycosylphosphatidyl inositol (GPI) to anchor the target to the yeast cell wall. atively, positioning can be accomplished by appending signal sequences that target the n(s) or other proteins of interest into the secretory pathway Via ocation into the asmic reticulum (ER) such that the antigen binds to a protein which is bound to the cell wall (e.g., cwp).
In one aspect, the spacer arm protein is a yeast protein. The yeast protein can consist of between about two and about 800 amino acids of a yeast protein. In one embodiment, the yeast protein is about 10 to 700 amino acids. In another embodiment, the yeast protein is about 40 to 600 amino acids. Other embodiments of the invention include the yeast n being at least 250 amino acids, at least 300 amino acids, at least 350 amino acids, at least 400 amino acids, at least 450 amino acids, at least 500 amino acids, at least 550 amino acids, at least 600 amino acids, or at least 650 amino acids. In one embodiment, the yeast protein is at least 450 amino acids in length. Another consideration for optimizing antigen surface expression, if that is desired, is whether the antigen and spacer arm ation should be expressed as a r or as dimer or as a trimer, or even more units connected together. This use of monomers, dimers, trimers, etc. allows for appropriate spacing or folding of the antigen such that some part, if not all, of the n is displayed on the surface of the yeast vehicle in a manner that makes it more genic.
Use of yeast proteins can stabilize the expression of fusion proteins in the yeast vehicle, prevents anslational modification of the expressed fusion protein, and/or targets the fusion protein to a particular compartment in the yeast (e.g., to be sed on the yeast cell surface). For delivery into the yeast secretory pathway, exemplary yeast proteins to use include, but are not limited to: Aga (including, but not limited to, Agal and/or Aga2); SUC2 (yeast invertase); alpha factor signal leader sequence; CPY; pr2p for its zation and retention in the cell wall; BUD genes for localization at the yeast cell bud during the l phase of daughter cell ion; Flolp; Pir2p; and Pir4p.
Other sequences can be used to target, retain and/or stabilize the protein to other parts of the yeast vehicle, for example, in the cytosol or the mitochondria or the endoplasmic reticulum or the nucleus. es of suitable yeast protein that can be used for any of the ments above include, but are not limited to, TK, AF, SEC7; phosphoenolpyruvate carboxykinase PCKl, phosphoglycerokinase PGK and triose phosphate isomerase TPI gene products for their repressible expression in glucose and cytosolic localization; the heat shock proteins SSAl, SSA3, SSA4, SSCl, whose expression is induced and whose proteins are more thermostable upon exposure of cells to heat treatment; the ondrial protein CYCl for import into mitochondria; ACTl.
Methods of producing yeast vehicles and expressing, combining and/or associating yeast vehicles with antigens and/or other proteins and/or agents of interest to produce yeast-based immunotherapy compositions are contemplated by the ion.
According to the present invention, the term "yeast vehicle-antigen x" or "yeast—antigen complex" is used generically to describe any association of a yeast vehicle with an antigen, and can be used interchangeably with "yeast-based immunotherapy composition" when such ition is used to elicit an immune response as described above. Such association includes expression of the antigen by the yeast (a recombinant yeast), introduction of an antigen into a yeast, physical attachment of the antigen to the yeast, and mixing of the yeast and antigen together, such as in a buffer or other solution or formulation. These types of complexes are described in detail below.
In one embodiment, a yeast cell used to prepare the yeast vehicle is transfected with a heterologous nucleic acid molecule ng a protein (e.g, the antigen) such that the protein is expressed by the yeast cell. Such a yeast is also referred to herein as a recombinant yeast or a recombinant yeast vehicle. The yeast cell can then be loaded into the dendritic cell as an intact cell, or the yeast cell can be killed, or it can be derivatized such as by ion of yeast spheroplasts, cytoplasts, ghosts, or subcellular particles, any of which is followed by loading of the derivative into the dendritic cell. Yeast spheroplasts can also be directly transfected with a recombinant nucleic acid molecule (e. g., the spheroplast is produced from a whole yeast, and then transfected) in order to produce a recombinant spheroplast that expresses an antigen or other protein.
In l, the yeast vehicle and antigen(s) and/or other agents can be associated by any technique described herein. In one aspect, the yeast e was loaded intracellularly with the antigen(s) and/or agent(s). In another aspect, the antigen(s) and/or agent(s) was covalently or valently ed to the yeast vehicle. In yet another , the yeast vehicle and the antigen(s) and/or agent(s) were associated by mixing. In another aspect, and in one embodiment, the antigen(s) and/or agent(s) is expressed recombinantly by the yeast vehicle or by the yeast cell or yeast spheroplast from which the yeast vehicle was derived.
A number of antigens and/or other proteins to be produced by a yeast vehicle of the t invention is any number of antigens and/or other proteins that can be reasonably produced by a yeast e, and typically ranges from at least one to at least about 6 or more, ing from about 2 to about 6 heterologous antigens and or other proteins.
Expression of an antigen or other protein in a yeast vehicle of the present invention is accomplished using techniques known to those skilled in the art. Briefly, a nucleic acid molecule encoding at least one desired antigen or other protein is inserted into an expression vector in such a manner that the nucleic acid molecule is ively linked to a transcription control sequence in order to be capable of effecting either constitutive or regulated sion of the nucleic acid molecule when transformed into a host yeast cell.
Nucleic acid molecules encoding one or more antigens and/or other proteins can be on one or more expression vectors operatively linked to one or more expression control sequences. ularly important expression control sequences are those which control transcription initiation, such as promoter and upstream activation sequences. Any suitable yeast er can be used in the present invention and a y of such promoters are known to those skilled in the art. Promoters for expression in Saccharomyces cerevisiae include, but are not limited to, promoters of genes encoding the ing yeast proteins: alcohol dehydrogenase I (ADHl) or II (ADH2), CUPl, phosphoglycerate kinase (PGK), triose 2012/024409 phosphate ase (TPI), translational elongation factor EF-l alpha (TEFZ), glyceraldehydephosphate dehydrogenase (GAPDH; also referred to as TDH3, for triose ate dehydrogenase), galactokinase (GALl), galactose-l-phosphate uridyl— erase (GAL7), UDP-galactose epimerase (GALIO), cytochrome c1 (CYCl), Sec7 protein (SEC7) and acid phosphatase , including hybrid ers such as APDH and CYCl/GALlO promoters, and including the ADH2/GAPDH promoter, which is induced when glucose concentrations in the cell are low (e.g., about 0.1 to about 0.2 percent), as well as the CUPl promoter and the TEF2 promoter. Likewise, a number of upstream activation sequences (UASs), also referred to as enhancers, are known. am activation sequences for expression in Saccharomyces cerevisiae include, but are not limited to, the UASs of genes encoding the following proteins: PCKl, TPI, TDH3, CYCl, ADHl, ADH2, SUC2, GALl, GAL7 and GALIO, as well as other UASs activated by the GAL4 gene product, with the ADH2 UAS being used in one aspect.
Since the ADH2 UAS is activated by the ADRl gene product, it may be preferable to overexpress the ADRl gene when a heterologous gene is operatively linked to the ADH2 UAS. Transcription termination sequences for expression in Saccharomyces cerevisiae include the termination ces of the a-factor, GAPDH, and CYCl genes.
Transcription control sequences to express genes in trophic yeast include the transcription control regions of the genes encoding alcohol oxidase and formate dehydrogenase.
Transfection of a nucleic acid molecule into a yeast cell according to the present invention can be lished by any method by which a nucleic acid molecule can be introduced into the cell and includes, but is not limited to, diffusion, active transport, bath sonication, electroporation, microinjection, lipofection, adsorption, and protoplast fusion. Transfected nucleic acid molecules can be integrated into a yeast chromosome or maintained on extrachromosomal vectors using techniques known to those skilled in the art. Examples of yeast vehicles carrying such nucleic acid molecules are disclosed in detail . As discussed above, yeast cytoplast, yeast ghost, and yeast membrane particles or cell wall preparations can also be produced recombinantly by transfecting intact yeast microorganisms or yeast spheroplasts with d nucleic acid molecules, producing the antigen n, and then further manipulating the microorganisms or spheroplasts using techniques known to those skilled in the art to produce cytoplast, ghost or subcellular yeast membrane extract or fractions f containing desired antigens or other proteins.
Effective conditions for the production of recombinant yeast vehicles and expression of the antigen and/or other protein by the yeast vehicle include an effective medium in which a yeast strain can be cultured. An effective medium is typically an aqueous medium comprising assimilable carbohydrate, nitrogen and phosphate sources, as well as appropriate salts, minerals, metals and other nutrients, such as vitamins and growth factors. The medium may comprise complex nutrients or may be a defined minimal medium. Yeast strains of the present invention can be ed in a variety of containers, including, but not limited to, bioreactors, Erlenmeyer flasks, test tubes, microtiter dishes, and Petri plates. Culturing is d out at a ature, pH and oxygen content appropriate for the yeast strain. Such culturing conditions are well within the expertise of one of ordinary skill in the art (see, for example, Guthrie et al. (eds), 1991, Methods in Enzymology, vol. 194, Academic Press, San Diego).
In some embodiments of the invention, yeast are grown under neutral pH conditions. As used herein, the general use of the term "neutral pH" refers to a pH range between about pH 5.5 and about pH 8, and in one aspect, between about pH 6 and about 8.
One of skill the art will appreciate that minor fluctuations (e.g., tenths or hundredths) can occur when measuring with a pH meter. As such, the use of neutral pH to grow yeast cells means that the yeast cells are grown in neutral pH for the majority of the time that they are in e. In one embodiment, yeast are grown in a medium ined at a pH level of at least 5.5 (126., the pH of the e medium is not allowed to drop below pH 5.5). In another aspect, yeast are grown at a pH level maintained at about 6, 6.5, 7, 7.5 or 8. The use of a neutral pH in culturing yeast promotes several biological effects that are ble characteristics for using the yeast as vehicles for immunomodulation. For example, culturing the yeast in neutral pH allows for good growth of the yeast t negative effect on the cell generation time (e.g, slowing of doubling time). The yeast can continue to grow to high ies t losing their cell wall pliability. The use of a neutral pH allows for the production of yeast with pliable cell walls and/or yeast that are more sensitive to cell wall digesting s (e.g., glucanase) at all harvest ies. This trait is desirable because yeast with flexible cell walls can induce different or improved immune responses as compared to yeast grown under more acidic conditions, e. g., by ing the secretion of cytokines by antigen ting cells that have phagocytosed the yeast (e.g., THl-type cytokines including, but not limited to, IFN-y, interleukin—l2 (IL- 12), and IL-2, as well as proinflammatory cytokines such as IL-6). In addition, greater accessibility to the antigens located in the cell wall is afforded by such culture methods.
WO 09404 In another aspect, the use of l pH for some antigens allows for release of the di- sulfide bonded antigen by treatment with dithiothreitol (DTT) that is not possible when such an antigen-expressing yeast is ed in media at lower pH (e.g., pH 5).
In one embodiment, control of the amount of yeast glycosylation is used to l the expression of antigens by the yeast, particularly on the surface. The amount of yeast glycosylation can affect the immunogenicity and nicity of the antigen expressed on the surface, since sugar moieties tend to be bulky. As such, the existence of sugar moieties on the surface of yeast and its impact on the three-dimensional space around the target antigen(s) should be ered in the modulation of yeast according to the invention. Any method can be used to reduce the amount of glycosylation of the yeast (or increase it, if desired). For example, one could use a yeast mutant strain that has been selected to have low glycosylation (e. g., mnnl, ochl and mnn9 mutants), or one could eliminate by mutation the glycosylation acceptor sequences on the target n.
Alternatively, one could use a yeast with abbreviated glycosylation patterns, e. g., Pichia.
One can also treat the yeast using methods that reduce or alter the glycosylation.
In one embodiment of the present invention, as an alternative to expression of an antigen or other n recombinantly in the yeast vehicle, a yeast vehicle is loaded intracellularly with the protein or peptide, or with ydrates or other molecules that serve as an antigen and/or are useful as immunomodulatory agents or ical response modifiers according to the invention. Subsequently, the yeast vehicle, which now contains the antigen and/or other proteins intracellularly, can be administered to an individual or loaded into a carrier such as a dendritic cell. Peptides and proteins can be inserted directly into yeast vehicles of the present invention by techniques known to those d in the art, such as by diffusion, active transport, liposome fusion, electroporation, phagocytosis, freeze-thaw cycles and bath sonication. Yeast vehicles that can be directly loaded with peptides, proteins, carbohydrates, or other molecules include intact yeast, as well as spheroplasts, ghosts or cytoplasts, which can be loaded with antigens and other agents after production. Alternatively, intact yeast can be loaded with the antigen and/or agent, and then spheroplasts, ghosts, cytoplasts, or subcellular particles can be prepared therefrom. Any number of antigens and/or other agents can be loaded into a yeast vehicle in this ment, from at least 1, 2, 3, 4 or any whole integer up to ds or thousands of antigens and/or other agents, such as would be provided by the loading of a microorganism or portions thereof, for example.
In another embodiment of the present ion, an n and/or other agent is physically attached to the yeast vehicle. Physical attachment of the antigen and/or other agent to the yeast vehicle can be accomplished by any method le in the art, including covalent and non-covalent association methods which include, but are not limited to, chemically crosslinking the antigen and/or other agent to the outer surface of the yeast vehicle or biologically linking the antigen and/or other agent to the outer surface of the yeast vehicle, such as by using an antibody or other binding partner. Chemical cross- g can be achieved, for example, by methods including glutaraldehyde linkage, photoaffinity labeling, treatment with carbodiimides, treatment with chemicals capable of g di-sulfide bonds, and treatment with other cross—linking chemicals standard in the art. Alternatively, a chemical can be contacted with the yeast vehicle that alters the charge of the lipid bilayer of yeast membrane or the ition of the cell wall so that the outer surface of the yeast is more likely to fuse or bind to antigens and/or other agent having particular charge characteristics. Targeting agents such as antibodies, binding es, soluble receptors, and other ligands may also be incorporated into an antigen as a fusion protein or otherwise associated with an antigen for binding of the antigen to the yeast vehicle.
When the antigen or other n is expressed on or physically attached to the surface of the yeast, spacer arms may, in one aspect, be lly selected to optimize antigen or other protein expression or content on the surface. The size of the spacer arm(s) can affect how much of the antigen or other protein is d for binding on the surface of the yeast. Thus, depending on which antigen(s) or other protein(s) are being used, one of skill in the art will select a spacer arm that effectuates appropriate spacing for the antigen or other protein on the yeast surface. In one embodiment, the spacer arm is a yeast protein of at least 450 amino acids. Spacer arms have been discussed in detail above.
In yet another embodiment, the yeast vehicle and the antigen or other protein are associated with each other by a more passive, non-specific or non-covalent binding mechanism, such as by gently mixing the yeast vehicle and the antigen or other protein together in a buffer or other suitable formulation (e. g., admixture).
In one ment of the ion, the yeast vehicle and the antigen or other protein are both loaded intracellularly into a carrier such as a tic cell or macrophage to form the therapeutic composition or vaccine of the t invention. Alternatively, an antigen or other protein can be loaded into a dendritic cell in the absence of the yeast vehicle.
In one embodiment, intact yeast (with or without expression of heterologous antigens or other proteins) can be ground up or processed in a manner to produce yeast cell wall preparations, yeast membrane particles or yeast fragments (z'.e., not intact) and the yeast fragments can, in some embodiments, be ed with or administered with other compositions that include antigens (e.g., DNA vaccines, protein t vaccines, killed or inactivated pathogens) to e immune responses. For example, tic treatment, chemical treatment or physical force (e.g., mechanical ng or sonication) can be used to break up the yeast into parts that are used as an adjuvant.
In one embodiment of the invention, yeast vehicles useful in the ion include yeast vehicles that have been killed or vated. Killing or vating of yeast can be accomplished by any of a y of suitable methods known in the art. For example, heat inactivation of yeast is a standard way of vating yeast, and one of skill in the art can monitor the structural changes of the target antigen, if desired, by standard methods known in the art. Alternatively, other methods of vating the yeast can be used, such as chemical, electrical, radioactive or UV methods. See, for example, the methodology disclosed in standard yeast culturing textbooks such as Methods of Enzymology, Vol. 194, Cold Spring Harbor Publishing (1990). Any of the inactivation strategies used should take the secondary, tertiary or quaternary structure of the target antigen into consideration and preserve such structure as to optimize its immunogenicity.
Yeast vehicles can be formulated into yeast-based immunotherapy compositions or products of the present invention, including preparations to be administered to a subject directly or first loaded into a carrier such as a dendritic cell, using a number of techniques known to those skilled in the art. For example, yeast vehicles can be dried by lyophilization. Formulations comprising yeast vehicles can also be prepared by packing yeast in a cake or a tablet, such as is done for yeast used in baking or brewing operations. In addition, yeast vehicles can be mixed with a pharmaceutically acceptable excipient, such as an isotonic buffer that is tolerated by a host or host cell.
Examples of such excipients include water, saline, Ringer's solution, dextrose solution, Hank's solution, and other aqueous physiologically ed salt solutions. eous vehicles, such as fixed oils, sesame oil, ethyl oleate, or triglycerides may also be used.
Other useful formulations include suspensions containing viscosity—enhancing , such as sodium carboxymethylcellulose, sorbitol, glycerol or dextran. Excipients can also contain minor amounts of additives, such as substances that enhance isotonicity and chemical stability. Examples of s include phosphate buffer, bicarbonate buffer and Tris buffer, while examples of preservatives include thimerosal, m- or o-cresol, formalin and benzyl alcohol. rd formulations can either be liquid injectables or solids which can be taken up in a suitable liquid as a suspension or solution for ion. Thus, in a non-liquid formulation, the excipient can comprise, for example, dextrose, human serum albumin, and/or preservatives to which sterile water or saline can be added prior to administration.
In one embodiment of the present ion, a composition can include additional agents, which may also be referred to as biological response modifier compounds, or the ability to produce such agents/modifiers. For example, a yeast vehicle can be transfected with or loaded with at least one antigen and at least one agent/biological response modifier compound, or a composition of the invention can be administered in conjunction with at least one biological response modifier. Biological se modifiers include adjuvants and other compounds that can modulate immune responses, which may be referred to as immunomodulatory compounds, as well as compounds that modify the biological activity of another compound or agent, such as a yeast—based immunotherapeutic, such biological activity not being limited to immune system effects.
Certain immunomodulatory compounds can stimulate a protective immune response whereas others can suppress a harmful immune response, and whether an immunomodulatory is useful in combination with a given yeast-based immunotherapeutic may depend, at least in part, on the disease state or condition to be treated or prevented, and/or on the individual who is to be treated. Certain biological response rs preferentially enhance a cell-mediated immune response whereas others preferentially enhance a l immune response (i.e., can stimulate an immune response in which there is an increased level of cell-mediated ed to humoral immunity, or vice versa.).
Certain biological response modifiers have one or more properties in common with the biological properties of yeast-based immunotherapeutics or e or complement the ical properties of based immunotherapeutics. There are a number of techniques known to those skilled in the art to measure stimulation or suppression of immune responses, as well as to differentiate ediated immune responses from humoral immune ses, and to differentiate one type of cell-mediated response from another (e.g, a TH17 response versus a THl response).
Agents/biological response modifiers useful in the invention may include, but are not limited to, nes, chemokines, hormones, lipidic derivatives, peptides, proteins, polysaccharides, small molecule drugs, antibodies and antigen binding fragments thereof (including, but not limited to, anti-cytokine dies, anti-cytokine receptor antibodies, anti-chemokine antibodies), vitamins, polynucleotides, nucleic acid binding moieties, aptamers, and growth tors. Some suitable agents include, but are not limited to, IL-1 or agonists of IL-1 or of IL-lR, anti-IL-l or other IL-l antagonists; IL-6 or agonists of IL-6 or of IL-6R, anti-IL-6 or other IL-6 antagonists; IL—12 or agonists of IL-12 or of IL-12R, anti-IL-12 or other IL-12 antagonists; IL-17 or agonists of IL-17 or of IL-17R, anti-IL-l7 or other IL-l7 antagonists; IL-21 or agonists of IL-21 or of IL-21R, L-Zl or other IL—2l antagonists; IL-22 or agonists of IL-22 or of IL—22R, anti-lL-22 or other IL- 22 antagonists; IL-23 or agonists of IL-23 or of IL-23R, anti-lL-23 or other IL-23 antagonists; IL-25 or agonists of IL-25 or of IL-25R, anti-IL-25 or other IL—25 antagonists; IL-27 or agonists of IL-27 or of IL—27R, anti-IL-27 or other IL-27 antagonists; type I eron (including IFN—u) or ts or antagonists of type I interferon or a receptor thereof; type II interferon (including IFN—y) or agonists or antagonists of type II interferon or a receptor thereof; anti-CD40 antibody, CD40L, anti- CTLA-4 antibody (e.g., to release anergic T cells); T cell co-stimulators (e.g., anti-CD137, D28, D40); alemtuzumab (e.g., CamPath®), denileukin diftitox (e.g., ONTAK®); anti—CD4; anti-CD25; anti—PD—l, D-Ll, anti-PD-LZ; agents that block FOXP3 (e.g., to abrogate the activity/kill CD4+/CD25+ T regulatory cells); Flt3 ligand, imiquimod (AldaraTM), granulocyte-macrophage colony stimulating factor (GM-CSF); ocyte-colony stimulating factor (G-CSF), sargramostim (Leukine®); hormones including without limitation prolactin and growth hormone; Toll-like receptor (TLR) agonists, including but not limited to TLR-2 agonists, TLR-4 agonists, TLR-7 agonists, and TLR-9 agonists; TLR nists, including but not limited to TLR-2 antagonists, TLR-4 antagonists, TLR—7 nists, and TLR-9 antagonists; anti—inflammatory agents and immunomodulators, including but not limited to, COX-2 inhibitors (e.g., xib, NSAIDS), orticoids, statins, and thalidomide and analogues thereof including IMiDTMs (which are structural and functional analogues of thalidomide (e.g., REVLIMID® (lenalidomide), D® (pomalidomide)); proinflammatory agents, such as fungal or bacterial ents or any proinflammatory cytokine or chemokine; therapeutic vaccines including, but not limited to, virus-based vaccines, ia-based vaccines, or antibody-based vaccines; and any other immunomodulators, potentiators, anti— inflammatory agents, and/or pro—inflammatory agents. Any combination of such agents is contemplated by the invention, and any of such agents combined with or administered in a protocol with (e.g., concurrently, sequentially, or in other formats with) a yeast-based immunotherapeutic is a composition encompassed by the invention. Such agents are well known in the art. These agents may be used alone or in combination with other agents described .
Agents can include agonists and antagonists of a given protein or peptide or domain thereof. As used herein, an "agonist" is any compound or agent, including without limitation small molecules, proteins, peptides, antibodies, nucleic acid g agents, etc., that binds to a receptor or ligand and produces or rs a response, which may include agents that mimic the action of a naturally occurring substance that binds to the receptor or ligand. An "antagonist" is any compound or agent, including without limitation small molecules, proteins, peptides, antibodies, nucleic acid binding , etc., that blocks or inhibits or reduces the action of an agonist.
Compositions of the invention can further include or can be administered with (concurrently, sequentially, or intermittently with) any other compounds or compositions that are useful for preventing or treating HBV infection or any compounds that treat or ameliorate any symptom ofHBV infection. A variety of agents are known to be useful for preventing and/or treating or ameliorating HBV ion. Such agents include, but are not limited to, anti-viral compounds, including, but not limited to, nucleotide analogue reverse transcriptase inhibitor (nRTIs). In one aspect of the invention, suitable anti-viral compounds include, but are not d to: tenofovir (VIREAD®), lamivudine (EPIVIR®), adefovir (HEPSERA®), telbivudine A®), entecavir (BARACLUDE®), and ations thereof, and/or interferons, such as interferon-(12a or pegylated interferon- (12a (PEGASYS®) or interferon-9t. These agents are typically stered for long periods of time (e.g., daily or weekly for up to one to five years or longer). In addition, compositions of the invention can be used er with other immunotherapeutic compositions, including prophylactic and/or therapeutic immunotherapy. For example, prophylactic vaccines for HBV have been commercially available since the early 1980’s.
These commercial vaccines are non-infectious, subunit viral vaccines providing d recombinant hepatitis B virus surface antigen (HBsAg), and can be administered beginning at birth. While no therapeutic therapeutic compositions have been approved in the US. for the treatment of HBV, such itions can include HBV protein or epitope subunit vaccines, HBV viral vector vaccines, nes, and/or other immunomodulatory agents (e.g, TLR ts, modulatory drugs).
The ion also includes a kit comprising any of the compositions described , or any of the individual components of the compositions described herein. s for Administration or Use 0: Comgosz'tions 01 the Invention Compositions of the invention, which can include any one or more (e.g, combinations of two, three, four, five, or more) yeast-based immunotherapeutic compositions described herein, HBV antigens including HBV proteins and fusion proteins, and/or recombinant nucleic acid molecules encoding such HBV proteins or fusion proteins described above, and other compositions comprising such yeast-based compositions, antigens, proteins, fusion proteins, or recombinant molecules described , can be used in a variety of in vivo and in vitro methods, including, but not limited to, to treat and/or prevent HBV infection and its sequelae, in diagnostic assays for HBV, or to produce antibodies against HBV.
One embodiment of the invention relates to a method to treat chronic tis B virus (HBV) infection, and/or to prevent, ameliorate or treat at least one symptom of chronic HBV infection, in an individual or population of individuals. The method includes the step of stering to an individual or a population of individuals who are chronically infected with HBV one or more immunotherapeutic compositions of the invention. In one aspect, the ition is an immunotherapeutic composition comprising one or more HBV antigens as described herein, which can include a yeast- based therapeutic composition. In one aspect, the composition includes a protein or fusion protein sing HBV antigens as described herein, and/or recombinant nucleic acid le encoding such protein or fusion protein. In one embodiment, the dual or population of individuals has chronic HBV infection. In one aspect, the dual or population of individuals is additionally treated with at least one other eutic compound useful for the treatment of HBV infection. Such eutic compounds include, but are not limited to, direct-acting antiviral drugs (e.g., those described above or elsewhere herein) and/or interferons and/or other immunotherapeutic or immunomodulatory agents. In one aspect, such therapeutic compounds include host- targeted therapeutics (e. g., cyclophilin inhibitors which can interfere with viral replication, or ry inhibitors that can interfere with the viral life cycle (re-infection)).
"Standard Of Care" or "SOC" generally refers to the current approved rd of care for the treatment of a specific disease. In c HBV infection, SOC may be one of several different approved therapeutic protocols, and include, but may not be limited to, interferon therapy and/or anti-viral therapy. Currently approved anti-viral drugs for the treatment of HBV infection include vir (VIREAD®), lamivudine (EPIVIR®), adefovir (HEPSERA®), telbivudine (TYZEKA®) and entecavir (BARACLUDE®). The anti-viral drugs prescribed most often for chronic HBV infection currently are tenofovir and entecavir. Interferon useful for the treatment of chronic HBV infection includes a type I interferon such as interferon-0t, including, but not limited to interferon-(x2 or pegylated interferon-(12 (e.g., PEGASYS®). In one embodiment, the interferon is a type III interferon, including without limitation, interferon-M, interferon-k2, and/or interferon-k3. The immunotherapeutic composition of the invention can be administered prior to, concurrently with, intermittently with, and/or after one or more anti— viral(s) and/or interferon and/or other immunotherapeutic or immunomodulatory .
The other therapeutic compounds may also be stered prior to or after treatment with the immunotherapeutic compositions of the ion.
HBV infection is typically diagnosed in an individual by detection of HBsAg (hepatitis B virus surface antigen) and/or HBeAg (e—antigen) in the blood of the infected individual. The detection of HBeAg in the serum s active viral replication, and clinical outcome of infection can be correlated with gen status, although long—term remission (or cure) is better predicted using HBsAg seroconversion when using current therapies (see below). Detection of IgM core antibody may also be used to detect acute HBV infection during the first 6-12 months of infection. tence of HBsAg in the blood for more than 6 months typically identifies chronic HBV infection. In addition, chronic HBV ion can be diagnosed by identifying HBV DNA (>2000 IU/ml), which can be combined with detection or identification of ed serum alanine ransferase (ALT) and/or aspartate aminotrasferase (AST) levels (e.g., more than twice the upper limit of normal).
] Recovery from the viral infection (complete response, or the endpoint for a treatment of HBV) is determined by HBsAg seroconversion, which is loss of HBeAg and HBsAg, respectively, and the pment of antibodies against the tis B surface antigen (anti-HBs) and/or antibodies t HBeAg. Clinical studies have defined seroconversion, or a protective antibody (anti-HBs) level as: (a) 10 or more sample ratio units (SRU) as determined by radioimmunoassay; (b) a positive result as determined by enzyme immunoassay; or (c) detection of an antibody concentration of 310 mIU/ml (10 SRU is comparable to 10 mIU/mL of antibody). Seroconversion can take years to develop in a chronically infected patient under current standard of care treatment (i.e., anti—viral drugs or interferon). Patients can also be monitored for loss or marked reduction of viral DNA (below detectable levels by PCR or <2000 IU/ml), normalization of serum alanine ransferase (ALT) levels, and improvement in liver inflammation and fibrosis. "ALT" is a well-validated measure of hepatic injury and serves as a surrogate for hepatic inflammation. In prior large hepatitis trials, reductions and/or ization of ALT levels (ALT normalization) have been shown to correlate with improved liver function and reduced liver fibrosis as determined by serial biopsy.
Another embodiment of the invention relates to a method to immunize an individual or population of individuals against HBV in order to prevent HBV infection, prevent chronic HBV infection, and/or reduce the severity of HBV infection in the individual or population of individuals. The method includes the step of stering to an dual or population of duals that is not infected with HBV (or ed not to be infected with HBV), a ition of the invention. In one aspect, the composition is an immunotherapeutic composition comprising one or more HBV antigens as described herein, ing one or more yeast-based immunotherapeutic compositions. In one aspect, the composition includes a fusion protein comprising HBV ns as described herein, or recombinant nucleic acid molecule encoding such fusion protein.
As used , the phrase "treat" HBV infection, or any permutation thereof (e.g., "treated for HBV infection", etc.) generally refers to ng or administering a composition of the invention once the infection (acute or chronic) has ed, with the goal of reduction or elimination of detectable viral titer (e.g., reduction of viral DNA (below detectable levels by PCR or <2000 IU/ml)), reaching seroconversion (development of antibodies against HBsAg and/or HBeAg and concurrent loss or reduction of these ns from the serum), ion in at least one symptom resulting from the infection in the individual, delaying or ting the onset and/or severity of symptoms and/or downstream sequelae caused by the infection, reduction of organ or physiological system damage (e.g., cirrhosis) resulting from the infection (e.g., reduction of abnormal ALT levels, reduction of liver inflammation, reduction of liver fibrosis), prevention and/or reduction in the frequency and incidence of hepatocellular carcinoma (HCC), improvement in organ or system function that was negatively impacted by the infection (normalization of serum ALT levels, improvement in liver inflammation, improvement in liver fibrosis), improvement of immune responses against the infection, improvement of long term memory immune responses against the infection, reduced reactivation of HBV virus, and/or ed general health of the individual or population of individuals.
In one aspect, a goal of treatment is sustained viral clearance for at least 6 months after the completion of therapy. In one aspect, a goal of treatment is the loss of detectable serum HBeAg and/or HBsAg proteins. In one aspect, a goal of treatment is the development of antibodies against the tis B surface antigen (anti-HBs) and/or antibodies against HBeAg. In one aspect, the goal of treatment is seroconversion, which may be defined by: (a) 10 or more sample ratio units (SRU) as determined by radioimmunoassay; (b) a positive result as determined by enzyme immunoassay; or (0) detection of an antibody concentration of 310 mIU/ml (10 SRU is comparable to 10 mIU/mL of antibody).
] To "prevent" HBV infection, or any permutation f (e.g., "prevention of HBV infection", etc.), generally refers to applying or administering a ition of the invention before an infection with HBV has occurred, with the goal of ting infection by HBV, preventing chronic infection by HBV (i.e., enabling an individual to clear an acute HBV infection without further intervention), or, should the infection later occur, at least reducing the severity, and/or length of infection and/or the physiological damage caused by the chronic infection, including preventing or reducing the severity or incidence of at least one symptom resulting from the infection in the individual, and/or ng or ting the onset and/or ty of symptoms and/or downstream sequelae caused by the infection, in an individual or population of individuals. In one , the present invention can be used to prevent chronic HBV infection, such as by enabling an individual who becomes acutely infected with HBV subsequent to administration of a composition of the invention to clear the infection and not become chronically infected.
The present invention includes the delivery (administration, immunization) of one or more therapeutic compositions of the invention, including a yeast-based immunotherapy composition, to a subject. The administration process can be performed ex viva or in viva, but is typically performed in viva. Ex viva administration refers to performing part of the regulatory step outside of the t, such as administering a composition of the present invention to a population of cells (dendritic cells) removed from a patient under conditions such that a yeast vehicle, antigen(s) and any other agents or compositions are loaded into the cell, and returning the cells to the t. The therapeutic composition of the present invention can be returned to a patient, or administered to a patient, by any suitable mode of administration.
] Administration of a ition can be systemic, mucosal and/or proximal to the location of the target site (e.g., near a site of ion). Suitable routes of administration will be apparent to those of skill in the art, depending on the type of condition to be prevented or d, the antigen used, and/or the target cell population or tissue. Various acceptable methods of administration e, but are not limited to, intravenous administration, intraperitoneal administration, intramuscular administration, intranodal administration, intracoronary administration, intraarterial administration (e.g, into a d ), subcutaneous administration, transderrnal delivery, intratracheal administration, intraarticular administration, intraventricular administration, inhalation (e.g, aerosol), intracranial, intraspinal, cular, aural, intranasal, oral, pulmonary administration, nation of a catheter, and direct ion into a tissue. In one aspect, routes of administration include: intravenous, intraperitoneal, subcutaneous, intradermal, intranodal, intramuscular, transdermal, inhaled, intranasal, oral, intraocular, intraarticular, intracranial, and intraspinal. Parenteral ry can include intradermal, intramuscular, intraperitoneal, leural, intrapulmonary, intravenous, subcutaneous, atrial catheter and venal catheter routes. Aural delivery can include ear drops, intranasal delivery can include nose drops or intranasal injection, and intraocular ry can include eye drops. l (inhalation) delivery can also be performed using methods standard in the art (see, for example, Stribling et a1., Proc. Natl. Acad. Sci. USA 277-11281, 1992). Other routes of administration that modulate mucosal immunity may be useful in the treatment of viral infections. Such routes include bronchial, intradermal, intramuscular, intranasal, other inhalatory, rectal, subcutaneous, topical, transdermal, vaginal and urethral routes. In one aspect, an therapeutic composition of the invention is administered subcutaneously.
With respect to the yeast—based immunotherapy compositions of the invention, in general, a suitable single dose is a dose that is capable of effectively providing a yeast vehicle and an n (if included) to a given cell type, tissue, or region of the patient body in an amount effective to elicit an n-specific immune response against one or more HBV antigens or epitopes, when administered one or more times over a suitable time . For example, in one embodiment, a single dose of a yeast vehicle of the present invention is from about 1 x 105 to about 5 x 107 yeast cell equivalents per kilogram body weight of the organism being administered the composition. In one aspect, a single dose of a yeast vehicle of the present invention is from about 0.1 Y.U. (1 x 106 cells) to about 100 Y.U. (1 x 109 cells) per dose (i.e., per organism), including any m dose, in increments of 0.1 x 106 cells (i.e., 1.1 x 106, 1.2 x 106, 1.3 x 106...). In one embodiment, doses include doses between 1 Y.U and 40 Y.U., doses between 1 Y.U. and 50 Y.U., doses 2012/024409 between 1 Y.U. and 60 Y.U., doses between 1 Y.U. and 70 Y.U., or doses between 1 Y.U. and 80 Y.U., and in one aspect, between 10 Y.U. and 40 Y.U., 50 Y.U., 60 Y.U., 70 Y.U., or 80 Y.U. In one embodiment, the doses are administered at different sites on the individual but during the same dosing period. For e, a 40 Y.U. dose may be administered via by injecting 10 Y.U. doses to four different sites on the individual during one dosing period, or a 20 Y.U. dose may be stered by injecting 5 Y.U. doses to four different sites on the individual, or by injecting 10 Y.U. doses to two different sites on the individual, during the same dosing period. The invention includes administration of an amount ofthe yeast-based immunotherapy composition (e.g., l, 2, 3, 4, 5, 6, 7, 8, 9 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20 Y.U. or more) at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different sites on an individual to form a single dose.
"Boosters" or "boosts" of a therapeutic composition are administered, for example, when the immune se t the antigen has waned or as needed to provide an immune response or induce a memory response t a particular antigen or antigen(s). Boosters can be administered from about 1, 2, 3, 4, 5, 6, 7, or 8 weeks apart, to monthly, to bimonthly, to rly, to annually, to several years after the original administration. In one embodiment, an administration schedule is one in which from about 1 X 105 to about 5 x 107 yeast cell equivalents of a composition per kg body weight of the sm is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times over a time period of from weeks, to , to years. In one embodiment, the doses are administered weekly for l, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more doses, followed by monthly doses as needed to achieve the d inhibition or elimination of the HBV virus. For example, the doses can be administered until the individual achieves seroconversion, until HBV DNA titers fall below 2000 IU/ml, and/or until ALT levels normalize. In one embodiment, the doses are administered in a 4-weekly protocol (every 4 weeks, or on day 1, week 4, week 8, week 12, etc., for between 2 and 10 doses or longer as determined by the clinician). Additional doses can be administered even after the individual achieves seroconversion, if desired, although such dosing may not be necessary.
With respect to administration of yeast-based immunotherapeutic itions described herein, a single composition can be administered to an individual or population of individuals or combination of such compositions can be administered.
For example, the invention provides several e protein" compositions or itions directed against a particular genotype, as well as multi—protein compositions and compositions that target multiple genotypes, or sub-genotypes. Accordingly, two or more compositions can be selected in a "spice rack" approach to most effectively prevent or treat HBV infection in a given dual or population of individuals.
In one aspect of the invention, one or more additional therapeutic agents are administered tially with the yeast-based immunotherapy composition. In another embodiment, one or more additional therapeutic agents are administered before the yeast- based immunotherapy ition is stered. In another embodiment, one or more additional therapeutic agents are administered after the based immunotherapy composition is administered. In one embodiment, one or more additional therapeutic agents are administered in alternating doses with the yeast-based therapy composition, or in a protocol in which the yeast-based composition is administered at prescribed intervals in between or with one or more consecutive doses of the additional agents, or vice versa. In one embodiment, the yeast-based immunotherapy composition is stered in one or more doses over a period of time prior to commencing the administration of the additional agents. In other words, the yeast-based immunotherapeutic composition is administered as a monotherapy for a period of time, and then the agent administration is added, either concurrently with new doses of yeast- based immunotherapy, or in an alternating fashion with yeast-based immunotherapy.
Alternatively, the agent may be administered for a period of time prior to ing administration of the yeast-based immunotherapy composition. In one aspect, the yeast is engineered to express or carry the agent, or a different yeast is engineered or produced to express or carry the agent.
In one aspect of the invention, when a treatment course of interferon or anti- viral compound therapy begins, additional doses of the immunotherapeutic composition are administered over the same period of time, or for at least a portion of that time, and may ue to be administered once the course of eron or anti—viral compound has ended. However, the dosing schedule for the therapy over the entire period may be, and is expected to typically be, different than that for the interferon or the anti-viral compound. For example, the immunotherapeutic composition may be administered on the same days or at least 3-4 days after the last given (most recent) dose of interferon or anti- viral (or any suitable number of days after the last dose), and may be administered daily, , ly, monthly, bimonthly, or every 3-6 months, or at longer intervals as determined by the physician. During an initial period of monotherapy administration of the immunotherapeutic composition, if utilized, the immunotherapeutic composition is preferably administered weekly for between 4 and 12 weeks, followed by monthly administration dless of when the additional interferon or anti-Viral therapy is added into the protocol). In one aspect, the immunotherapeutic composition is administered weekly for four or five weeks, followed by monthly administration thereafter, until conclusion of the complete treatment protocol.
In aspects of the invention, an immunotherapeutic composition and other agents can be administered together (concurrently). As used herein, concurrent use does not arily mean that all doses of all compounds are administered on the same day at the same time. Rather, concurrent use means that each of the therapy ents (e.g., immunotherapy and eron therapy, or immunotherapy and anti-Viral therapy) are d at approximately the same period (within hours, or up to 1-7 days of each other) and are administered over the same general period of time, noting that each component may have a different dosing schedule (e.g., interferon , immunotherapy monthly, anti-viral daily or weekly). In addition, before or after the concurrent administration period, any one of the agents or immunotherapeutic compositions can be administered without the other agent(s).
It is contemplated by the present invention that the use of an immunotherapeutic ition of the invention with an iral such as tenofovir or entecaVir will enable a shorter time course for the use of the anti-Viral drug. Similar results are expected when combining an immunotherapeutic of the invention with interferon. Dosing requirements for the anti-Viral or interferon may also be reduced or modified as a result of combination with the immunotherapeutic of the ion to generally e the nce of the patient for the drug. In addition, it is contemplated that the immunotherapeutic composition of the invention will enable seroconversion or sustained Viral responses for patients in whom anti-Viral therapy alone fails to achieve these endpoints. In other words, more patients will achieve nversion when an immunotherapeutic composition of the invention is combined with an anti-Viral or interferon than will achieve seroconversion by using anti-Virals or interferon alone. Under current SOC for HBV infection, anti-Virals may be administered for 6 months to one year, two years, three years, four years, five years, or longer (e.g, indefinitely). By combining such y with an immunotherapeutic composition of the invention, the time for the administration of the iral may be reduced by several months or years. It is contemplated that use of the immunotherapeutic compositions of the present invention, as a monotherapy or in combination with anti-Viral and/or immunomodulatory approaches will be effective to achieve loss of HBsAg and/or HBeAg; HBeAg seroconversion, HBsAg seroconversion, or te seroconversion; and in many individuals, sustained viral clearance for at least 6 months after the tion of therapy. In some patients, immunotherapy according to the present invention, when used as a monotherapy or in combination with anti-viral and/or immunomodulatory approaches, may achieve loss of HBsAg and/or HBeAg, but not achieve seroconversion (development of anti—HBs or anti— HBeAg). In this scenario, it is an embodiment of the invention to additionally use, alone or in combination with the yeast-based immunotherapy of the invention and/or anti-virals or other immunomodulatory agents, an agent such as the current prophylactic recombinant HBV subunit vaccine, in order to e te se in the patient.
As used herein, the term "anti—viral" refers to any compound or drug, typically a small—molecule inhibitor or antibody, which s one or more steps in the virus life cycle with direct anti-viral therapeutic effects. In one embodiment of the invention, the anti-viral compound or drug to be administered in the same therapeutic protocol with an immunotherapeutic composition of the invention is selected from tenofovir D®), dine (EPIVIR®), adefovir (HEPSERA®), telbivudine A®) and entecavir (BARACLUDE®), or any analog or derivative f, or any composition comprising or containing such compound, drug, analog or derivative.
Tenofovir (tenofovir disoproxil fumarate or TDF), or )(6-amino-9H— purinyl)propanyl]oxy}methyl)phosphonic acid, is a nucleotide analogue reverse transcriptase inhibitor (nRTIs). For the ent of HBV ion, tenofovir is typically administered to adults as a pill taken at a dose of 300 mg (tenofovir disproxil fumarate) once daily. Dosage for pediatric patients is based on body weight of the patient (8 mg per kg body weight, up to 300 mg once daily) and may be provided as tablet or oral powder.
Lamivudine, or 2',3'-dideoxy-3'-thiacytidine, ly called 3TC, is a potent nucleoside analog reverse transcriptase inhibitor (nRTI). For the treatment of HBV infection, lamivudine is administered as a pill or oral solution taken at a dose of 100mg once a day (1 .4-2mg/lb. twice a day for children 3 months to 12 years old).
Adefovir (adefovir dipivoxil), or 9-[2-[[bis[(pivaloyloxy)methoxy]- phosphinyl]-methoxy]ethyl]adenine, is an orally-administered nucleotide analog reverse riptase inhibitor (ntRTI). For the treatment of HBV infection, adefovir is administered as a pill taken at a dose of 10 mg once daily.
Telbivudine, or l—(2-deoxy-B-L-erythro-pentofuranosyl)methylpyrimidine— 2,4(1H,3H)-dione, is a synthetic thymidine nucleoside analogue (the L—isomer of 2012/024409 thymidine). For the treatment of HBV ion, telbivudine is administered as a pill or oral solution taken at a dose of 600 mg once daily. [003 13] Entecavir, or 2-Amino—9— [(l S ,3R,4S)hydroxy(hydroxymethyl)—2— methylidenecyclopentyl]—6,9-dihydro-3H-purin—6-one, is a nucleoside analog (guanine analogue) that inhibits e transcription, DNA replication and transcription of the virus.
For the treatment of HBV ion, entecavir is administered as a pill or oral solution taken at a dose of 0.5 mg once daily (1 mg daily for lamivudine-refractory or telbivudine resistance mutations).
In one embodiment of the ion, the eron to be administered in a therapeutic protocol with an immunotherapeutic composition of the invention is an interferon, and in one aspect, interferon-0L, and in one aspect, interferon-cab (administered by subcutaneous injection 3 times per week); or pegylated interferon-(x2a (e.g.
PEGASYS®). As used herein, the term "interferon" refers to a cytokine that is typically produced by cells of the immune system and by a wide y of cells in response to the presence of double-stranded RNA. Interferons assist the immune response by inhibiting viral replication within host cells, activating natural killer cells and macrophages, increasing antigen presentation to lymphocytes, and ng the resistance of host cells to viral infection. Type I interferons include interferon-u. Type III interferons include interferon-9t. Interferons useful in the methods of the present invention include any type I or type III interferon, including interferon-0L, interferon-(12, and in one aspect, longer lasting forms of interferon, ing, but not d to, pegylated interferons, interferon fusion proteins (interferon fused to albumin), and controlled-release formulations comprising interferon (e.g, interferon in microspheres or interferon with polyaminoacid rticles). One interferon, PEGASYS®, pegylated interferon-02a, is a covalent conjugate of recombinant interferon-u2a (approximate molecular weight [MW] 20,000 daltons) with a single branched bis-monomethoxy polyethylene glycol (PEG) chain (approximate MW 40,000 daltons). The PEG moiety is linked at a single site to the interferon-0L moiety via a stable amide bond to lysine. Pegylated interferon-(12a has an approximate molecular weight of 60,000 daltons.
] Interferon is typically stered by uscular or subcutaneous injection, and can be administered in a dose of between 3 and 10 million units, with 3 million units being preferred in one embodiment. Doses of interferon are administered on a regular schedule, which can vary from 1, 2, 3, 4, 5, or 6 times a week, to weekly, biweekly, every three weeks, or monthly. A l dose of interferon that is currently available is provided weekly, and that is a preferred dosing schedule for interferon, according to the present invention. For the treatment of HBV, pegylated interferon-0t2a is currently administered subcutaneously once a week at a dose of 180 mg (1.0 ml viral or 0.5 ml prefilled syringe), for a total of 48 weeks. The dose amount and timing can be varied according to the preferences and recommendations of the physician, as well as according to the recommendations for the particular interferon being used, and it is within the abilities of those of skill in the art to determine the proper dose. It is contemplated that by using interferon therapy together with an immunotherapeutic ition of the invention, the dose strength and/or number of doses of interferon (length of time on interferon and/or intervals between doses of interferon) can be reduced.
In the method of the present invention, compositions and therapeutic compositions can be stered to , ing any vertebrate, and particularly to any member of the Vertebrate class, Mammalia, including, without limitation, primates, rodents, livestock and domestic pets. ock include mammals to be ed or that produce useful products (e.g., sheep for wool production). Mammals to treat or protect include humans, dogs, cats, mice, rats, goats, sheep, cattle, horses and pigs.
An "individual" is a vertebrate, such as a mammal, including without limitation a human. Mammals e, but are not limited to, farm animals, sport animals, pets, primates, mice and rats. The term "individual" can be used interchangeably with the term "animal", "subject" or nt".
General Techniques Useful in the Invention The practice of the present invention will employ, unless otherwise indicated, conventional ques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, nucleic acid chemistry, and immunology, which are well known to those skilled in the art. Such techniques are explained fully in the literature, such as, Methods of Enzmology, Vol. 194, Guthrie et al., eds., Cold Spring Harbor Laboratory Press (1990); Biology and activities of yeasts, Skinner, et al., eds., Academic Press (1980); Methods in yeast cs : a laboratory course manual, Rose et al., Cold Spring Harbor Laboratory Press (1990); The Yeast Saccharomyces: Cell Cycle and Cell Biology, e et al., eds., Cold Spring Harbor Laboratory Press (1997); The Yeast romyces: Gene Expression, Jones et al., eds., Cold Spring Harbor Laboratory Press (1993); The Yeast romyces: Genome Dynamics, Protein Synthesis, and Energetics, Broach et al., eds., Cold Spring Harbor Laboratory Press ; Molecular Cloning: A Laboratog Manual, second edition (Sambrook et al., 1989) and Molecular Cloning: A Laboratory Manual, third edition (Sambrook and Russel, 2001), ly referred to herein as ook"); Current Protocols in Molecular Biology (F.M. Ausubel et al., eds., 1987, including supplements through 2001); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Harlow and Lane (1988), Antibodies, A Laboratog m, Cold Spring Harbor Publications, New York; Harlow and Lane (1999) Mg Antibodies: A Laboratog Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (jointly referred to herein as "Harlow and Lane"), Beaucage et al. eds., Current Protocols in Nucleic Acid try, John Wiley & Sons, Inc., New York, 2000); Casarett and Doull’s Toxicology The Basic Science of Poisons, C. Klaassen, ed., 6th edition (2001), and Vaccines, S. Plotkin and W. Orenstein, eds., 3rd edition (1999).
General Definitions A "TARMOGEN®" Immune, Inc., Louisville, Colorado) generally refers to a yeast vehicle expressing one or more heterologous antigens extracellularly (on its surface), intracellularly (internally or lically) or both extracellularly and intracellularly. TARMOGEN® products have been generally described (see, e. g., US.
Patent No. 5,830,463). Certain yeast-based immunotherapy compositions, and methods of making and generally using the same, are also bed in detail, for e, in US.
Patent No. 5,830,463, US. Patent No. 7,083,787, US. Patent No. 7,736,642, Stubbs et al., Nat. Med. 7:625-629 (2001), Lu et al., Cancer Research 4—5088 (2004), and in Bernstein et al., e 2008 Jan 24;26(4):509-21, each of which is incorporated herein by reference in its entirety.
As used herein, the term "analog" refers to a chemical compound that is structurally similar to another compound but s slightly in composition (as in the replacement of one atom by an atom of a ent element or in the presence of a particular functional group, or the replacement of one functional group by another functional group). Thus, an analog is a nd that is similar or comparable in on and appearance, but has a different structure or origin with respect to the reference compound.
The terms "substituted", "substituted derivative" and "derivative", when used to describe a compound, means that at least one hydrogen bound to the unsubstituted compound is replaced with a different atom or a chemical moiety.
Although a derivative has a similar physical structure to the parent compound, the derivative may have ent chemical and/or biological ties than the parent compound. Such properties can include, but are not limited to, sed or decreased activity of the parent compound, new activity as compared to the parent compound, enhanced or decreased bioavailability, ed or decreased efficacy, enhanced or sed stability in vitro and/or in vivo, and/or ed or decreased absorption properties.
In general, the term "biologically active" indicates that a compound (including a protein or peptide) has at least one detectable activity that has an effect on the metabolic or other ses of a cell or organism, as measured or observed in viva (i.e., in a natural physiological environment) or in vitro (126., under laboratory conditions).
According to the present invention, the term "modulate" can be used interchangeably with "regulate" and refers generally to upregulation or downregulation of a particular activity. As used herein, the term "upregulate" can be used generally to describe any of: elicitation, tion, increasing, augmenting, boosting, improving, enhancing, amplifying, promoting, or providing, with respect to a particular activity.
Similarly, the term "downregulate" can be used generally to be any of: decreasing, reducing, inhibiting, ameliorating, diminishing, ing, ng, or preventing, with respect to a ular activity.
In one embodiment of the present ion, any of the amino acid sequences described herein can be produced with from at least one, and up to about 20, additional logous amino acids flanking each of the C- and/or N—terminal ends of the specified amino acid sequence. The resulting protein or polypeptide can be referred to as "consisting essentially of‘ the specified amino acid sequence. According to the present ion, the logous amino acids are a sequence of amino acids that are not naturally found (126., not found in nature, in vivo) flanking the specified amino acid sequence, or that are not related to the function of the specified amino acid sequence, or that would not be encoded by the nucleotides that flank the naturally occurring nucleic acid sequence ng the specified amino acid sequence as it occurs in the gene, if such nucleotides in the naturally occurring sequence were translated using standard codon usage for the organism from which the given amino acid sequence is derived. Similarly, the phrase "consisting essentially of‘, when used with reference to a nucleic acid sequence herein, refers to a c acid sequence encoding a specified amino acid sequence that can be flanked by from at least one, and up to as many as about 60, additional heterologous nucleotides at each of the 5' and/or the 3' end of the nucleic acid sequence encoding the specified amino acid sequence. The heterologous nucleotides are not naturally found (i.e., not found in nature, in viva) flanking the nucleic acid sequence encoding the specified amino acid sequence as it occurs in the natural gene or do not encode a protein that imparts any additional function to the protein or changes the function of the protein having the specified amino acid ce.
According to the present invention, the phrase "selectively binds to" refers to the ability of an antibody, antigen-binding fragment or binding partner of the present invention to preferentially bind to specified proteins. More specifically, the phrase "selectively binds" refers to the specific binding of one protein to another (e.g., an antibody, fragment thereof, or binding partner to an antigen), n the level of binding, as ed by any standard assay (e.g, an immunoassay), is statistically significantly higher than the background control for the assay. For example, when performing an immunoassay, controls typically e a reaction well/tube that contain antibody or antigen binding nt alone (i.e., in the e of antigen), wherein an amount of reactivity (e.g., non-specific g to the well) by the dy or antigen-binding fragment thereof in the absence of the antigen is considered to be background. Binding can be measured using a variety of methods standard in the art including enzyme immunoassays (e.g., ELISA, immunoblot assays, etc.).
Reference to a protein or polypeptide used in the present invention includes full-length proteins, fusion proteins, or any fragment, domain, conformational e, or homologue of such proteins, including functional domains and immunological s of proteins. More specifically, an isolated protein, ing to the present invention, is a protein (including a polypeptide or peptide) that has been removed from its natural milieu (i.e., that has been subject to human manipulation) and can include purified proteins, partially purified proteins, recombinantly produced proteins, and synthetically produced proteins, for e. As such, "isolated" does not reflect the extent to which the protein has been d. Preferably, an isolated protein of the present ion is produced recombinantly. According to the present ion, the terms cation" and "mutation" can be used interchangeably, particularly with regard to the modifications/mutations to the amino acid sequence of proteins or portions thereof (or nucleic acid sequences) bed herein.
As used herein, the term "homologue" is used to refer to a protein or peptide which differs from a naturally occurring protein or peptide (i.e., the "prototype" or "wild— type" protein) by minor modifications to the naturally ing protein or peptide, but which maintains the basic protein and side chain structure of the naturally occurring form.
Such changes include, but are not limited to: changes in one or a few amino acid side chains; changes one or a few amino acids, including deletions (e.g., a truncated version of the n or peptide) insertions and/or substitutions; changes in stereochemistry of one or a few atoms; and/or minor derivatizations, including but not limited to: methylation, ylation, phosphorylation, ation, oylation, prenylation, palmitation, amidation and/or addition of glycosylphosphatidyl inositol. A homologue can have enhanced, decreased, or substantially similar properties as compared to the naturally occurring protein or peptide. A homologue can include an agonist of a protein or an antagonist of a protein. Homologues can be produced using techniques known in the art for the production of proteins including, but not limited to, direct modifications to the isolated, naturally occurring protein, direct protein synthesis, or ations to the nucleic acid sequence encoding the protein using, for example, classic or recombinant DNA techniques to effect random or targeted nesis.
A gue of a given protein may comprise, consist essentially of, or consist of, an amino acid sequence that is at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91% identical, or at least about 92% identical, or at least about 93% identical, or at least about 94% identical, or at least about 95% identical, or at least about 96% identical, or at least about 97% cal, or at least about 98% identical, or at least about 99% cal (or any percent ty between 45% and 99%, in whole integer increments), to the amino acid sequence of the reference protein. In one embodiment, the homologue comprises, consists essentially of, or consists of, an amino acid sequence that is less than 100% identical, less than about 99% identical, less than about 98% identical, less than about 97% identical, less than about 96% identical, less than about 95% identical, and so on, in increments of 1%, to less than about 70% identical to the naturally occurring amino acid sequence of the reference protein.
A homologue may include proteins or domains of proteins that are "near full- length", which means that such a gue differs from the full-length protein, onal domain or immunological domain (as such protein, functional domain or immunological domain is described herein or otherwise known or described in a publicly available sequence) by the addition of or deletion of l, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids from the N— and/or the C-terminus of such full—length protein or ength functional domain or full—length immunological domain.
] As used herein, unless ise specified, reference to a percent (%) identity refers to an evaluation of homology which is performed using: (1) a BLAST 2.0 Basic BLAST homology search using blastp for amino acid searches and blastn for c acid searches with standard default parameters, n the query sequence is filtered for low complexity regions by default (described in Altschul, S.F., , T.L., Schaaffer, A.A., Zhang, J ., Zhang, Z., Miller, W. & Lipman, D]. (1997) "Gapped BLAST and PS1- BLAST: a new generation of protein database search programs." Nucleic Acids Res. :3389-3402, incorporated herein by reference in its entirety); (2) a BLAST 2 alignment (using the parameters described below); (3) and/or PSI-BLAST with the standard default parameters (Position—Specific Iterated BLAST. It is noted that due to some differences in the rd parameters n BLAST 2.0 Basic BLAST and BLAST 2, two specific sequences might be recognized as having significant homology using the BLAST 2 program, whereas a search performed in BLAST 2.0 Basic BLAST using one of the sequences as the query sequence may not identify the second sequence in the top matches.
In addition, PSI-BLAST provides an automated, o-use version of a "profile" search, which is a sensitive way to look for sequence homologues. The program first performs a gapped BLAST database . The PSI—BLAST program uses the information from any significant alignments returned to construct a position-specific score matrix, which replaces the query sequence for the next round of database searching. Therefore, it is to be understood that percent identity can be ined by using any one of these programs.
Two specific sequences can be aligned to one another using BLAST 2 ce as described in Tatusova and Madden, (1999), "Blast 2 sequences - a new tool for comparing protein and nucleotide sequences", FEMS Microbiol Lett. 174:247-250, incorporated herein by nce in its entirety. BLAST 2 sequence alignment is performed in blastp or blastn using the BLAST 2.0 algorithm to perform a Gapped BLAST search (BLAST 2.0) between the two sequences allowing for the introduction of gaps (deletions and insertions) in the resulting alignment. For es of y herein, a BLAST 2 sequence alignment is performed using the standard default parameters as follows.
For blastn, using 0 BLOSUM62 matrix: Reward for match = 1 Penalty for mismatch = -2 Open gap (5) and extension gap (2) penalties gap x_dropoff (50) 61M (10) word size (11) filter (on) 2012/024409 For blastp, using 0 BLOSUM62 matrix: Open gap (11) and extension gap (1) penalties gap x_dropoff (50) expect (10) word size (3) filter (on).
An ed nucleic acid molecule is a nucleic acid le that has been removed from its natural milieu (i.e., that has been t to human manipulation), its l milieu being the genome or chromosome in which the nucleic acid molecule is found in nature. As such, "isolated" does not necessarily reflect the extent to which the nucleic acid molecule has been purified, but indicates that the molecule does not include an entire genome or an entire chromosome in which the nucleic acid molecule is found in nature. An isolated c acid molecule can include a gene. An isolated nucleic acid molecule that includes a gene is not a fragment of a some that includes such gene, but rather includes the coding region and regulatory s associated with the gene, but no additional genes that are naturally found on the same chromosome. An isolated nucleic acid molecule can also include a ied nucleic acid sequence flanked by (i.e., at the 5' and/or the 3' end of the sequence) additional nucleic acids that do not ly flank the specified nucleic acid sequence in nature (i.e., heterologous sequences). Isolated nucleic acid molecule can include DNA, RNA (e.g., mRNA), or tives of either DNA or RNA (e.g, cDNA). Although the phrase "nucleic acid molecule" primarily refers to the physical nucleic acid molecule and the phrase "nucleic acid sequence" ily refers to the sequence of nucleotides on the nucleic acid molecule, the two phrases can be used interchangeably, especially with respect to a nucleic acid molecule, or a nucleic acid sequence, being capable of encoding a protein or domain of a protein.
A recombinant nucleic acid molecule is a molecule that can include at least one of any nucleic acid sequence ng any one or more proteins described herein operatively linked to at least one of any transcription control sequence capable of ively regulating expression of the nucleic acid molecule(s) in the cell to be transfected. Although the phrase "nucleic acid molecule" primarily refers to the physical nucleic acid molecule and the phrase "nucleic acid sequence" primarily refers to the sequence of nucleotides on the nucleic acid molecule, the two phrases can be used interchangeably, especially with respect to a c acid molecule, or a nucleic acid sequence, being capable of encoding a protein. In addition, the phrase "recombinant molecule" primarily refers to a nucleic acid molecule operatively linked to a transcription control sequence, but can be used interchangeably with the phrase ic acid molecule" which is administered to an animal.
A recombinant nucleic acid molecule includes a inant vector, which is any nucleic acid sequence, typically a logous sequence, which is operatively linked to the isolated nucleic acid molecule encoding a fusion protein of the present invention, which is capable of enabling inant production of the fusion protein, and which is capable of delivering the nucleic acid molecule into a host cell according to the present invention. Such a vector can contain nucleic acid sequences that are not naturally found adjacent to the isolated nucleic acid molecules to be inserted into the vector. The vector can be either RNA or DNA, either prokaryotic or eukaryotic, and preferably in the present invention, is a virus or a plasmid. Recombinant vectors can be used in the cloning, sequencing, and/or otherwise manipulating of nucleic acid molecules, and can be used in delivery of such molecules (e.g., as in a DNA composition or a viral vector-based composition). inant vectors are preferably used in the expression of nucleic acid molecules, and can also be referred to as sion vectors. Preferred inant s are capable of being expressed in a transfected host cell.
In a recombinant le of the present ion, nucleic acid molecules are operatively linked to expression vectors containing regulatory sequences such as transcription control sequences, translation control sequences, origins of replication, and other regulatory sequences that are compatible with the host cell and that control the expression of nucleic acid molecules of the present ion. In particular, recombinant molecules of the present invention include nucleic acid les that are operatively linked to one or more expression control sequences. The phrase "operatively " refers to linking a nucleic acid molecule to an expression control sequence in a manner such that the molecule is expressed when transfected (i.e., transformed, transduced or transfected) into a host cell. ing to the present invention, the term "transfection" is used to refer to any method by which an exogenous nucleic acid molecule (i.e., a recombinant nucleic acid molecule) can be inserted into a cell. The term "transformation" can be used interchangeably with the term "transfection" when such term is used to refer to the introduction of nucleic acid molecules into microbial cells, such as algae, bacteria and yeast. In ial systems, the term "transformation" is used to describe an ted change due to the acquisition of exogenous nucleic acids by the microorganism and is essentially synonymous with the term "transfection.' Therefore, transfection techniques include, but are not limited to, ormation, chemical treatment of cells, particle bombardment, electroporation, microinjection, ction, adsorption, infection and protoplast fusion.
The following experimental results are provided for purposes of illustration and are not ed to limit the scope of the invention.
EXAMPLES Example 1 The following e describes the production of a yeast-based immunotherapeutic composition for the treatment or prevention of hepatitis B virus (HBV) infection.
In this ment, yeast (e.g, Saccharomyces cerevisiae) were engineered to express various HBV e-core fusion proteins, each having the basic structure shown in Fig. 2, under the control of the copper-inducible promoter, CUPI , or the TEF2 promoter. In each case, the HBV fusion n was a single polypeptide of approximately 595 amino acids, with the following sequence elements fused in frame from N— to inus, represented by SEQ ID NO:34 (1) an N—terminal peptide to impart resistance to proteasomal degradation and stabilize expression (positions 1 to 6 of SEQ ID NO:34); 2) a two amino acid spacer (Thr-Ser) to introduce a SpeI restriction enzyme site; 3) the amino acid sequence of a near full-length (minus position 1) HBV genotype C large (L) e antigen (e.g., positions 9 to 407 of SEQ ID NO:34, ponding to positions 2-400 of SEQ ID NO:11, which differs from SEQ ID NO:34 at positions 350-351 of SEQ ID NO:ll, where a Leu-Val sequence in SEQ ID NO:11 is replaced with a Gln-Ala ce at positions 357-358 of SEQ ID N034); 4) the amino acid sequence of an HBV core antigen (e.g., positions 408 to 589 of SEQ ID N034 or ons 31-212 of SEQ ID N09); and 5) a hexahistidine tag (positions 590—595 of SEQ ID NO:34). A nucleic acid sequence encoding the fusion n of SEQ ID NO:34 (codon optimized for yeast expression) is represented herein by SEQ ID NO:33. Positions 28-54 of SEQ ID NO:34 se the hepatocyte receptor portion of large (L) surface protein. SEQ ID NO:34 contains multiple epitopes or domains that are believed to enhance the immunogenicity of the fusion protein. For example, at positions 209-220, positions 389-397, positions 360- 367, and positions 499—506, with respect to SEQ ID NO:34, comprise known MHC Class I binding and/or CTL epitopes. Positions 305-328 of SEQ ID NO:34 comprise an antibody epitope. This fusion protein and corresponding yeast-based immunotherapeutic 2012/024409 comprising this protein can be generally referred to herein as "Score", "MADEAP—Score", "M-Score", or "GI-13002".
Briefly, DNA encoding nearly full length large surface antigen (L) fused to full length core antigen was codon zed for expression in yeast, and then digested with EcoRI and NotI and inserted behind the CUP] promoter (pGI—100), or the TEF2 promoter (pTK57-1), in yeast 2 um expression vectors. The fusion n encoded by these constructs is represented herein by SEQ ID NO:34 (encoded by tide sequence SEQ ID N033) and has an expected imate molecular weight of 66 kDa. The resulting plasmids were introduced into Saccharomyces cerevisiae W3030L yeast by m acetate/polyethylene glycol transfection, and primary transfectants were selected on solid minimal plates lacking uracil (UDM; uridine dropout medium). Colonies were re—streaked onto UDM or ULDM (uridine and leucine dropout medium) and allowed to grow for 3 days at 30°C. Liquid cultures lacking uridine (U2 medium: 20g/L glucose; 6.7 g/L of yeast nitrogen base containing um e; 0.04 mg/mL each of histidine, leucine, tryptophan, and adenine) or lacking uridine and leucine (UL2 medium: 20g/L glucose; 6.7 g/L of yeast nitrogen base containing ammonium sulfate; and 0.04 mg/mL each of his, tryptophan, and adenine) were inoculated from plates and starter cultures were grown for 20h at 30°C, 250 rpm. pH buffered media containing 4.2g/L of Bis-Tris (BT- U2; BT—UL2) was also inoculated to evaluate growth of the yeast under neutral pH conditions. Primary cultures were used to inoculate final es of the same formulation and growth was continued until a density or 1.1 to 4.0 YU/mL was reached.
For TEF2 strains (constitutive sion), cells were harvested, washed and heat killed at 56°C for 1h in PBS. Live cells were also processed for comparison. For CUPI strains (inducible expression), expression was induced in the same medium with 0.5 mM copper sulfate for 5h at 30°C, 250 rpm. Cells were ted, washed and heat killed at 56°C for 1h in PBS. Live cells were also processed for comparison.
After heat kill of TEF2 and CUP] cultures, cells were washed three times in PBS. Total n expression was measured by a TCA precipitation/nitrocellulose binding assay and antigen expression was ed by western blot using an anti-his tag monoclonal antibody. The antigen was quantified by interpolation from a standard curve of inant, hexa—histidine tagged NS3 protein that was processed on the same western blot. Results are shown in Fig. 16 (heat-killed) and Fig. 17 (live yeast). These figures show that the yeast—based immunotherapy composition of the invention ses the HBV surface—core fusion protein well using both promoters, and can be identified by W0 09404 Western blot in both heat-killed and live yeast cells. The calculated antigen expression by this yeast-based therapeutic was ~5000 ng protein per Y.U. (Yeast Unit; One Yeast Unit (Y.U.) is 1 x 107 yeast cells or yeast cell equivalents) or 76 pmol protein per Y.U.
Example 2 The following e describes the production of another yeast-based immunotherapeutic composition for the treatment or prevention of hepatitis B virus (HBV) infection.
Yeast (e.g., Saccharomyces siae) were engineered to express various HBV fusion proteins, each having the structure schematically shown in Fig. 3, under the control of the copper-inducible er, CUPI or the TEF2 promoter. In each case, the fusion protein was a single polypeptide of approximately 945 amino acids, with the following sequence elements fiJsed in frame from N— to C-terminus, ented by SEQ ID NO:36: (1) an N—terminal peptide to impart resistance to proteasomal ation and stabilize sion (positions 1 to 5 of SEQ ID NO:36); 2) the amino acid sequence of an HBV genotype C cyte receptor domain of the pre-Sl portion of HBV large (L) surface protein (unique to L) (e.g., positions 21—47 of SEQ ID NO:11 or positions 6 to 32 of SEQ ID NO:36); 3) the amino acid sequence of a full-length HBV genotype C small (S) surface antigen (e.g., positions 176 to 400 of SEQ ID NO:11 or positions 33 to 257 of SEQ ID NO:36); 4) a two amino acid spacer/linker (Leu—Glu) to facilitate cloning and manipulation of the sequences (positions 258 and 259 of SEQ ID NO:36); 5) the amino acid sequence of a portion of the HBV genotype C polymerase including the reverse transcriptase domain (e.g, positions 247 to 691 of SEQ ID NO:10 or positions 260 to 604 of SEQ ID NO:36); 6) an HBV genotype C core protein (e.g., positions 31-212 of SEQ ID NO:9 or ons 605 to 786 of SEQ ID NO:36); 7) the amino acid sequence of an HBV pe C X antigen (e.g, positions 2 to 154 of SEQ ID NO:12 or positions 787 to 939 of SEQ ID NO:36); and 8) a hexahistidine tag (positions 940 to 945 of SEQ ID NO:36).
This fusion protein and corresponding yeast-based immunotherapeutic comprising this protein can be generally referred to herein as "MADEAP-Spex", "M-Spex", or "GI- 13005".
A nucleic acid sequence encoding the fusion protein of SEQ ID NO:36 (codon optimized for yeast expression) is represented herein by SEQ ID NO:35. SEQ ID NO:36 has an expected approximate molecular weight of 106-107 kDa. SEQ ID NO:36 ns le epitopes or domains that are believed to enhance the immunogenicity of the fusion protein, including several described above for SEQ ID NO:34. In addition, the reverse transcriptase domain used in this fusion protein contains several amino acid positions that are known to become mutated as a drug-resistance response to treatment with anti-viral drugs, and therefore, may be mutated in this fusion protein in order to provide a therapeutic or prophylactic immunotherapeutic that targets specific drug resistance (escape) mutations. These amino acid positions are, with respect to SEQ ID NO:36, at amino acid position: 432 (Val, known to mutate to a Leu after lamivudine y); on 439 (Leu, known to mutate to a Met after lamivudine therapy); position 453 (Ala, known to mutate to a Thr after tenofovir therapy); position 463 (Met, known to mutate to an Ile or Val after lamivudine therapy); and position 495 (Asn, known to mutate to Thr after adefovir therapy).
To create a second yeast-based immunotherapeutic utilizing a ent N— terrninal e in the antigen, yeast (e.g., Saccharomyces cerevisiae) were engineered to express various HBV fusion proteins, also having the basic structure schematically shown in Fig. 3, under the control of the copper-inducible promoter, CUP], or the TEF2 promoter. In this second case, an alpha factor prepro sequence sented by SEQ ID NO:89) was used in place of the synthetic N—terminal peptide described above in the fusion represented by SEQ ID NO:36. Briefly, the new fiJsion protein was a single polypeptide with the following ce elements fused in frame from N— to C-terminus, represented by SEQ ID NO:92: (1) an N-terminal peptide to impart resistance to proteasomal degradation and stabilize or enhance sion (SEQ ID NO:89, positions 1 to 89 of SEQ ID N0192); 2) a two amino acid /linker (Thr-Ser) to facilitate cloning and manipulation of the sequences (positions 90 to 91 of SEQ ID NO:92); 3) the amino acid sequence of an HBV genotype C hepatocyte receptor domain of the pre-Sl portion of HBV large (L) surface protein e to L) (e.g., positions 21-47 of SEQ ID NO:11 or positions 92 to 118 of SEQ ID NO:92); 4) the amino acid sequence of a ength HBV genotype C small (S) surface n (e.g., positions 176 to 400 of SEQ ID NO:11 or positions 119 to 343 of SEQ ID NO:92); 5) a two amino acid spacer/linker (Leu-Glu) to facilitate cloning and manipulation of the sequences (e.g., positions 344 to 345 of SEQ ID NO:92); 6) the amino acid sequence of a n of the HBV genotype C polymerase including the reverse transcriptase domain (e.g., positions 247 to 691 of SEQ ID NO:10 or positions 346 to 690 of SEQ ID NO:92); 7) an HBV genotype C core protein (e.g., positions 31-212 of SEQ ID NO:9 or positions 691 to 872 of SEQ ID ; 8) the amino acid ce of an HBV genotype C X antigen (e.g., positions 2 to 154 of SEQ ID NO:12 or positions 873 to 1025 of SEQ ID NO:92); and 9) a hexahistidine tag (e.g., positions 1026 to 1031 of SEQ ID NO:92). This fusion protein and corresponding yeastbased immunotherapeutic comprising this protein can be generally referred to herein as "alpha-Spex", "a-Spex", or GI—13004".
A nucleic acid sequence encoding the fusion protein of SEQ ID NO:92 (codon optimized for yeast expression) is represented herein by SEQ ID NO:91. SEQ ID NO:92 has an expected approximate lar weight of 123 kDa. SEQ ID NO:92 contains multiple epitopes or domains that are believed to enhance the immunogenicity of the fusion protein, including several described above for SEQ ID N034 and SEQ ID NO:36.
In addition, the reverse transcriptase domain used in this fusion protein contains several amino acid positions that are known to become mutated as a drug-resistance response to treatment with anti-viral drugs, and therefore, may be d in this fusion protein in order to provide a therapeutic or prophylactic immunotherapeutic that targets specific drug resistance e) mutations. These amino acid positions are, with respect to SEQ ID NO:92, at amino acid position: 518 (Val, known to mutate to a Leu after lamivudine therapy); position 525 (Leu, known to mutate to a Met after dine therapy); position 539 (Ala, known to mutate to a Thr after tenofovir therapy); position 549 (Met, known to mutate to an Ile or Val after lamivudine therapy); and on 581 (Asn, known to mutate to Thr after adefovir therapy).
To create these immunotherapeutic compositions comprising the amino acid sequences ented by SEQ ID N036 and SEQ ID NO:92, DNA encoding the above- described conserved regions of surface antigen ocyte receptor region of pre-Sl or large surface antigen, and full-length small surface antigen) and the e transcriptase region of rase were fused to full length core and full length X antigen. The DNA was codon-optimized for expression in yeast and then digested with EcoRI and Not] and inserted behind the CUPI promoter (pGI-100) or the TEF2 promoter (pTK57-1) in yeast 2 um sion vectors. The resulting plasmids were introduced into Saccharomyces siae W3030t yeast by m acetate/polyethylene glycol transfection, and primary transfectants were selected on solid minimal plates lacking Uracil (UDM; uridine dropout medium). Colonies were re-streaked onto UDM or ULDM (uridine and leucine dropout medium) and allowed to grow for 3 days at 30°C.
] Liquid cultures lacking uridine (U2) or lacking e and leucine (UL2) were inoculated from plates and starter cultures were grown for 20h at 30°C, 250 rpm. pH buffered Media ning 4.2g/L of Bis-Tris (BT-U2; BT-UL2) were also inoculated to evaluate growth of the yeast under neutral pH conditions (data not shown). Primary cultures were used to inoculate final es of the same formulation and growth was continued until a density or 1.1 to 4.0 YU/mL was reached. For TEF2 strains (constitutive expression), cells were harvested, washed and heat killed at 56°C for 1h in PBS. For CUP] strains (inducible expression), expression was induced in the same medium with 0.5 mM copper sulfate for 5h at 30°C, 250 rpm. Cells were harvested, washed and heat killed at 56°C for 1h in PBS. Live cells were also processed for comparison (data not shown).
After heat kill of TEF2 and CUPI cultures, cells were washed three times in PBS. Total protein expression was measured by a TCA precipitation/nitrocellulose binding assay and n expression was measured by western blot using an is tag monoclonal antibody. The n was quantified by interpolation from a standard curve of recombinant, hexa-histidine tagged NS3 protein that was processed on the same western blot.
For the yeast-based immunotherapeutic expressing the fusion protein represented by SEQ ID NO:36 (GI-13005), results are shown in Fig. 18. Fig. 18 shows that the yeast-based immunotherapy composition of the invention expresses the fusion protein well using both promoters, and can be identified by Western blot in heat-killed yeast cells (expression was also achieved in live yeast cells, data not shown). The calculated antigen expression by this yeast-based immunotherapeutic was ~1200 ng protein per Y.U. or 11 pmol protein per Y.U., for growth in UL2.
For the yeast-based immunotherapeutic sing the fusion n represented by SEQ ID NO:92 (GI-13004), results are shown in Fig. 19. Fig. 19 shows expression of this yeast-based immunotherapy ition under the control of the CUPI promoter (identified in Fig. 19 as Alpha-SPEX) as compared to a yeast-based immunotherapeutic that expresses an unrelated antigen (Control Yeast) and to the yeast- based immunotherapeutic ition sing an HBV fusion protein represented by SEQ ID NO:36 (SPEX). Fig. 19 shows that the yeast-based immunotherapeutics expresses the relevant fusion proteins well, and can be identified by Western blot in heat- killed yeast cells. The calculated antigen expression by this yeast-based therapeutic -SPEX) was ~5000 ng protein per Y.U. or 41 pmol protein per Y.U. for growth in UL2.
Example 3 The following example describes the production of additional yeast-based therapeutic composition for the ent or prevention of tis B Virus (HBV) infection.
In this experiment, yeast (e.g., Saccharomyces cerevisiae) are engineered to express various HBV polymerase-core fusion proteins, as shown schematically in Fig. 4, under the control of the copper-inducible er, CUPI or the TEF2 promoter. In each case, the fusion protein is a single polypeptide of approximately 527 amino acids, with the following sequence elements fiised in frame from N— to C—terminus, represented by SEQ ID NO:38: (1) an N—terminal peptide to impart resistance to proteasomal degradation and stabilize expression (SEQ ID NO:37; positions 1 to 6 of SEQ ID NO:38); 2) the amino acid sequence of a portion of the HBV genotype C polymerase including the e transcriptase domain (e.g., ons 347 to 691 of SEQ ID NO:10 or positions 7 to 351 of SEQ ID ; 3) an HBV genotype C core protein (e.g, positions 31 to 212 of SEQ ID NO:9 or positions 352 to 533 of SEQ ID N038); and 4) a hexahistidine tag (e.g, positions 534 to 539 of SEQ ID NO:38). SEQ ID NO:38 has a predicted lar weight of approximately 58 kDa. The sequence also contains epitopes or domains that are ed to enhance the immunogenicity of the fusion protein. In additional constructs, the N—terminal peptide of SEQ ID NO:37 is replaced with a different synthetic N-terminal peptide represented by a homologue of SEQ ID NO:37 that meets the same basic structural requirements of SEQ ID NO:37 as described in detail in the specification, or the N—terminal peptide of SEQ ID NO:37 is ed with the inal peptide of SEQ ID NO:89 or SEQ ID N090, and in r construct, the N-terminal peptide is omitted and a methionine is included at position one.
In another ment, yeast (e.g, Saccharomyces cerevisiae) are ered to express various HBV X-core fusion proteins as shown schematically in Fig. 5 under the control of the copper-inducible promoter, CUPI or the TEF2 promoter. In each case, the fusion protein is a single polypeptide of approximately 337 amino acids with the following sequence elements filS6d in frame from N— to C-terminus, represented by SEQ ID NO:39 (1) an N-terminal peptide to impart resistance to proteasomal degradation and stabilize expression (SEQ ID NO:37; positions 1 to 6 of SEQ ID NO:39); 2) the amino acid sequence of a near full—length (minus position 1) HBV genotype C X antigen (e.g, positions 2 to 154 of SEQ ID NO:12 or positions 7 to 159 of SEQ ID NO:39); 3) an HBV genotype C core protein (e.g., positions 31 to 212 of SEQ ID NO:9 or positions 160 to 341 of SEQ ID NO:39); and 4) a hexahistidine tag (positions 342 to 347 of SEQ ID NO:39).
SEQ ID NO:39 has a predicted approximate molecular weight of 37 kDa. The sequence also contains epitopes or domains that are believed to enhance the immunogenicity of the fusion protein. In additional constructs, the N-terminal peptide of SEQ ID NO:37 is replaced with a different synthetic N-terminal peptide represented by a homologue of SEQ ID NO:37 that meets the same basic structural requirements of SEQ ID NO:37 as described in detail in the specification, or the N-terminal peptide of SEQ ID NO:37 is replaced with the N—terminal peptide of SEQ ID NO:89 or SEQ ID N090, and in another construct, the inal peptide is omitted and a methionine is ed at position one.
In another ment, yeast (e.g, Saccharomyces siae) are engineered to express various HBV polymerase proteins as shown tically in Fig. 6 under the control of the copper-inducible promoter, CUPI or the TEF2 promoter. In each case, the fusion protein is a single polypeptide with the following sequence elements fused in frame from N— to C-terminus, represented by SEQ ID NO:40 (1) an N-terminal peptide to impart resistance to proteasomal ation and stabilize expression (SEQ ID NO:37, or positions 1 to 6 of SEQ ID NO:40; 2) the amino acid sequence of a n of the HBV genotype C polymerase including the reverse transcriptase domain (e.g., positions 347 to 691 of SEQ ID NO:10 or positions 7 to 351 of SEQ ID ; and 3) a hexahistidine tag ions 352 to 357 of SEQ ID . The sequence also contains epitopes or domains that are believed to enhance the imrnunogenicity of the fusion protein. In addition, in one embodiment, the sequence of this construct can be modified to uce one or more or all of the following anti-Viral resistance mutations: rtM204l, M, rtM204V, rtVl73L, T, rtAl94T (positions given with respect to the ength amino acid sequence for HBV rase). In one embodiment, six different immunotherapy compositions are created, each one containing one of these mutations. In other embodiments, all or some of the mutations are included in a single fusion protein. In additional constructs, the N- terminal peptide of SEQ ID NO:37 is replaced with a different synthetic N-terminal peptide represented by a homologue of SEQ ID NO:37 that meets the same basic structural requirements of SEQ ID NO:37 as described in detail in the specification, or the N-terminal peptide of SEQ ID NO:37 is replaced with the N-terminal peptide of SEQ ID NO:89 or SEQ ID NO:90, and in another construct, the N—terminal peptide is d and a methionine is included at position one.
In another experiment, yeast (e.g, Saccharomyces cerevisiae) are engineered to express various HBV polymerase-surface-core fusion proteins as shown schematically in Fig. 7 under the control of the copper-inducible promoter, CUPI or the TEFZ promoter.
In each case, the fusion protein is a single polypeptide with the following sequence elements fused in frame from N— to C-terminus, represented by SEQ ID NO:41: (1) an N- terminal peptide to impart resistance to somal degradation and stabilize expression (e.g, positions 1 to 5 of SEQ ID NO:41); 2) an amino acid sequence of the amino HBV cyte receptor domain of the pre-Sl portion of HBV large (L) surface protein (unique to L) (e.g, positions 21-47 of SEQ ID NO:11 or positions 6 to 32 of SEQ ID NO:41); 3) the amino acid sequence of an HBV small (S) surface protein (e.g., positions 176 to 400 of SEQ ID NO:11 or positions 33 to 257 of SEQ ID NO:41); 4) a two amino acid /linker to facilitate cloning and manipulation of the sequences (e.g., positions 258 and 259 of SEQ ID N0241); 5) the amino acid sequence of an HBV polymerase comprising the reverse transcriptase domain (e.g., positions 247 to 691 of SEQ ID NO:10 or positions 260 to 604 of SEQ ID NO:41); 6) the amino acid ce of an HBV core protein (e.g., ons 31-212 of SEQ ID N09 or positions 605 to 786 of SEQ ID NO:41); and 7) a hexahistidine tag (e.g., positions 787 to 792 of SEQ ID NO:4l). The sequence also ns epitopes or domains that are believed to enhance the genicity of the fusion protein. In addition, in one embodiment, the sequence of this uct can be modified to introduce one or more or all of the following anti—viral resistance mutations: rtM204l, rtL180M, rtM204V, rtV173L, rtN236T, rtA194T (positions given with respect to the full-length amino acid sequence for HBV polymerase).
In one embodiment, six different immunotherapy compositions are created, each one containing one of these mutations. In other embodiments, all or some of the mutations are included in a single fusion n. In one embodiment, this construct also contains one or more anti—viral resistance mutations in the surface antigen. In additional constructs, the N- terminal peptide ented by positions 1 to 5 of SEQ ID NO:41 is replaced with a different synthetic N—terminal peptide represented by a homologue of positions 1 to 5 of SEQ ID NO:41 that meets the same basic structural requirements of positions 1 to 5 of SEQ ID NO:41 (or of SEQ ID NO:37) as described in detail in the specification, or the N- al peptide of positions 1 to 5 of SEQ ID NO:41 is replaced with the N-terminal peptide of SEQ ID NO:89 or SEQ ID N090, and in another construct, the N-terminal peptide is omitted and a methionine is ed at position one.
To produce any of the above-described fusion proteins and yeast-based immunotherapy itions expressing such proteins, briefly, DNA ng the fusion protein is codon optimized for expression in yeast and then digested with EcoRI and NotI and inserted behind the CUP] promoter (pGI—100) or the TEF2 promoter (pTK57—1) in yeast 2 um expression vectors. The resulting plasmids are introduced into Saccharomyces siae W3030L yeast by Lithium acetate/polyethylene glycol transfection, and primary transfectants are selected on solid l plates lacking Uracil (UDM; uridine dropout medium). es are re—streaked onto UDM or ULDM (uridine and leucine dropout medium) and allowed to grow for 3 days at 30°C.
Liquid cultures lacking uridine (U2) or lacking uridine and e (UL2) are inoculated from plates and starter cultures were grown for 20h at 30°C, 250 rpm. pH buffered Media containing 4.2g/L of Bis—Tris (BT-UZ; BT-UL2) can also be inoculated to evaluate growth of the yeast under l pH ions. Primary cultures are used to inoculate final cultures of the same ation and growth is continued until a density or 1.1 to 4.0 YU/mL is reached. For TEF2 strains (constitutive expression), cells are harvested, washed and heat killed at 56°C for 1h in PBS. For CUP] strains (inducible expression), expression is induced in the same medium with 0.5 mM copper sulfate for 5h at 30°C, 250 rpm. Cells are harvested, washed and heat killed at 56°C for 1h in PBS.
Live cells are also processed for comparison.
After heat kill of TEF2 and CUP] cultures, cells are washed three times in PBS. Total protein expression is measured by a TCA precipitation/nitrocellulose g assay and protein expression is measured by western blot using an anti-his tag monoclonal antibody. Fusion n is quantified by interpolation from a rd curve of recombinant, hexa—histidine tagged NS3 protein that was processed on the same western blot.
Example 4 The following example describes the production of additional yeast-based immunotherapeutic itions for the treatment or prevention of hepatitis B virus (HBV) infection.
This example describes the production of four different yeast-based immunotherapeutic compositions, each one designed to express one HBV protein. These "single HBV protein yeast immunotherapeutics" can be used in combination or in sequence with each other and/or in combination or in sequence with other based immunotherapeutics, such as those described in any of Examples l-3 and 5-8, including multi-HBV protein yeast-based immunotherapeutics described herein. In addition, a "single HBV protein yeast immunotherapeutic", such as those described in this example, can be produced using the HBV sequence for any given genotype or sub-genotype, and additional HBV surface n yeast-based immunotherapeutics can be produced using the HBV sequences for any one or more additional genotypes or sub—genotypes, in order to provide a "spice rack" of different HBV ns and pes and/or subgenotypes, each of which is provided in the context of a yeast-based immunotherapeutic of the invention, or in an immunization/administration strategy that includes at least one yeast- based immunotherapeutic of the invention.
In this example, the following four yeast-based therapeutic products are produced: face Antigen. Saccharomyces cerevisiae are engineered to express an HBV surface protein under the l of the copper-inducible promoter, CUP], or the TEF2 promoter. In each case, the fusion protein is a single polypeptide with the following sequence elements filSGd in frame from N- to C-terminus, represented by SEQ ID NO:93: 1) an N—terminal peptide of SEQ ID NO:89 (positions 1-89 of SEQ ID N0293); 2) the amino acid sequence of a near full-length (minus position 1) HBV genotype C large (L) surface antigen (e.g., positions 2-400 of SEQ ID NO:ll or positions 90 to 488 of SEQ ID NO:93); and 3) a hexahistidine tag (e.g., positions 489 to 494 of SEQ ID .
Alternatively, the N—terrninal peptide can be replaced with SEQ ID NO:37 or a homologue f or another N—terminal peptide described herein.
HBV Polymerase Antigen. Saccharomyces cerevisiae are engineered to s the following HBV Polymerase n under the control of the copper-inducible promoter, CUP], or the TEF2 promoter. In each case, the fusion protein is a single polypeptide with the following sequence ts fused in frame from N— to inus, ented by SEQ ID NO:94: 1) an N-terminal peptide of SEQ ID NO:89 (positions 1- 89 of SEQ ID NO:94); 2) the amino acid sequence of a portion of the HBV genotype C polymerase including the reverse transcriptase domain (e.g., positions 347 to 691 of SEQ ID NO:10 or positions 90 to 434 of SEQ ID NO:94); and 3) a hexahistidine tag (e.g., positions 435 to 440 of SEQ ID NO:94). Alternatively, the N—terminal peptide can be replaced with SEQ ID NO:37 or a homologue thereof or another N-terminal peptide described .
HBV Core Antigen. Saccharomyces cerevisiae are engineered to express the following HBV Core protein under the control of the copper-inducible er, CUP], or the TEF2 promoter. In each case, the fusion protein is a single polypeptide with the following sequence elements fiJsed in frame from N— to C-terminus, represented by SEQ ID NO:95: 1) an N—terminal peptide of SEQ ID NO:89 ions 1-89 of SEQ ID NO:95); 2) the amino acid sequence of a portion of the HBV genotype C Core protein (e.g, positions 31 to 212 of SEQ ID NO:9 or positions 90 to 271 of SEQ ID N095); and 3) a hexahistidine tag (e.g., positions 272 to 277 of SEQ ID NO:95). Alternatively, the N- terminal peptide can be replaced with SEQ ID NO:37 or a homologue thereof or another N—terminal peptide described herein.
HBV X Antigen. Saccharomyces cerevisiae are engineered to express the following HBV X antigen under the control of the copper—inducible promoter, CUPI , or the TEF2 promoter. In each case, the fusion protein is a single ptide with the following sequence elements fitsed in frame from N— to C—terminus, represented by SEQ ID NO:96: 1) an N—terminal peptide of SEQ ID NO:89 (positions 1-89 of SEQ ID NO:96); 2) the amino acid sequence of a portion of the HBV genotype C X n (e.g, positions 2 to 154 of SEQ ID NO:12 or positions 90 to 242 of SEQ ID NO:96); and 3) a hexahistidine tag (e.g., positions 243 to 248 of SEQ ID NO:96). Alternatively, the N- terrninal peptide can be replaced with SEQ ID NO:37 or a homologue thereof or r N—terminal peptide bed herein.
To create these immunotherapeutic compositions, , DNA ng the fusion protein is codon zed for expression in yeast and then digested with EcoRI and Not] and inserted behind the CUP] promoter (pGI-lOO) or the TEF2 promoter (pTK57-1) in yeast 2 um expression vectors. The resulting plasmids are introduced into Saccharomyces cerevisiae W3030t yeast by m acetate/polyethylene glycol transfection, and primary transfectants are selected on solid minimal plates lacking uracil (UDM; uridine dropout medium). Colonies are re-streaked onto UDM or ULDM (uridine and leucine dropout medium) and allowed to grow for 3 days at 30°C.
Liquid cultures lacking uridine (U2) or lacking uridine and leucine (UL2) are inoculated from plates and starter cultures were grown for 20h at 30°C, 250 rpm. pH buffered Media containing 4.2g/L of Bis-Tris (BT-U2; BT-UL2) may also be inoculated to te growth of the yeast under neutral pH conditions. Primary es are used to inoculate final cultures of the same formulation and growth is continued until a density or 1.1 to 4.0 YU/mL is reached. For TEF2 strains (constitutive expression), cells are ted, washed and heat killed at 56°C for 1h in PBS. For CUPI strains (inducible expression), expression is induced in the same medium with 0.5 mM copper sulfate for 5h at 30°C, 250 rpm. Cells are harvested, washed and heat killed at 56°C for 1h in PBS. Live cells are also processed for ison.
After heat kill of TEF2 and CUP] cultures, cells are washed three times in PBS. Total protein expression is ed by a TCA precipitation/nitrocellulose binding assay and protein expression is measured by western blot using an anti-his tag monoclonal antibody. Fusion n is quantified by interpolation from a standard curve of recombinant, hexa—histidine tagged NS3 protein that was processed on the same n blot.
Example 5 The following example bes the production of several different yeast- based immunotherapeutic itions for the treatment or tion of tis B virus (HBV) infection.
This example bes the production of yeast-based immunotherapeutics expressing proteins that have been ed to achieve one or more of the following goals: (1) produce a multi-antigen HBV construct that comprises less than about 690 amino acids (corresponding to less than two thirds of the HBV ), in order to produce a yeast- based immunotherapeutic clinical product that is compliant with the guidelines of the Recombinant DNA Advisory Committee (RAC), if necessary; (2) e a multi-antigen HBV construct containing a zed number of known T cell epitopes associated with immune responses to acute/self—limiting HBV infections and/or chronic HBV infections; (3) produce a multi—antigen HBV construct containing T cell epitopes that are most conserved among genotypes; and/or (4) produce a multi-antigen HBV construct modified to correspond more closely to one or more consensus sequences, consensus epitopes, and/or epitope(s) from particular genotypes. The modifications demonstrated in this example can be applied individually or together to any other yeast-based immunotherapeutic described or contemplated herein.
In one experiment, a yeast-based immunotherapeutic composition that comprises a yeast expressing a fusion protein meeting the requirements of the goals specified above, and comprising portions of each of the HBV major proteins: HBV surface antigen, polymerase, core and X antigen, was designed. To design this fusion protein, individual HBV antigens within the fusion were d in size (as compared to full-length), and the fusion segments were individually modified to maximize the inclusion of known T cell epitopes corresponding to those identified in Table 5. Inclusion of T cell epitopes in this fusion protein was prioritized as follows: Epitopes identified in immune responses to both acute/self—limiting HBV infectionsw c HBV infections > Epitopes identified in immune responses to acute/self—limiting HBV infections > es identified in immune responses to chronic HBV infections Artificial junctions were also minimized in the design of each t of this fusion protein because, without being bound by theory, it is believed that natural evolution has resulted in: i) contiguous sequences in the virus that express well; and ii) an immunoproteasome in antigen ting cells that can properly digest and present those sequences to the immune system. Accordingly, a fusion n with many unnatural junctions may be less useful in a yeast-based immunotherapeutic as ed to one that retains more of the natural HBV protein sequences.
To uct a segment comprising HBV surface antigen for use in a fusion protein, a full-length large (L) surface antigen protein from HBV genotype C was reduced in size by truncation of the N— and C-terminal sequences ions 1 to 119 and positions 369 to 400 of large antigen were removed, as compared to a fiJll-length L e antigen protein, such as that represented by SEQ ID NO:11). The remaining n was selected, in part, to maximize the inclusion of known MHC Class I T cell epitopes corresponding to those identified in Table 5, using the prioritization for inclusion of T cell epitopes described above. The resulting surface antigen segment is represented by SEQ ID NO:97.
To construct the segment comprising HBV polymerase for use in a fusion protein, substantial portions of a full-length polymerase from HBV genotype C, which is a very large protein of about 842 amino acids, were eliminated by focusing on inclusion of the active site domain (from the RT domain), which is the most conserved region of the protein among HBV genotypes and isolates. The RT domain also includes several sites where drug resistance mutations have been known to occur; thus, this portion of the construct can be further d in other versions, as needed, to target escape mutations of targeted therapy. In fusion proteins including fewer HBV proteins, the size of the polymerase segment can be ed, if desired. The selected portion of the HBV polymerase was included to maximize known T cell epitopes, using the prioritization strategy discussed above. Sequence of full-length polymerase that was ore ated included sequence outside of the RT domain, and sequences within the RT domain that contained no known T cell epitopes, or that included two epitopes identified in less than 17% or 5%, respectively, of genotype A ts where these epitopes were identified (see d et al., 2008 and Table 5). All but one of the remaining T cell epitopes in the HBV polymerase genotype C segment were perfect s to the published epitopes from the genotype A analysis, and the one epitope with a single amino acid mismatch was d to correspond to the published epitope. The resulting HBV polymerase antigen segment is represented by SEQ ID NO:98.
To construct the segment comprising HBV core n for use in a fusion protein, a full-length Core protein (e. g., similar to positions 31-212 of SEQ ID NO:9) from HBV genotype C was modified as follows: i) a single amino acid within a T cell epitope of the genotype C-derived protein was modified to create a perfect match to a known T cell epitope described in Table 5; ii) seven amino acids of the N—terminus, which did not n a T cell epitope, were removed, preserving some flanking amino acids inal to the first known T cell epitope in the protein; and iii) the 24 C terminal amino acids of Core were removed, which does not delete known epitopes, but which does remove an exceptionally positively charged C-terminus. A positively charged C-terminus is a good candidate for removal from an n to be expressed in yeast, as such sequences may, in some constructs, be toxic to yeast by competitive interference with natural yeast RNA binding ns which often are ne rich (positively charged). Accordingly, removal of this portion of Core is acceptable. The resulting HBV Core antigen segment is represented by SEQ ID NO:99.
To construct a segment comprising HBV X antigen for use in a fusion protein, a ength X antigen from HBV genotype C (e.g., r to SEQ ID NO:12) was truncated at the N— and C-terminus to produce a segment of X antigen that includes most of the known T cell epitopes from Table 5, which are clustered in the X n. Two of the epitopes were modified by single amino acid changes to pond to the published T cell epitope sequences, and sequence flanking the T cell es at the ends of the segment was retained to tate efficient processing and presentation of the correct epitopes by an antigen presenting cell. The resulting HBV X antigen segment is represented by SEQ ID NO: 100.
To construct a complete fusion protein containing all four HBV protein segments, the four HBV segments described above were linked (surface-pol-core-X) to form a single n that optimizes the inclusion of T cell epitopes spanning all proteins encoded by the HBV genome, and that is expected to meet criteria for viral proteins for anticipated clinical use.
Two different fusion proteins were tely d, each with a different N- terminal peptide added to enhance and/or stabilize expression of the fusion protein in yeast.
In addition, a hexahistidine peptide was included at the C-terminus to aid with the identification of the protein. As for all of the other proteins used in the yeast-based immunotherapeutic compositions described herein, in additional constructs, the N-terminal peptide of SEQ ID NO:37 or SEQ ID NO:89 utilized in this example can be replaced with WO 09404 a different synthetic N-terminal peptide (e.g., a homologue of SEQ ID NO:37 that meets the same basic structural requirements of SEQ ID NO:37 as described in detail in the specification), or with a homologue of the N—terminal peptide of SEQ ID N0289 or SEQ ID N090, and in another construct, the N—terminal peptide is omitted and a methionine is included at position one. In addition, linker sequences of one, two, three or more amino acids may be added between segments of the fusion n, if desired. Also, while these constructs were designed using HBV ns from genotype C as the ne, any other HBV pe, sub-genotype, or HBV proteins from ent strains or isolates can be used to design these protein segments, as exemplified in Example 7. Finally, if one or more segments are excluded from the fusion protein as described , then the sequence from the remaining segments can be expanded to e onal T cell epitopes and flanking regions of the proteins (e.g., see Example 8).
To produce yeast-based immunotherapeutic compositions comprising a fusion protein constructed of the HBV ts bed above, yeast (e.g, Saccharomyces cerevisiae) are engineered to express various HBV surface-polymerase-core-X fusion proteins, optimized as discussed above, under the control of the -inducible er, CUP] or the TEF2 promoter.
In one construct, the fusion protein is a single polypeptide with the following sequence elements fused in frame from N— to C-terminus, represented by SEQ ID NO: 101: (1) an N—terminal peptide that is an alpha factor prepro sequence, to impart resistance to proteasomal degradation and stabilize expression represented by SEQ ID NO:89 (positions 1-89 of SEQ ID ); (2) an optimized portion of an HBV large (L) surface antigen represented by SEQ ID NO:97 (positions 90 to 338 of SEQ ID NO:101); (3) an optimized portion of the reverse transcriptase (RT) domain of HBV polymerase represented by SEQ ID NO:98 (positions 339 to 566 of SEQ ID NO:101); (4) an optimized portion of HBV Core protein represented by SEQ ID NO:99 (positions 567 to 718 of SEQ ID NO: 101); (5) an optimized portion of HBV X antigen represented by SEQ ID NO: 100 (positions 719 to 778 of SEQ ID NO:101); and (6) a hexahistidine tag (e.g., positions 779 to 784 of SEQ ID NO:101).
In a second construct, the fusion protein is a single polypeptide with the following sequence elements fused in frame from N— to C-terminus, represented by SEQ ID NO:102: (1) an N—terminal peptide that is a synthetic N—terminal peptide designed to impart resistance to proteasomal degradation and ize expression represented by SEQ ID NO:37 (positions 1-6 of SEQ ID NO:102); (2) an optimized portion of an HBV large (L) e antigen represented by positions 2 to 248 of SEQ ID NO:97 ions 7 to 254 of SEQ ID NO:102); (3) an zed portion of the reverse transcriptase (RT) domain of HBV polymerase represented by SEQ ID NO:98 (positions 255 to 482 of SEQ ID NO:102); (4) an optimized portion of HBV Core protein represented by SEQ ID NO:99 (positions 483 to 634 of SEQ ID NO:102); (5) an optimized portion of HBV X antigen represented by SEQ ID NO:100 ions 635 to 694 of SEQ ID NO:102); and (6) a hexahistidine tag (e.g., positions 695 to 700 of SEQ ID NO:102).
Yeast—based immunotherapy itions expressing these fusion proteins are produced using the same protocol described in detail in Example 1-4.
Example 6 The ing example describes the production of additional yeast-based HBV immunotherapeutic compositions that maximize the targeting of HBV genotypes and/or sub-genotypes in conjunction with conserved antigen and/or epitope inclusion within a single composition, in order to e single compositions with the potential to treat a large number of individuals or populations of individuals.
To prepare a construct comprising multiple different pes within the same yeast-based therapeutic, yeast (e.g, Saccharomyces cerevisiae) are engineered to express an HBV fission protein under the control of a suitable promoter, such as the copper-inducible promoter, CUP], or the TEF2 promoter. The n is a single polypeptide comprising four Core antigens, each one from a different genotype (HBV genotypes A, B, C and D), represented by SEQ ID NO:105: 1) an N-terminal methionine at position 1 of SEQ ID NO:105; 2) the amino acid sequence of a near full- length Core protein from HBV genotype A (e.g., positions 31 to 212 of SEQ ID NO] or positions 2 to 183 of SEQ ID NO: 105); 3) the amino acid sequence of a near ength Core protein from HBV genotype B (e.g., positions 30 to 212 of SEQ ID NO:5 or positions 184 to 395 of SEQ ID NO: 105); 4) the amino acid sequence of a near full-length Core protein from HBV genotype C (e.g., positions 30 to 212 of SEQ ID N029 or positions 396 to 578 of SEQ ID NO: 105); 5) the amino acid sequence of a near full-length Core protein from HBV genotype D (e.g., positions 30 to 212 of SEQ ID NO:13 or positions 579 to 761 of SEQ ID NO: 105); and 5) a hexahistidine tag (e.g., ons 762 to 767 of SEQ ID NO: 105). The sequence also ns epitopes or domains that are believed to enhance the immunogenicity of the fusion protein. The N—terminal methionine at position 1 can be substituted with SEQ ID NO:37 or a homologue thereof, or with an alpha prepro sequence of SEQ ID NO:89 or SEQ ID NO:90, or a homologue thereof, or 2012/024409 any other suitable N-terminal sequence if desired. In addition, linker sequences can be ed between HBV proteins to facilitate cloning and manipulation of the construct, if desired. This is an exemplary construct, as any other combination of HBV pes and/or subgenotypes can be substituted into this design as desired to construct a single n yeast-based HBV immunotherapeutic product with broad clinical ability and efficient design for manufacturing. The amino acid sequence of SEQ ID NO:105 also contains several known T cell epitopes, and certain epitopes have been modified to correspond to the published sequence for the given epitope, which can be identified by comparison ofthe sequence to the es shown in Table 5, for e.
To prepare a construct comprising more than one HBV antigen and more than one pe within the same yeast-based therapeutic, yeast (e.g., Saccharomyces siae) are engineered to express an HBV fusion protein under the control of a suitable promoter, such as the copper-inducible promoter, CUP], or the TEF2 promoter. The protein is a single polypeptide comprising two Core antigens and two X antigens, each one of the pair from a different genotype (HBV genotypes A and C), represented by SEQ ID NO:106: 1) an N-terminal methionine at position 1 of SEQ ID NO:106; 2) the amino acid sequence of a near full-length Core protein from HBV genotype A (e.g., positions 31 to 212 of SEQ ID NO] or positions 2 to 183 of SEQ ID NO:106); 3) the amino acid sequence of a full-length X antigen from HBV genotype A (e.g., positions SEQ ID NO:4 or positions 184 to 337 of SEQ ID NO:106); 4) the amino acid sequence of a near full- length Core protein from HBV genotype C (e.g., positions 30 to 212 of SEQ ID N09 or positions 338 to 520 of SEQ ID NO:106); 5) the amino acid sequence of a fiill-length X antigen from HBV genotype C (e.g., SEQ ID NO:8 or positions 521 to 674 of SEQ ID NO:106); and 5) a hexahistidine tag (e.g., positions 675 to 680 of SEQ ID N02106). The sequence also contains epitopes or s that are believed to enhance the immunogenicity of the fusion protein. The N—terminal nine at position 1 can be substituted with SEQ ID NO:37 or a gue thereof, or with an alpha prepro sequence of SEQ ID NO:89 or SEQ ID NO:90, or a homologue thereof. The amino acid sequence of SEQ ID NO:106 also contains several known T cell es, and certain epitopes have been modified to correspond to the published sequence for the given epitope, which can be identified by comparison of the sequence to the epitopes shown in Table 5, for example.
Yeast-based immunotherapy compositions expressing these fusion proteins are produced using the same protocol described in detail in Example 1-4.
Example 7 The following example describes the production of additional yeast-based HBV therapeutic compositions that utilize consensus sequences for HBV genotypes, r maximizing the targeting of HBV pes and/or sub—genotypes in conjunction with conserved antigen and/or e inclusion, in order to provide itions with the potential to treat a large number of individuals or populations of individuals using one composition.
To design several constructs that include HBV segments from each of surface protein, core, polymerase, and X antigen, the fusion protein structure described in e 5 for SEQ ID NO:101 and SEQ ID N02102 (and therefore the subparts of these fusion proteins represented by SEQ ID NO:97 (Surface antigen), SEQ ID NO:98 (Polymerase), SEQ ID NO:99 (Core n), and SEQ ID NO:100 (X antigen)) was used as a template. With reference to consensus sequences for each of HBV genotype A, B, C and D that were built from multiple sources of HBV sequences (e.g., Yu and Yuan et a1, 2010, for S, Core and X, where consensus sequences were generated from 322 HBV sequences, or for Pol (RT), from the Stanford University HIV Drug Resistance Database, HBVseq and HBV Site e Notes), sequences in the template structure were replaced with consensus sequences corresponding to the same positions, unless using the consensus sequence altered one of the known acute imiting T cells epitopes or one of the known polymerase escape on sites, in which case, these positions followed the published sequence for these es or mutation sites. onal antigens could be constructed based solely on consensus sequences or using other published epitopes as they become known.
A first construct based on a consensus sequence for HBV genotype A was designed as follows. Using SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99 and SEQ ID NO:100, which were designed to reduce the size of the fusion segments (as compared to full-length), to maximize the ion of known T cell epitopes corresponding to those identified in Table 5 (priority as discussed above), and to minimize artificial junctions, new fusion segments were d based on a consensus sequence for HBV genotype A.
The new surface antigen segment is represented by positions l-249 of SEQ ID NO:lO7.
The new polymerase (RT) segment is represented by positions 7 of SEQ ID NO:107. The new Core segment is represented by positions 478-629 of SEQ ID N02107.
The new X antigen segment is represented by positions 630-689 of SEQ ID NO: 107. This complete fusion protein is a single polypeptide with the following sequence elements fused in frame from N— to C-terminus, wherein the HBV sequences are represented by SEQ ID NO: 107 (non—HBV sequences denoted as "optional" were not included in the base sequence of SEQ ID NO:lO7, but were actually added to the fusion protein described in this example): (1) an optional N—terminal peptide that is a synthetic inal peptide designed to impart resistance to proteasomal degradation and stabilize expression represented by SEQ ID NO:37; (2) an optional linker peptide of Thr—Ser; (3) an optimized portion of an HBV large (L) surface antigen represented by ons 1 to 249 of SEQ ID NO:lO7, which is a consensus sequence for HBV genotype A utilizing the design strategy discussed above; (4) an optimized portion of the reverse riptase (RT) domain of HBV polymerase represented by positions 250 to 477 of SEQ ID NO:lO7, which is a consensus sequence for HBV genotype A utilizing the design strategy discussed above; (5) an optimized portion of HBV Core protein represented by ons 478 to 629 of SEQ ID NO:lO7, which is a sus sequence for HBV genotype A utilizing the design strategy discussed above; (6) an optimized portion of HBV X antigen represented by positions 630 to 689 of SEQ ID NO:lO7, which is a consensus ce for HBV pe A utilizing the design strategy discussed above; and (7) an optional hexahistidine tag (six histidine residues following position 689 of SEQ ID NO:lO7). A yeast-based immunotherapy ition expressing this complete fusion n is also ed to herein as GI-13010.
The fusion protein and corresponding based immunotherapeutic can also be referred to herein as "SPEXv2-A" or "Spex-A".
A second uct based on a consensus sequence for HBV genotype B was designed as follows. Using SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99 and SEQ ID NO:lOO, which were designed to reduce the size of the fusion ts (as compared to full-length), to maximize the inclusion of known T cell epitopes corresponding to those identified in Table 5 (priority as discussed above), and to minimize artificial junctions, new fusion segments were created based on a consensus sequence for HBV genotype B.
The new surface antigen segment is represented by ons l—249 of SEQ ID NO:lO8.
The new polymerase (RT) segment is represented by positions 250-477 of SEQ ID NO:lO8. The new Core segment is ented by positions 478-629 of SEQ ID NO:lO8.
The new X antigen segment is represented by positions 630-689 of SEQ ID NO: 108. This fusion protein is a single polypeptide with the following sequence elements fused in frame from N— to C-terminus, represented by SEQ ID NO:lO8 (non-HBV sequences denoted as "optional" were not included in the base sequence of SEQ ID , but were actually added to the fusion protein described in this example): (1) an optional N-terminal peptide that is a synthetic N-terminal peptide designed to impart resistance to proteasomal degradation and stabilize expression represented by SEQ ID NO:37; (2) an optional linker peptide of Thr-Ser; (3) an optimized portion of an HBV large (L) surface n represented by ons 1 to 249 of SEQ ID NO:108, which is a consensus sequence for HBV genotype B ing the design strategy discussed above; (4) an optimized portion of the e transcriptase (RT) domain of HBV polymerase represented by ons 250 to 477 of SEQ ID NO: 108, which is a consensus sequence for HBV genotype B utilizing the design gy discussed above; (5) an optimized portion of HBV Core protein represented by positions 478 to 629 of SEQ ID NO:108, which is a consensus sequence for HBV genotype B utilizing the design strategy discussed above; (6) an zed portion ofHBV X n represented by positions 630 to 689 of SEQ ID NO:108, which is a consensus sequence for HBV genotype B utilizing the design strategy discussed above; and (7) an optional hexahistidine tag. A yeast-based immunotherapy composition expressing this complete fusion protein is also referred to herein as GI-l3011. The fusion protein and corresponding yeast-based immunotherapeutic can also be referred to herein as "SPEXv2—B" or "Spex-B".
A third uct based on a consensus sequence for HBV genotype C was designed as follows. Using SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99 and SEQ ID NO:100, which were designed to reduce the size of the fusion segments (as compared to full-length), to maximize the inclusion of known T cell es corresponding to those identified in Table 5 (priority as discussed above), and to minimize ial junctions, new fusion segments were created based on a consensus sequence for HBV genotype C.
The new surface antigen segment is represented by positions l-249 of SEQ ID NO:109.
The new polymerase (RT) segment is represented by positions 250-477 of SEQ ID NO:109. The new Core segment is ented by positions 478-629 of SEQ ID NO:109.
The new X antigen segment is represented by positions 630-689 of SEQ ID NO:109. This fusion protein is a single polypeptide with the ing sequence elements fused in frame from N— to C-terminus, ented by SEQ ID NO:109 (non-HBV sequences denoted as "optional" were not included in the base sequence of SEQ ID NO:109, but were actually added to the fusion n described in this example): (1) an optional N-terminal peptide that is a synthetic N-terminal peptide designed to impart resistance to proteasomal degradation and ize expression represented by SEQ ID N0237; (2) an optional linker peptide of Thr-Ser; (3) an optimized portion of an HBV large (L) surface antigen represented by positions 1 to 249 of SEQ ID NO:109, which is a consensus sequence for HBV pe C utilizing the design strategy discussed above; (4) an optimized portion of the reverse transcriptase (RT) domain of HBV polymerase represented by positions 250 to 477 of SEQ ID NO: 109, which is a consensus sequence for HBV genotype C utilizing the design strategy discussed above; (5) an optimized n of HBV Core protein represented by positions 478 to 629 of SEQ ID NO:109, which is a consensus sequence for HBV genotype C utilizing the design strategy discussed above; (6) an optimized portion ofHBV X antigen represented by positions 630 to 689 of SEQ ID NO:109, which is a consensus sequence for HBV genotype C utilizing the design strategy discussed above; and (7) an al hexahistidine tag. A yeast-based immunotherapy ition expressing this complete fusion protein is also referred to herein as GI-l3012. The fusion protein and corresponding yeast-based immunotherapeutic can also be referred to herein as "SPEXv2-C" or "Spex-C".
A fourth construct based on a consensus sequence for HBV genotype D was designed as follows. Using SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99 and SEQ ID NO:100, which were designed to reduce the size of the fusion segments (as compared to full-length), to maximize the inclusion of known T cell es corresponding to those identified in Table 5 (priority as sed above), and to minimize artificial junctions, new fusion segments were created based on a consensus sequence for HBV genotype D.
The new surface antigen segment is ented by positions 1-249 of SEQ ID NO:110.
The new polymerase (RT) segment is represented by positions 250—477 of SEQ ID NO:110. The new Core t is ented by ons 478-629 of SEQ ID .
The new X n segment is represented by positions 630-689 of SEQ ID NO:110, This fusion protein is a single polypeptide with the following sequence elements fused in frame from N— to C-terminus, represented by SEQ ID NO:110 (non-HBV ces denoted as "optional" were not included in the base ce of SEQ ID NO:110, but were actually added to the fusion protein described in this example): (1) an optional N-terminal peptide that is a synthetic N-terminal peptide designed to impart resistance to proteasomal degradation and stabilize expression represented by SEQ ID NO:37; (2) an optional linker e of Thr-Ser; (3) an optimized portion of an HBV large (L) surface antigen represented by positions 1 to 249 of SEQ ID NO: 110, which is a consensus sequence for HBV genotype D utilizing the design strategy discussed above; (4) an optimized portion of the e transcriptase (RT) domain of HBV polymerase represented by ons 250 to 477 of SEQ ID NO: 110, which is a sus sequence for HBV genotype D utilizing the design strategy discussed above; (5) an optimized portion of HBV Core protein represented by positions 478 to 629 of SEQ ID NO: 110, which is a consensus sequence 2012/024409 for HBV genotype D utilizing the design strategy sed above; (6) an optimized n of HBV X antigen represented by positions 630 to 689 of SEQ ID NO: 110, which is a consensus sequence for HBV genotype D ing the design strategy discussed above; and (7) an optional hexahistidine tag. A yeast-based immunotherapy composition expressing this complete fusion protein is also referred to herein as GI-13013. A yeast— based immunotherapy composition expressing a similar fusion protein (containing SEQ ID NO:110), except that the N—terminal peptide of SEQ ID NO:37 is substituted with the alpha factor sequence of SEQ ID NO:89, is referred to herein as 14. The fusion protein and corresponding yeast-based immunotherapeutic can also be referred to herein as "SPEXv2-D", "Spex-D", or "M-SPEXv2-D" (for GI-13013) or "a-SPEXv2-D" for (GI- 13014).
Additional HBV fusion proteins for use in a yeast-based immunotherapeutic were designed using the application of consensus sequences for four HBV genotypes to demonstrate how alterations similar to those made in the fusion proteins described above (SEQ ID NOs:lO7-110) can be made in a different HBV fusion protein, such as that described by SEQ ID NO:34, which contains HBV Surface proteins and HBV Core ns. To design these additional HBV antigens and corresponding yeast-based immunotherapy compositions, the fusion protein structure described above for SEQ ID NOZ34 (and ore the subparts of these fusion proteins (Surface antigen and Core) was used as a template. As above for the constructs described above, consensus sequences for each of HBV genotype A, B, C and D were built from multiple sources of HBV ces (e.g, Yu and Yuan et al, 2010, for S and Core), and sequences in the template structure were replaced with consensus sequences corresponding to the same positions, unless using the consensus sequence altered one of the known acute imiting T cells es or one of the known polymerase escape mutation sites, in which case, these positions followed the published sequence for these epitopes or on sites.
A first construct based on a consensus sequence for HBV genotype A was designed as follows. Using SEQ ID NO:34 as a template, a new fusion protein was created based on a consensus sequence for HBV genotype A, represented here by SEQ ID . This fusion protein is a single polypeptide with the following ce elements fused in frame from N— to C-terminus, represented by SEQ ID NO:112 (non-HBV sequences denoted as "optional" are not included in the base sequence of SEQ ID NO:112, but were actually added to the fusion protein described in this example): (1) an al N- terminal peptide that is a synthetic N—terminal peptide designed to impart resistance to W0 2012/109404 proteasomal degradation and stabilize expression represented by SEQ ID N037; (2) an optional linker peptide of Thr-Ser; (3) a consensus sequence for HBV genotype A large (L) surface antigen represented by positions 1 to 399 of SEQ ID NO:112; 4) the amino acid sequence of a consensus sequence for HBV genotype A core antigen ented by ons 400 to 581 of SEQ ID NO:112; and (5) an optional hexahistidine tag. A c acid sequence encoding the fusion protein comprising SEQ ID NO:112 (codon optimized for yeast expression) is represented herein by SEQ ID . A yeast-based immunotherapy composition expressing this fusion protein is also referred to herein as GI- 13006. The fusion protein and corresponding yeast-based immunotherapeutic can also be referred to herein as —A".
A second uct based on a consensus sequence for HBV genotype B was designed as follows. Using SEQ ID NO:34 as a template, a new fusion protein was created based on a consensus sequence for HBV genotype B, ented here by SEQ ID . This fusion protein is a single polypeptide with the following sequence elements fused in frame from N- to C—terminus, represented by SEQ ID NO:1 l4 (non—HBV sequences denoted as "optional" are not included in the base sequence of SEQ ID NO:114, but were actually added to the fusion protein described in this example): (1) an optional N— terminal peptide that is a synthetic N—terminal peptide designed to impart resistance to proteasomal degradation and stabilize sion represented by SEQ ID NO:37; (2) an optional linker peptide of Thr-Ser; (3) a consensus sequence for HBV genotype B large (L) surface antigen represented by positions I to 399 of SEQ ID NO:114; 4) the amino acid sequence of a consensus sequence for HBV genotype B core antigen ented by positions 400 to 581 of SEQ ID NO:114; and (5) an optional hexahistidine tag. A nucleic acid sequence ng the fusion protein comprising SEQ ID NO:114 (codon optimized for yeast sion) is represented herein by SEQ ID NO:113. A based immunotherapy composition expressing this fusion protein is also referred to herein as GI- 13007. The fusion protein and corresponding based immunotherapeutic can also be referred to herein as "Score-B".
A third construct based on a consensus sequence for HBV genotype C was designed as follows. Using SEQ ID NO:34 as a template, a new fusion protein was created based on a consensus sequence for HBV genotype C, represented here by SEQ ID NO:116. This fusion protein is a single polypeptide with the following sequence elements fused in frame from N- to C-terminus, represented by SEQ ID NO:116 (non-HBV sequences denoted as "optional" are not included in the base ce of SEQ ID NO:116, but were actually added to the fusion n described in this example): (1) an optional N- terminal e that is a synthetic inal peptide designed to impart resistance to proteasomal degradation and stabilize expression represented by SEQ ID N0237; (2) an optional linker peptide of Thr-Ser; (3) a consensus sequence for HBV genotype C large (L) surface antigen represented by ons 1 to 399 of SEQ ID NO:116; 4) the amino acid sequence of a consensus sequence for HBV genotype C core antigen represented by positions 400 to 581 of SEQ ID NO:116; and (5) an optional hexahistidine tag. A nucleic acid sequence encoding the fusion protein comprising SEQ ID NO:116 (codon optimized for yeast sion) is ented herein by SEQ ID NO:115. A yeast-based immunotherapy composition expressing this fusion protein is also referred to herein as GI— 13008. The fusion protein and corresponding yeast-based immunotherapeutic can also be referred to herein as "Score-C".
] A fourth construct based on a sus sequence for HBV genotype D was designed as follows. Using SEQ ID NO:34 as a template, a new fusion protein was created based on a consensus sequence for HBV genotype D, represented here by SEQ ID NO:118. This fusion protein is a single polypeptide with the following sequence elements fused in frame from N— to C-terminus, represented by SEQ ID NO:118 (non-HBV sequences denoted as "optional" are not included in the base ce of SEQ ID NO:118, but were actually added to the fusion protein described in this example): (1) an optional N— terminal peptide that is a synthetic N—terminal peptide designed to impart resistance to proteasomal ation and stabilize expression represented by SEQ ID NO:37; (2) an optional linker peptide of Thr-Ser; (3) a consensus sequence for HBV genotype D large (L) surface n represented by ons 1 to 399 of SEQ ID NO:118; 4) the amino acid sequence of a consensus sequence for HBV genotype D core n represented by positions 400 to 581 of SEQ ID NO:118; and (5) an optional hexahistidine tag. The amino acid sequence of the complete fusion protein comprising SEQ ID NO:118 and the N— and C-terminal peptides and linker peptide is represented herein by SEQ ID NO:151. A nucleic acid sequence encoding the fusion protein comprising SEQ ID NO:118 or SEQ ID NO:151 (codon optimized for yeast expression) is represented herein by SEQ ID NO:117.
A yeast-based therapy composition expressing this fusion protein is also ed to herein as 09. The fusion proteins and corresponding based immunotherapeutic can also be referred to herein as "Score-D".
The yeast-based immunotherapy compositions of GI-13010 (comprising SEQ ID NO:107), GI-13011 (comprising SEQ ID NO:108), GI-13012 (comprising SEQ ID WO 09404 NO:109), GI—l3013 (comprising SEQ ID NO:110), GI—l3006 (comprising SEQ ID ), GI-l3007 (comprising SEQ ID NO:114), GI-l3008 (comprising SEQ ID NO:116) and 09 (comprising SEQ ID ) were produced as described for other compositions above. Briefly, DNA encoding the fusion protein was codon optimized for expression in yeast and then inserted behind the CUPI promoter (pGI—lOO) in yeast 2 um expression vectors. The resulting plasmids were introduced into Saccharomyces cerevisiae W3030L yeast by Lithium acetate/polyethylene glycol transfection. Yeast transformants of each plasmid were isolated on solid minimal plates g uracil (UDM; uridine dropout medium). Colonies were re-streaked onto ULDM (uridine and leucine dropout medium) and allowed to grow for 3 days at 30°C. Liquid starter cultures g uridine and leucine (UL2; formulation provided in Example 1) were inoculated from plates and starter cultures were grown for 18h at 30°C, 250 rpm.
Primary cultures were used to inoculate final cultures ofUL2 and growth continued until a density of 2 YU/mL was reached. Cultures were induced with 0.5 mM copper sulfate for 3h and then cells were washed in PBS, heat-killed at 56°C for lh, and washed three times in PBS. Total protein t was measured by a TCA precipitation/nitrocellulose binding assay and HBV n expression was measured by western blot using an anti-his tag monoclonal antibody.
The results are shown in Fig. 20. The lanes in the blot shown in Fig. 20 contain protein from the ing yeast-based immunotherapeutics: Lane 1 (v1.0; Score) = GI-l3002 (expressing SEQ ID NO:34); Lane 2 (v2.0; ScA) = GI-l3006 (expressing SEQ ID NOzllZ); Lane 3 (v2.0; ScB) = GI-l3007 (expressing SEQ ID NO:114), Lane 4 (v2.0; ScC) = 08 (expressing SEQ ID ); Lane 5 (v2.0; ScD) = GI-l3009 (expressing SEQ ID NO:118); Lane 6 (v1.0; Sp) = GI-13005 (expressing SEQ ID N0236); Lane 7 (v1.0; a-Sp) = GI-l3004 (expressing SEQ ID NO:92); Lane 8 (v2.0; SpA) = GI- 13010 (expressing SEQ ID NO:107); Lane 9 (v2.0; SpB) = GI-l3011 (expressing SEQ ID NO:108); Lane 10 (v2.0; SpC) = 12 (expressing SEQ ID NO:109), Lane 11 (v2.0; SpD) = GI-l3013 (expressing SEQ ID NO:110).
The results show that each of the HBV antigens comprising the combination of surface antigen and core e" antigens), i.e., GI-l3002 (Score), 06 (ScA; Score-A), GI-13007 (ScB; Score-B), GI-l3008 (ScC; Score-C), and GI-l3009 (ScD; Score-D) expressed ly in yeast. Typical Score v2.0 expression levels in these and similar experiments were in the range of approximately 90 to 140 pmol/YU (116., 5940 ng/YU to 9240 ng/YU). Expression levels of the HBV antigens comprising all four HBV proteins (surface, polymerase, core and X, or "Spex") was le. Specifically, expression of the antigens from 10 (SpA; Spex-A), GI-l3011 (SpB; Spex-B), GI- 13012 (SpC; Spex-D) and GI—13013 (SpD; Spex—D) was substantially lower than expression of the "Score" antigens, as well as the antigens from GI—l3005 (Sp; Spex) and GI-13004 (a-Sp; ). Expression of the antigen in GI—l3012 (SpC; Spex-C) was barely detectable. Taken together, these results indicate that as a group, HBV antigens comprising surface antigen and core express very well in yeast, whereas HBV antigens comprising all of surface antigen, polymerase, core and X have variable expression in yeast, and generally express less well than the " antigens.
Example 8 The following example describes the tion of additional yeast-based HBV immunotherapeutic compositions that utilize consensus sequences for HBV genotypes, and additionally demonstrate the use of alternate configurations/arrangements of HBV protein ts within a fusion n in order to modify or improve the expression of an HBV antigen in yeast and/or improve or modify the immunogenicity or other functional ute of the HBV antigen.
In this example, new fusion proteins were designed that append X antigen and/or rase antigens to the N— or C-terminus of the combination of e antigen fused to core. These constructs were designed in part based on the rationale that because the fusion proteins arranged in the configuration generally referred to herein as "Score" (e.g., SEQ ID NO:34, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116 and SEQ ID NO:118) express very well in yeast, it may be advantageous to utilize this base configuration (i.e., surface n fused to core protein) to produce HBV antigens comprising additional HBV protein components. Such strategies may improve expression of protein antigens and/or e or modify the functionality of such antigens in the context of immunotherapy. For example, without being bound by theory, the inventors proposed that the expression of an HBV antigen using three or all four HBV proteins could be improved by constructing the fusion protein using surface-core (in order) as a base, and then appending the other antigens to this construct.
Accordingly, to exemplify this ment of the invention, eight new fusion proteins were designed and constructed, and yeast—based immunotherapy products expressing these proteins were produced. In each case, the fusion protein used a surface- core fusion protein as a base that was d from segments of the fusion protein represented by SEQ ID NO:118, which is a surface-core fusion n described in Example 7 utilizing a sus sequence for HBV genotype D and optimized to maximize the use of conserved immunological epitopes. All possible arrangements of a polymerase segment and/or an X n segment were appended to this base ration, utilizing segments derived from the fitsion protein represented by SEQ ID NO:110, which is a multi-protein HBV fusion protein described in e 7 that was constructed to reduce the size of the protein segments, maximize the use of conserved immunological epitopes, and utilize a consensus sequence for HBV genotype D. While these eight resulting antigens are based on a consensus sequence for HBV genotype D, it would be straightforward to produce a fusion protein having a similar l structure using the corresponding fusion segments from the fusion proteins represented by SEQ ID NO:107 and/or SEQ ID NO:112 (genotype A), SEQ ID NO:108 and/or SEQ ID NO:114 (genotype B), SEQ ID NO:109 and/or SEQ ID NO:116 (genotype C), or using the corresponding sequences from a different HBV genotype, sub-genotype, consensus sequence or .
To produce the first composition, yeast (e.g, Saccharomyces cerevisiae) were engineered to express a new HBV fusion protein, schematically illustrated in Fig. 8, under the control of the copper—inducible promoter, CUPI . The resulting yeast-HBV immunotherapy composition can be referred to herein as GI-13015. This fusion protein, also referred to herein as "Score-Pol" and represented by SEQ ID NO:120, comprises, in order, surface antigen, core protein, and polymerase ces, as a single polypeptide with the following sequence elements fused in frame from N— to C-terminus BV sequences denoted as "optional" were not included in the base sequence of SEQ ID NO:120, but were actually added to the fusion protein described in this example): (1) an optional N—terminal peptide that is a synthetic N—terminal peptide ed to impart ance to proteasomal degradation and stabilize expression represented by SEQ ID NO:37; (2) an optional linker e of Thr—Ser; (3) the amino acid sequence of a near full-length (minus position 1) consensus sequence for HBV genotype D large (L) surface antigen represented by positions 1 to 399 of SEQ ID NO: 120 (corresponding to positions 1 to 399 of SEQ ID NO:118); (4) the amino acid sequence of a consensus sequence for HBV genotype D core n represented by positions 400 to 581 of SEQ ID NO:120 (corresponding to positions 400 to 581 of SEQ ID NO:118); (5) an optimized portion of the reverse transcriptase (RT) domain of HBV polymerase using a consensus ce for HBV genotype D, represented by positions 582 to 809 of SEQ ID NO:120 (corresponding to positions to 250 to 477 of SEQ ID NO:110); and (6) an optional hexahistidine tag.
SEQ ID NO:120 contains multiple T cell epitopes (human and murine), which can be found in Table 5. A nucleic acid ce encoding the fusion protein of SEQ ID NO:120 (codon-optimized for expression in yeast) is represented herein by SEQ ID NO:119.
] To produce the second composition, yeast (e.g., romyces cerevisiae) were engineered to express a new HBV fusion protein, schematically rated in Fig. 9, under the control of the copper-inducible promoter, CUP]. The resulting yeast-HBV immunotherapy composition can be ed to herein as GI-13016. This fusion protein, also referred to herein as "Score-X" and represented by SEQ ID , comprises, in order, surface antigen, core, and X antigen ces, as a single polypeptide with the following sequence elements fused in frame from N- to C-terminus (non-HBV sequences denoted as "optional" were not included in the base sequence of SEQ ID NO:122, but were actually added to the fusion protein described in this example): (1) an optional N— terrninal e that is a synthetic N—terminal peptide designed to impart resistance to proteasomal degradation and stabilize expression represented by SEQ ID NO:37; (2) an optional linker peptide of Thr-Ser; (3) the amino acid sequence of a near full-length (minus position 1) consensus sequence for HBV pe D large (L) surface antigen represented by positions 1 to 399 of SEQ ID NO:122 (corresponding to positions 1 to 399 of SEQ ID NO:118); 4) the amino acid sequence of a consensus sequence for HBV genotype D core antigen represented by positions 400 to 581 of SEQ ID NO:122 (corresponding to positions 400 to 581 of SEQ ID NO:118); (5) an optimized portion of HBV X antigen using a consensus sequence for HBV genotype D, represented by positions 582 to 641 of SEQ ID NO:122 (corresponding to ons 630 to 689 of SEQ ID NO:110); and (6) an al hexahistidine tag. SEQ ID NO:122 contains le T cell epitopes (human and murine), which can be found in Table 5. A nucleic acid sequence encoding the fusion protein comprising SEQ ID NO:122 (codon-optimized for expression in yeast) is represented herein by SEQ ID NO: 121.
To produce the third composition, yeast (e.g., Saccharomyces cerevisiae) were engineered to express a new HBV fusion protein, schematically illustrated in Fig. 10, under the control of the copper-inducible promoter, CUPI. The resulting yeast-HBV immunotherapy ition can be referred to herein as GI-13017. This fusion protein, also referred to herein as "Score-Pol-X" and represented by SEQ ID NO:124 comprises, in order, surface antigen, core, polymerase and X antigen sequences, as a single polypeptide with the following ce ts fused in frame from N— to C-terminus (non-HBV sequences denoted as "optional" were not included in the base sequence of SEQ ID NO:124, but were actually added to the fusion protein described in this example): (1) an optional N—terminal peptide that is a synthetic N-terminal peptide designed to impart ance to proteasomal degradation and stabilize expression represented by SEQ ID NO:37; (2) an optional linker peptide of Thr-Ser; (3) the amino acid ce of a near ength (minus position 1) consensus sequence for HBV genotype D large (L) surface antigen represented by positions 1 to 399 of SEQ ID NO: 124 (corresponding to positions 1 to 399 of SEQ ID NO:118); 4) the amino acid ce of a consensus sequence for HBV genotype D core antigen represented by positions 400 to 581 of SEQ ID NO:124 (corresponding to positions 400 to 581 of SEQ ID NO:118); (5) an optimized n of the reverse transcriptase (RT) domain of HBV polymerase using a consensus sequence for HBV genotype D, represented by positions 582 to 809 of SEQ ID NO:124 (corresponding to positions to 250 to 477 of SEQ ID NO:110); (6) an optimized portion of HBV X n using a consensus ce for HBV pe D, represented by positions 810 to 869 of SEQ ID NO:124 (corresponding to positions 630 to 689 of SEQ ID NO:110); and (7) an optional hexahistidine tag. SEQ ID NO:124 contains multiple T cell epitopes (human and murine), which can be found in Table 5. A nucleic acid sequence encoding the fusion protein comprising SEQ ID NO:124 (codon-optimized for expression in yeast) is represented herein by SEQ ID NO: 123.
To produce the fourth composition, yeast (e.g., Saccharomyces cerevisiae) were engineered to express a new HBV fusion protein, schematically rated in Fig. 11, under the control of the copper-inducible promoter, CUP]. The resulting yeast-HBV immunotherapy ition can be referred to herein as GI-13018. This fusion protein, also referred to herein as "Score-X-Pol" and represented by SEQ ID NO: 126 comprises, in order, surface antigen, core, X antigen, and polymerase sequences, as a single polypeptide with the ing sequence elements fused in frame from N— to C-terminus BV sequences denoted as "optional" were not included in the base sequence of SEQ ID NO:126, but were actually added to the fusion protein described in this example): (1) an optional N—terminal peptide that is a synthetic N-terminal peptide designed to impart resistance to proteasomal degradation and ize expression ented by SEQ ID NO:37; (2) an optional linker peptide of Thr-Ser; (3) the amino acid sequence of a near ength (minus position 1) consensus sequence for HBV genotype D large (L) surface antigen represented by positions 1 to 399 of SEQ ID NO:126 (corresponding to positions 1 to 399 of SEQ ID NO:118); 4) the amino acid sequence of a consensus sequence for HBV genotype D core antigen represented by positions 400 to 581 of SEQ ID NO:126 2012/024409 (corresponding to positions 400 to 581 of SEQ ID NO:118); (5) an optimized portion of HBV X antigen using a consensus sequence for HBV genotype D, represented by positions 582 to 641 of SEQ ID NO:126 (corresponding to positions 630 to 689 of SEQ ID NO:110); (5) an optimized portion of the e transcriptase (RT) domain of HBV polymerase using a consensus sequence for HBV genotype D, represented by positions 642 to 869 of SEQ ID NO:126 (corresponding to positions to 250 to 477 of SEQ ID NO:1 10); and (7) an al stidine tag. SEQ ID NO:126 contains multiple T cell epitopes (human and murine), which can be found in Table 5. A nucleic acid sequence encoding the fusion protein comprising SEQ ID NO:126 (codon-optimized for expression in yeast) is represented herein by SEQ ID NO: 125.
] To produce the fifth composition, yeast (e.g, Saccharomyces cerevisiae) were engineered to express a new HBV fusion protein, tically illustrated in Fig. 12, under the control of the copper-inducible promoter, CUP]. The resulting yeast-HBV immunotherapy composition can be referred to herein as GI-l3019. This fusion protein, also ed to herein as core" and represented by SEQ ID NO:128 comprises, in order, polymerase, surface antigen, and core sequences, as a single polypeptide with the following sequence elements fused in frame from N- to C-terminus (non-HBV sequences denoted as "optional" were not included in the base sequence of SEQ ID NO: 128, with the ion of the Leu-Glu linker between the polymerase segment and the surface antigen segment in the construct exemplified here, but were ly added to the fusion protein described in this example): (1) an al N—terminal peptide that is a synthetic N- terminal peptide designed to impart resistance to proteasomal ation and stabilize sion represented by SEQ ID NO:37; (2) an optional linker peptide of Thr-Ser; (3) an optimized portion of the reverse transcriptase (RT) domain of HBV polymerase using a consensus sequence for HBV genotype D, represented by positions 1 to 228 of SEQ ID NO:120 (corresponding to positions to 250 to 477 of SEQ ID ); (4) a linker peptide (optional) of u, represented by positions 229 to 230 of SEQ ID NO: 128; (5) the amino acid sequence of a near full-length (minus position 1) consensus sequence for HBV genotype D large (L) surface antigen represented by positions 231 to 629 of SEQ ID NO:128 (corresponding to positions 1 to 399 of SEQ ID NO:118); (6) the amino acid ce of a consensus sequence for HBV genotype D core antigen represented by positions 630 to 811 of SEQ ID NO:128 (corresponding to positions 400 to 581 of SEQ ID NO:118); and (7) an optional hexahistidine tag. SEQ ID NO:128 contains multiple T cell epitopes (human and murine), which can be found in Table 5. A nucleic acid sequence encoding the fusion protein comprising SEQ ID NO:128 (codon-optimized for expression in yeast) is represented herein by SEQ ID NO:127.
To produce the sixth composition, yeast (e.g., Saccharomyces cerevisiae) were engineered to express a new HBV fusion protein, schematically illustrated in Fig. 13, under the control of the copper-inducible promoter, CUP]. The resulting yeast-HBV immunotherapy composition can be ed to herein as GI—13020. This fusion protein, also referred to herein as re" and represented by SEQ ID NO:130 comprises, in order, X antigen, surface antigen, and core ces, as a single polypeptide with the following sequence elements fused in frame from N— to C-terminus, (non-HBV sequences denoted as "optional" were not included in the base sequence of SEQ ID NO: 130, with the exception of the Leu—Glu linker between the X segment and the surface antigen segment in the construct exemplified here, but were actually added to the fusion protein described in this example): (1) an al N-terminal peptide that is a synthetic N-terminal peptide designed to impart resistance to proteasomal degradation and stabilize expression represented by SEQ ID NO:37; (2) an optional linker peptide of Thr-Ser; (3) an optimized portion of HBV X antigen using a sus sequence for HBV genotype D, represented by positions 1 to 60 of SEQ ID NO:130 (corresponding to positions 630 to 689 of SEQ ID NO:110); (4) a linker peptide (optional) of Leu—Glu, represented by ons 61 to 62 of SEQ ID NO:130; (5) the amino acid sequence of a near full-length (minus on 1) consensus sequence for HBV pe D large (L) surface antigen represented by ons 63 to 461 of SEQ ID NO:130 (corresponding to ons 1 to 399 of SEQ ID NO:118); (6) the amino acid sequence of a consensus sequence for HBV genotype D core antigen represented by positions 462 to 643 of SEQ ID NOzl30 (corresponding to positions 400 to 581 of SEQ ID N02118); and (7) an optional hexahistidine tag. SEQ ID NO:130 ns multiple T cell epitopes (human and murine), which can be found in Table 5. The amino acid sequence of the complete fusion protein comprising SEQ ID NO:130 and the N- and inal peptides and linkers is represented herein by SEQ ID NOzlSO. A nucleic acid sequence encoding the fiasion protein sing SEQ ID NO:130 or SEQ ID NO:150 (codon-optimized for expression in yeast) is represented herein by SEQ ID NO:129.
To produce the seventh composition, yeast (e.g., romyces cerevisiae) were engineered to s a new HBV fusion n, schematically illustrated in Fig. 14, under the control of the copper-inducible promoter, CUP]. The resulting yeast-HBV immunotherapy composition can be referred to herein as GI—1302l. This fusion protein, also referred to herein as "Pol-X-Score" and ented by SEQ ID NOzl32 comprises, in order, polymerase, X antigen, surface antigen, and core, as a single polypeptide with the following sequence elements fused in frame from N- to C-terminus (non-HBV sequences denoted as "optional" were not included in the base sequence of SEQ ID NO:l32, but were actually added to the fusion protein described in this example): (1) an optional N— terrninal peptide that is a synthetic N—terminal peptide designed to impart resistance to proteasomal degradation and stabilize expression represented by SEQ ID NO:37; (2) an optional linker peptide of Thr-Ser; (3) an optimized portion of the reverse transcriptase (RT) domain of HBV polymerase using a consensus sequence for HBV genotype D, represented by positions 1 to 228 of SEQ ID NO:132 (corresponding to positions to 250 to 477 of SEQ ID NO:110); (4) an optimized portion ofHBV X antigen using a consensus ce for HBV pe D, represented by positions 229 to 288 of SEQ ID NO:132 (corresponding to positions 630 to 689 of SEQ ID NO:1 10); (5) the amino acid sequence of a near full-length (minus position 1) consensus sequence for HBV genotype D large (L) surface antigen represented by positions 289 to 687 of SEQ ID NOzl32 (corresponding to positions 1 to 399 of SEQ ID NOzll8); (6) the amino acid sequence of a sus ce for HBV genotype D core antigen ented by positions 688 to 869 of SEQ ID NO:132 (corresponding to positions 400 to 581 of SEQ ID NO:118); and (7) an optional stidine tag. SEQ ID NO: 132 contains multiple T cell epitopes (human and murine), which can be found in Table 5. A nucleic acid sequence encoding the fusion protein comprising SEQ ID NO:l32 (codon-optimized for expression in yeast) is represented herein by SEQ ID NO: 131.
To produce the eighth composition, yeast (e.g., romyces cerevisiae) were engineered to express a new HBV fusion protein, schematically rated in Fig. 15, under the control of the copper-inducible promoter, CUP]. The resulting HBV immunotherapy composition can be referred to herein as GI-13022. This fusion protein, also referred to herein as "X-Pol-Score" and represented by SEQ ID NOzl34 comprises, in order, X antigen, polymerase, e antigen, and core protein, as a single ptide with the following sequence elements fused in frame from N- to C-terminus (non-HBV sequences d as "optional" were not included in the base sequence of SEQ ID NO:134, but were actually added to the fusion protein described in this example): (1) an optional N—terminal peptide that is a tic N-terminal peptide designed to impart resistance to proteasomal degradation and stabilize expression represented by SEQ ID NO:37; (2) an optional linker peptide of Thr-Ser; (3) an optimized portion of HBV X antigen using a sus sequence for HBV genotype D, represented by positions 1 to 60 of SEQ ID NO:134 (corresponding to positions 630 to 689 of SEQ ID NO:110); (4) an optimized portion of the reverse transcriptase (RT) domain of HBV polymerase using a sus sequence for HBV genotype D, represented by positions 61 to 288 of SEQ ID NO:134 (corresponding to positions to 250 to 477 of SEQ ID ); (5) the amino acid sequence of a near full-length (minus position 1) consensus sequence for HBV genotype D large (L) surface antigen represented by positions 289 to 687 of SEQ ID NO:134 (corresponding to positions 1 to 399 of SEQ ID NO:118); (6) the amino acid ce of a consensus sequence for HBV pe D core antigen represented by ons 688 to 869 of SEQ ID NO: 134 (corresponding to positions 400 to 581 of SEQ ID NO:118); and (7) an optional hexahistidine tag. SEQ ID NO: 134 contains multiple T cell epitopes (human and murine), which can be found in Table 5. A nucleic acid sequence encoding the fusion protein comprising SEQ ID NO:134 (codon-optimized for expression in yeast) is represented herein by SEQ ID NO: 133.
To produce each of the yeast—based immunotherapy compositions described above, yeast transformants of each plasmid were isolated on solid l plates lacking uracil (UDM; e dropout medium). Colonies were re—streaked onto ULDM and UDM plates and allowed to grow for 3 days at 300C. Liquid r es lacking uridine and leucine (UL2) or lacking e (U2) were inoculated from plates and starter cultures were grown for 18h at 30°C, 250 rpm. Primary cultures were used to inoculate intermediate cultures of U2 or UL2 and growth was continued until a density of approximately 2 YU/mL was reached. Intermediate cultures were used to inoculate final cultures to a density of 0.05 YU/mL and these were incubated until the cell density reached l-3 YU/mL. Final cultures were then induced with 0.5 mM copper sulfate for 3h and cells were washed in PBS, heat killed at 56°C for 1h, and washed three times in PBS.
Total protein content was measured with a TCA precipitation/nitrocellulose g assay and HBV antigen expression was measured by Western blot using an anti—his tag monoclonal antibody. Lysates from two yeast immunotherapeutic itions described in Example 7 as GI-13008 (SEQ ID NO:116; "Score-C") or GI-13009 (SEQ ID NO:118; "Score-D") were used as a basis of comparison to a yeast expressing the base surface-core antigen product.
Fig. 21 is a blot showing the expression of all eight constructs in yeast cultured in UL2 medium (1 ug of protein loaded) as compared to expression of the construct in the yeast immunotherapeutic described in e 7 as GI-13009 (SEQ ID NO:118; "Score-D"). Referring to Fig. 21, lanes 1 and 2 contain molecular weight markers, and lanes 4-6 contain recombinant hexahistidine tagged NS3 protein that was processed on the same blot in order to quantify antigen by interpolation from a standard curve generated from these lanes. Lanes 7-14 contain lysates from each yeast-based immunotherapeutic d by number (e.g., GI-13015) grown in UL2 medium, and lane ns the lysate from the GI-13009 comparison. Additional n blots from yeast cultured in U2 medium, as well as additional blots evaluating different amounts of protein loading on the gel are not shown here, but overall, the results indicated that all eight antigens were expressed to detectable levels in at least one growth medium.
The overall expression results are summarized in Fig. 22 as a bar graph for those cultures that had detectable expression of target antigen in U2 or UL2 medium as compared to expression of antigens in GI-13008 (Score-C) and GI-13009 -D).
Referring to Fig. 22, the HBV antigens are denoted below each bar using the reference to antigen arrangement in the fusion protein as described for each construct above, along with the medium used to culture the corresponding yeast that expressed the n (i.e., "Pol-Score-U2" refers to the HBV antigen that is a polymerase-surface—core fusion protein, represented by SEQ ID N02128 and expressed by GI—l3019 in U2 ). The results indicated that expression of the antigen denoted "X-Score" (expressed by GI-13020; SEQ ID NO: 130) was particularly robust, at ~122 pmol/YU, which was approximately 79—80% of the expression level obtained for Score-C (GI-13008) or Score—D (GI-13009) on a molar basis r medium). Expression of the antigens expressed by GI-l3015 (Score- Pol; SEQ ID NOzl20), GI-l3016 (Score-X; SEQ ID NO:122), GI-l3017 (Score-Pol-X; SEQ ID ) and GI-l3018 -X-Pol; SEQ ID NO:126) in U2 medium was below the level of quantification in this ment, although each of these antigens were expressed when the same yeast-based immunotherapeutic was grown in UL2 medium (see Fig. 22). In general, antigen configurations containing the rase reverse transcriptase (RT) domain accumulated to lower levels than those containing only S-core with or without the addition of X antigen. Taking the data shown in this and prior Examples as a whole, the antigen rations of surface-core ("Score" or "SCORE"; all similar constructs) and X-surface-core ("X-Score" or "X-SCORE"; GI-l3020) were the t expressing antigen configurations among all based HBV immunotherapeutics tested.
Example 9 WO 09404 The following example describes preclinical experiments in mice to demonstrate the safety, immunogenicity, and in viva efficacy of based HBV immunotherapy compositions of the ion.
To te the based HBV immunotherapy compositions in preclinical studies, a variety of in vitra and in viva assays that detect induction of antigen-specific lymphocytes by yeast-based HBV therapy compositions of the ion were employed, including lymphocyte proliferation, cell-mediated cytotoxicity, cytokine secretion, and protection from tumor challenge (e.g., killing of tumors engineered to express HBV proteins in viva).
To support these studies, based HBV immunotherapy compositions described in Examples 1 and 2 were used initially, with onal studies performed using yeast-based HBV immunotherapy compositions bed in 7 and 8 or elsewhere herein.
However, these studies can be readily applied to any yeast-based HBV therapy composition of the invention, and the results ed herein can be extrapolated to other HBV compositions comprising the same antigen base or similar antigen constructs. The results of these initial experiments are described below.
As a general protocol that can be adapted for any yeast-based HBV immunotherapy composition, mice (e.g, female BALB/c and/or C57BL/6 mice) are injected with a suitable amount of a yeast-based HBV immunotherapy composition, e.g, 4—5 YU (administered subcutaneously in 2-2.5 YU injections at 2 different injection sites).
Optionally, an injection of anti-CD40 antibody is administered the day ing the yeast compositions. Mice are immunized weekly or biweekly, for 1, 2, or 3 doses, and a final booster dose is optionally administered 3-4 weeks after the last weekly or biweekly dose.
Mice are sacrificed 7-9 days after the final injection. Spleen cell suspensions, and/or lymph node suspensions, pooled fiom each group, are prepared and subjected to in vitra stimulation (IVS) conditions utilizing HBV-specific stimuli in the form of HBV peptides and/or HBV antigens, which may include yeast expressing HBV antigens. Control cultures are stimulated with non-HBV peptides, which can include an ovalbumin peptide, or a non-relevant viral peptide (e.g, a peptide from HIV). Standard assays are employed to evaluate immune responses induced by administration of yeast-based HBV immunotherapy compositions and include lymphocyte proliferation as assessed by 3H— thymidine incorporation, cell-mediated xicity assays (CTL ) employing 51Cr- d target cells (or other targets labeled for overnight CTL), quantification of cytokine ion by cytokine assay or ELISPOT (e.g., IFN—y, IL-12, TNF-(x, 1L-6, and/or IL-2, WO 09404 2012/024409 etc.), and protection from tumor nge (e.g., in viva challenge with tumor cells recombinantly ered to express HBV ns).
Yeast-based HBV immunotherapy compositions are expected to be immunogenic as demonstrated by their ability to elicit HBV antigen—specific T cell responses as measured by the assays described above.
In initial experiments, two of the yeast-based HBV immunotherapy products described in Examples 1 and 2 were tested in lymphocyte proliferation assays (LPA) to determine whether immunization with these products elicits antigen-specific CD4+ T cell proliferation. More specifically, the yeast-based immunotherapy product (GI-13002) expressing a fusion protein represented by SEQ ID NO:34 under the control of the CUP] promoter, also known as "SCORE" and more specifically described in Example 1 above, and the yeast—based therapy product (GI—13004) expressing a fusion protein represented by SEQ ID NO:92 under the control of the CUP] promoter and also known as "a—SPEX" and more specifically described in Example 2, were each used to immunize mice and evaluate CD4+ T cells c for the surface and/or Core antigens that are targeted in both products using lymphocyte proliferation assays (LPAs).
Female BALB/c mice were immunized three times weekly with 5 YU of " or a-SPEX subcutaneously at 2 different sites on the mouse (2.5 YU/flank).
Control mice were vaccinated with empty vector yeast (denoted "YVEC") or nothing (denoted "Na'1've"). One week after the third immunization, mice were humanely ced and spleens and periaortal and inguinal draining lymph nodes (LNs) were removed and sed to single cell suspensions. LN cells from the two types of nodes were pooled and stimulated in vitro (IVS) with a mixture of recombinant core and surface antigen ("S/Core mix") or a class II restricted mimetope peptide (GYHGSSLY, SEQ ID NO:103, denoted "Class II SAg mimetope peptide"), previously reported to elicit proliferation of T cells from SAg-immunized BALB/c mice hyaksha et al (1995).
PNAS 92: 1575-1579).
Spleen cells were subjected to CD4+ T cell enrichment by Magnetic Activated Cell Sorting (MACS) and incubated with the same antigens as described for LN. After 4 days incubation, IVS cultures were pulsed with tritiated (3H) thymidine for 18h, and cellular DNA was harvested on glass fiber microfilters. The level of incorporated 3H— thymidine was measured by llation counting. Replicate LN cultures from SCORE- immunized mice were assayed in parallel. eron gamma (IFN-y) production by ELISpot was used as an additional means to assess T cell activation.
TIS510927NZPR compared to the placebo controlled group. Patients receiving yeast-based HBV immunotherapy are expected to show an improvement in viral loss (e.g., viral negativity as measured by PCR). Improvements in ALT normalization are expected in patients receiving yeast-based HBV immunotherapy.
] While various embodiments of the present invention have been described in detail, it is apparent that cations and adaptations of those ments will occur to those skilled in the art. It is to be expressly understood, however, that such modifications and adaptations are within the scope of the present invention, as set forth in the following claims. [00503A] Unless the context clearly requires otherwise, throughout the ption and the claims, the words "comprise", "comprising", and the like, are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense, that is to say, in the sense of "including, but not limited to". [00503B] The reference to any prior art in the ication is not, and should not be taken as, an acknowledgement or any form of suggestion that the prior art forms part of the common general knowledge in New Zealand. 304153833 TIS510927NZPR

Claims (167)

What is claimed
1. An immunotherapeutic composition comprising: a) a yeast vehicle; and b) a fusion protein comprising HBV ns, wherein the HBV antigens consist of: i) an HBV surface antigen having an amino acid sequence that is at least 90% identical to ons 1 to 399 of SEQ ID NO:118, positions 1 to 399 SEQ ID NO:116, positions 1 to 399 SEQ ID NO: 114 or ons 1 to 399 SEQ ID NO:112; and ii) an HBV core antigen having an amino acid sequence that is at least 90% identical to positions 400 to 581 of SEQ ID NO:118, positions 400 to 581 SEQ ID NO:116, positions 400 to 581 SEQ ID NO: 114 or positions 400 to 581 SEQ ID ; wherein the composition s an HBV-specific immune response and wherein the fusion protein is expressed by the yeast vehicle.
2. The immunotherapeutic composition of Claim 1, wherein the HBV ns are ed in the ing order, from N- to C-terminus, in the fusion protein: HBV surface antigen, HBV core antigen.
3. The immunotherapeutic composition of Claim 1, wherein the HBV antigens consist of an amino acid sequence that is at least 90% identical to SEQ ID NO:118, SEQ ID NO:116, SEQ ID NO: 114 or SEQ ID NO:112.
4. The immunotherapeutic composition of Claim 1, wherein the fusion protein comprises an amino acid sequence of SEQ ID NO:118, SEQ ID NO:116, SEQ ID NO: 114 or SEQ ID NO:112.
5. The immunotherapeutic composition of Claim 1, wherein the fusion protein comprises an amino acid sequence of SEQ ID NO:151.
6. The immunotherapeutic ition of Claim 1, wherein the fusion protein consists of an amino acid sequence of SEQ ID NO:151.
7. The immunotherapeutic composition of Claim 1, wherein the amino acid sequence of the HBV surface antigen consists of positions 1 to 399 of SEQ ID NO:118, positions 1 to 304153833 TIS510927NZPR 399 of SEQ ID NO:116, positions 1 to 399 of SEQ ID NO: 114 or positions 1 to 399 of SEQ ID NO:112.
8. The immunotherapeutic composition of Claim 1, wherein the amino acid sequence of the HBV core antigen consists of positions 400 to 581 of SEQ ID NO:118, positions 400 to 581 of SEQ ID NO:116, positions 400 to 581 of SEQ ID NO: 114 or positions 400 to 581 of SEQ ID NO:112.
9. The therapeutic composition of Claim 1, wherein the yeast vehicle is from Saccharomyces cerevisiae.
10. The immunotherapeutic ition of Claim 1, wherein the yeast vehicle is a whole yeast.
11. The immunotherapeutic composition of Claim 10, wherein the whole yeast is activated.
12. The immunotherapeutic composition of Claim 10, wherein the whole yeast is killed.
13. The therapeutic composition of Claim 11, wherein the whole yeast has been lyophilized.
14. The immunotherapeutic composition of Claim 10, wherein the yeast vehicle is from Saccharomyces cerevisiae.
15. The immunotherapeutic composition of Claim 1, comprising a pharmaceutically able excipient.
16. A yeast expressing a fusion n comprising HBV antigens, wherein the HBV antigens consist of: a) an HBV e antigen having an amino acid ce that is at least 90% identical to positions 1 to 399 of SEQ ID NO:118, positions 1 to 399 of SEQ ID NO:116, positions 1 to 399 of SEQ ID NO: 114 or positions 1 to 399 of SEQ ID NO:112; and b) an HBV core antigen having an amino acid ce that is at least 90% identical to positions 400 to 581 of SEQ ID NO:118, positions 400 to 581 of SEQ ID NO:116, positions 400 to 581 of SEQ ID NO: 114 or positions 400 to 581 of SEQ ID NO:112.
17. The yeast of Claim 16, wherein the amino acid sequence of the HBV surface antigen consists of positions 1 to 399 of SEQ ID NO:118, positions 1 to 399 of SEQ ID NO:116, positions 1 to 399 of SEQ ID NO: 114 or positions 1 to 399 of SEQ ID NO:112. 304153833 TIS510927NZPR
18. The yeast of Claim 16, n the amino acid sequence of the HBV core antigen consists of positions 400 to 581 of SEQ ID NO:118, positions 400 to 581 of SEQ ID NO:116, positions 400 to 581 of SEQ ID NO: 114 or positions 400 to 581 of SEQ ID NO:112.
19. The yeast of Claim 16, wherein the HBV antigens consist of an amino acid sequence that is at least 90% identical to SEQ ID NO:118, SEQ ID NO:116, SEQ ID NO: 114 or SEQ ID NO:112.
20. The yeast of Claim 16, wherein the fusion protein consists of an amino acid sequence of SEQ ID NO:151.
21. The yeast of Claim 16, wherein the yeast is from Saccharomyces cerevisiae.
22. A fusion protein comprising HBV antigens, n the fusion n comprises an amino acid sequence that is at least 90% identical to an amino acid sequence of SEQ ID NO:118, SEQ ID NO:116, SEQ ID NO: 114, SEQ ID NO:112 or SEQ ID NO:151.
23. The fusion protein of Claim 22, wherein the fusion protein comprises an amino acid sequence that is at least 90% cal to SEQ ID NO:118, SEQ ID NO:116, SEQ ID NO: 114, or SEQ ID NO:112.
24. The fusion protein of Claim 22, wherein the fusion protein comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:151.
25. The fusion protein of Claim 22, wherein the fusion protein consists of an amino acid sequence of SEQ ID NO:151.
26. Use of : a) a yeast vehicle comprising a fusion protein; b) the fusion protein comprising HBV antigens, n the HBV ns consist of: i) an HBV e antigen having an amino acid sequence that is at least 90% cal to positions 1 to 399 of SEQ ID NO:118, positions 1 to 399 of SEQ ID NO:116, positions 1 to 399 of SEQ ID NO: 114 or positions 1 to 399 of SEQ ID NO:112; and ii) an HBV core antigen having an amino acid sequence that is at least 90% identical to positions 400 to 581 of SEQ ID NO:118, positions 400 to 581 of SEQ ID NO:116, positions 400 to 581 of SEQ ID NO: 114 or positions 400 to 581 of SEQ ID NO:112, 304153833 TIS510927NZPR in the manufacture of an immunotherapeutic composition for the ent of hepatitis B virus (HBV) ion or at least one symptom resulting from HBV infection in a subject.
27. The use of Claim 26, wherein the composition is formulated for stration in a dose from 0.1 Y.U. to 100 Y.U.
28. The use of Claim 26, wherein the composition is formulated for administration in a dose from about 2 Y.U. to about 80 Y.U.
29. The use of Claim 26, wherein the composition is formulated for administration in a dose of 2 Y.U., 10 Y.U., 40 Y.U. or 80 Y.U.
30. The use of Claim 26, wherein the composition is composition is formulated for administration weekly.
31. The use of Claim 26, n the composition is ated for stration bi-weekly.
32. The use of Claim 26, wherein the composition formulated for administration triweekly.
33. The use of Claim 26, wherein the composition is formulated for administration monthly.
34. The use of Claim 26, wherein the composition is formulated for administration at more than one site on the subject.
35. The use of Claim 26, wherein the amino acid sequence of the HBV e n is at least 95% identical to positions 1 to 399 of SEQ ID NO:118, positions 1 to 399 of SEQ ID NO:116, positions 1 to 399 of SEQ ID NO: 114 or positions 1 to 399 of SEQ ID NO:112.
36. The use of Claim 26, wherein the amino acid sequence of the HBV surface antigen consists of positions 1 to 399 of SEQ ID NO:118, positions 1 to 399 of SEQ ID NO:116, positions 1 to 399 of SEQ ID NO: 114 or positions 1 to 399 of SEQ ID NO:112.
37. The use of Claim 26, wherein the amino acid ce of the HBV core antigen is at least 95% identical to positions 400 to 581 of SEQ ID NO:118, positions 400 to 581 of SEQ ID NO:116, positions 400 to 581 of SEQ ID NO: 114 or positions 400 to 581 of SEQ ID NO:112.
38. The use of Claim 26, wherein the amino acid sequence of the HBV core antigen consists of positions 400 to 581 of SEQ ID NO:118, positions 400 to 581 of SEQ ID 304153833 TIS510927NZPR , positions 400 to 581 of SEQ ID NO: 114 or positions 400 to 581 of SEQ ID
39. The use of Claim 26, wherein the HBV antigens consist of: i) an HBV surface antigen having an amino acid sequence that is at least 95% identical to positions 1 to 399 of SEQ ID NO:118, positions 1 to 399 of SEQ ID NO:116, positions 1 to 399 of SEQ ID NO: 114 or positions 1 to 399 of SEQ ID NO:112; and ii) an HBV core antigen having an amino acid sequence that is at least 95% identical to positions 400 to 581 of SEQ ID NO:118, ons 400 to 581 of SEQ ID NO:116, positions 400 to 581 of SEQ ID NO: 114 or ons 400 to 581 of SEQ ID NO:112.
40. The use of Claim 26, wherein the HBV antigens consist of the amino acid sequence that is at least 90% identical to SEQ ID NO:118, SEQ ID , SEQ ID NO: 114 or SEQ ID NO:112.
41. The use of Claim 26, wherein the HBV antigens consist of the amino acid sequence that is at least 95% identical to SEQ ID NO:118, SEQ ID NO:116, SEQ ID NO: 114 or SEQ ID NO:112.
42. The use of Claim 26, n the HBV antigens consist of the amino acid sequence of SEQ ID NO:118, SEQ ID NO:116, SEQ ID NO: 114 or SEQ ID NO:112.
43. The use of Claim 26, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO:118, SEQ ID NO:116, SEQ ID NO: 114 or SEQ ID NO:112.
44. The use of Claim 26, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO:151.
45. The use of Claim 26, wherein the fusion protein consists of the amino acid sequence of SEQ ID NO:151.
46. The use of Claim 26, wherein the HBV antigens are arranged in the following order, from N- to C-terminus, in the fusion protein: HBV surface antigen, HBV core antigen.
47. The use of Claim 26, wherein the yeast vehicle is a whole yeast.
48. The use of Claim 47, n the whole yeast is heat-inactivated.
49. The use of Claim 47, wherein the whole yeast has been lyophilized.
50. The use of Claim 47, n the whole yeast is from Saccharomyces siae.
51. The use of Claim 26, wherein the yeast vehicle is from Saccharomyces cerevisiae.
52. Use of: 304153833 927NZPR a) a yeast comprising a fusion n; b) the fusion protein sing an amino acid sequence that is at least 90% cal to SEQ ID NO:118, SEQ ID NO:116, SEQ ID NO: 114 or SEQ ID NO:112, in the manufacture of an immunotherapeutic composition for the treatment of hepatitis B virus (HBV) infection in a subject.
53. The use of Claim 52, wherein the fusion protein ses an amino acid sequence that is at least 95% identical to SEQ ID NO:118, SEQ ID NO:116, SEQ ID NO: 114 or SEQ ID NO:112.
54. The use of Claim 52, wherein the fusion protein comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:151.
55. The use of Claim 52, wherein the fusion protein comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:118, SEQ ID NO:116, SEQ ID NO: 114 or SEQ ID NO:112.
56. The use of Claim 52, wherein the composition is formulated for administration in a dose of 2 Y.U., 10 Y.U., 40 Y.U. or 80 Y.U.
57. The use of Claim 52, wherein the composition is is formulated for administration monthly.
58. The use of Claim 52, wherein the composition is formulated for administration at more than one site on the subject.
59. The use of Claim 52, wherein the yeast is a whole heat-inactivated yeast from romyces cerevisiae.
60. Use of: a) a whole, heat-inactivated yeast from Saccharomyces siae sing a fusion protein; b) the fusion protein expressed by the yeast, and comprising SEQ ID NO:118, SEQ ID NO:116, SEQ ID NO: 114 or SEQ ID NO:112, in the manufacture of an immunotherapeutic composition for the treatment of chronic hepatitis B virus (HBV) infection in a subject.
61. The use of claim 60, wherein the fusion protein comprises SEQ ID NO:151.
62. Use of: a) a yeast vehicle comprising a fusion protein 304153833 TIS510927NZPR b) the fusion protein sing HBV antigens, wherein the HBV antigens consist of: i) an HBV surface antigen having an amino acid sequence that is at least 90% identical to positions 1 to 399 of SEQ ID , positions 1 to 399 of SEQ ID NO:116, positions 1 to 399 of SEQ ID NO: 114 or ons 1 to 399 of SEQ ID NO:112; and ii) an HBV core antigen having an amino acid sequence that is at least 90% identical to positions 400 to 581 of SEQ ID , positions 400 to 581 of SEQ ID , positions 400 to 581 of SEQ ID NO: 114 or positions 400 to 581 of SEQ ID , in the manufacture of an immunotherapeutic composition for the treatment of hepatitis B virus (HBV) infection in a subject, wherein the immunotherapeutic composition is ated for administration with an anti-viral nd.
63. The use of claim 62, wherein the anti-viral compound is a nucleotide analogue reverse transcriptase inhibitor.
64. The use of claim 62, wherein the anti-viral compound is tenofovir disoproxil fumarate (TDF).
65. The use of Claim 62, wherein the immunotherapeutic composition is formulated for a subject that has received an anti-viral compound prior to administration of the immunotherapeutic composition.
66. The use of Claim 65, wherein the immunotherapeutic composition is formulated for a subject that has received an anti-viral compound at least one year prior to administration of the immunotherapeutic composition.
67. The use of Claim 62, wherein the immunotherapeutic composition is formulated for a subject wherein the subject is naive to any prior anti-viral treatment for HBV.
68. The use of Claim 62, wherein the HBV infection is a chronic HBV infection.
69. The use of Claim 62, wherein the immunotherapeutic composition is formulated for administration in a dose from 0.1 Y.U. to 100 Y.U.
70. The use of Claim 62, wherein the immunotherapeutic composition is ated for administration in a dose from about 2 Y.U. to about 80 Y.U. 304153833 TIS510927NZPR
71. The use of Claim 62, wherein the immunotherapeutic composition is formulated for stration in a dose of 2 Y.U., 10 Y.U., 40 Y.U. or 80 Y.U.
72. The use of Claim 62, wherein the immunotherapeutic composition is formulated for administration weekly.
73. The use of Claim 62, wherein the therapeutic composition is formulated for administration bi-weekly.
74. The use of Claim 62, wherein the immunotherapeutic composition is formulated for administration tri-weekly.
75. The use of Claim 62, wherein the immunotherapeutic composition is ated for administration monthly.
76. The use of Claim 62, n the immunotherapeutic composition is formulated for administration at more than one site on the t.
77. The use of Claim 62, wherein the immunotherapeutic composition is formulated to result in a loss of detectable HBsAg proteins following administration of the immunotherapeutic composition and an anti-viral compound to the t.
78. The use of Claim 62, wherein the immunotherapeutic composition is formulated to result in ance of antibodies in serum of the subject against an HBV antigen following administration of the immunotherapeutic composition and an anti-viral compound to the subject.
79. The use of Claim 62, wherein the immunotherapeutic composition is formulated to result in a reduced HBV viral load in the t following administration of an anti-viral compound and the immunotherapeutic composition to the subject.
80. The use of Claim 62, wherein the therapeutic composition is formulated to result in reduced liver damage in the subject following administration of an anti-viral compound and the immunotherapeutic composition to the subject.
81. The use of Claim 62, n the amino acid sequence of the HBV surface antigen is at least 95% identical to positions 1 to 399 of SEQ ID NO:118, positions 1 to 399 of SEQ ID NO:116, positions 1 to 399 of SEQ ID NO: 114 or positions 1 to 399 of SEQ ID .
82. The use of Claim 62, wherein the amino acid sequence of the HBV surface antigen consists of positions 1 to 399 of SEQ ID NO:118, positions 1 to 399 of SEQ ID NO:116, positions 1 to 399 of SEQ ID NO: 114 or positions 1 to 399 of SEQ ID NO:112. 304153833 TIS510927NZPR
83. The use of Claim 62, wherein the amino acid ce of the HBV core antigen is at least 95% identical to ons 400 to 581 of SEQ ID NO:118, positions 400 to 581 of SEQ ID NO:116, positions 400 to 581 of SEQ ID NO: 114 or positions 400 to 581 of SEQ ID NO:112.
84. The use of Claim 62, wherein the amino acid sequence of the HBV core antigen consists of positions 400 to 581 of SEQ ID NO:118, positions 400 to 581 of SEQ ID NO:116, positions 400 to 581 of SEQ ID NO: 114 or positions 400 to 581 of SEQ ID NO:112.
85. The use of Claim 62, wherein the HBV antigens consist of: i) an HBV surface antigen having an amino acid sequence that is at least 95% identical to positions 1 to 399 of SEQ ID NO:118, positions 1 to 399 of SEQ ID NO:116, positions 1 to 399 of SEQ ID NO: 114 or positions 1 to 399 of SEQ ID NO:112; and ii) an HBV core antigen having an amino acid sequence that is at least 95% identical to ons 400 to 581 of SEQ ID NO:118, positions 400 to 581 of SEQ ID NO:116, positions 400 to 581 of SEQ ID NO: 114 or positions 400 to 581 of SEQ ID NO:112.
86. The use of Claim 62, wherein the HBV antigens consist of the amino acid sequence that is at least 90% identical to SEQ ID NO:118, SEQ ID , SEQ ID NO: 114 or SEQ ID NO:112.
87. The use of Claim 62, wherein the HBV antigens t of the amino acid sequence that is at least 95% identical to SEQ ID NO:118, SEQ ID NO:116, SEQ ID NO: 114 or SEQ ID .
88. The use of Claim 62, wherein the HBV antigens consist of the amino acid sequence of SEQ ID NO:118, SEQ ID NO:116, SEQ ID NO: 114 or SEQ ID .
89. The use of Claim 62, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO:118, SEQ ID , SEQ ID NO: 114 or SEQ ID NO:112.
90. The use of Claim 62, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO:151.
91. The use of Claim 62, wherein the fusion protein consists of the amino acid sequence of SEQ ID NO:151. 304153833 TIS510927NZPR
92. The use of Claim 62, wherein the HBV antigens are arranged in the following order, from N- to C-terminus, in the fusion protein: HBV surface antigen, HBV core n.
93. The use of Claim 62, wherein the yeast vehicle is a whole yeast.
94. The use of Claim 93, wherein the whole yeast is heat-inactivated.
95. The use of Claim 93, wherein the whole yeast has been lyophilized.
96. The use of Claim 93, wherein the whole yeast is from Saccharomyces cerevisiae.
97. The use of Claim 62, wherein the yeast vehicle is from Saccharomyces cerevisiae.
98. Use of: a) a yeast vehicle comprising a fusion n b) the fusion protein sing HBV antigens, wherein the HBV antigens consist of: i) an HBV surface antigen having an amino acid sequence that is at least 90% identical to positions 1 to 399 of SEQ ID NO:118, positions 1 to 399 of SEQ ID NO:116, positions 1 to 399 of SEQ ID NO: 114 or positions 1 to 399 of SEQ ID NO:112; and ii) an HBV core antigen having an amino acid sequence that is at least 90% identical to ons 400 to 581 of SEQ ID NO:118, ons 400 to 581 of SEQ ID , positions 400 to 581 of SEQ ID NO: 114 or ons 400 to 581 of SEQ ID NO:112, in the manufacture of an immunotherapeutic composition for the treatment of hepatitis B virus (HBV) infection in a subject ing anti-viral ent.
99. The use of claim 98, wherein the anti-viral treatment comprises a nucleotide analogue reverse transcriptase inhibitor.
100. The use of claim 98, wherein the anti-viral treatment comprises tenofovir disoproxil fumarate (TDF).
101. The use of claim 98, wherein the immunotherapeutic composition is formulated for administration to a subject that has been receiving anti-viral treatment at least one year prior to administration of the immunotherapeutic composition.
102. The use of Claim 98, wherein the immunotherapeutic composition is formulated for administration in a dose of 2 Y.U., 10 Y.U., 40 Y.U. or 80 Y.U. 304153833 TIS510927NZPR
103. The use of Claim 98, wherein the immunotherapeutic composition is formulated for administration monthly.
104. The use of Claim 98, wherein the immunotherapeutic ition is formulated for administration at more than one site on the subject.
105. The use of Claim 98, wherein the immunotherapeutic composition is formulated to result in a loss of detectable HBsAg ns following administration of the therapeutic composition to the subject receiving iral treatment.
106. The use of Claim 98, wherein the immunotherapeutic composition is formulated to result in the appearance of antibodies in serum of the subject against an HBV antigen following administration of the therapeutic composition and antiviral compound to the subject.
107. The use of Claim 98, wherein the immunotherapeutic ition is formulated to reduce HBV viral load in the subject following administration of the immunotherapeutic composition to the subject receiving anti-viral treatment.
108. The use of Claim 98, wherein the immunotherapeutic composition is formulated to reduce liver damage in the subject following administration of the immunotherapeutic composition to the subject receiving anti-viral treatment.
109. The use of Claim 98, wherein the amino acid sequence of the HBV surface antigen is at least 95% cal to positions 63-461 of SEQ ID NO:130.
110. The use of Claim 98, wherein the amino acid sequence of the HBV surface antigen consists of positions 1 to 399 of SEQ ID , positions 1 to 399 of SEQ ID NO:116, positions 1 to 399 of SEQ ID NO: 114 or positions 1 to 399 of SEQ ID NO:112.
111. The use of Claim 98, wherein the amino acid sequence of the HBV core antigen is at least 95% identical to positions 400 to 581 of SEQ ID NO:118, positions 400 to 581 of SEQ ID NO:116, positions 400 to 581 of SEQ ID NO: 114 or positions 400 to 581 of SEQ ID NO:112.
112. The use of Claim 98, wherein the amino acid sequence of the HBV core antigen ts of positions 400 to 581 of SEQ ID NO:118, positions 400 to 581 of SEQ ID NO:116, positions 400 to 581 of SEQ ID NO: 114 or positions 400 to 581 of SEQ ID NO:112.
113. The use of Claim 98, wherein the HBV antigens consist of: a) an HBV surface antigen having an amino acid sequence that is at least 95% identical to positions 1 to 399 of SEQ ID NO:118, positions 1 to 304153833 TIS510927NZPR 399 of SEQ ID NO:116, positions 1 to 399 of SEQ ID NO: 114 or positions 1 to 399 of SEQ ID NO:112; and b) an HBV core antigen having an amino acid sequence that is at least 95% identical to positions 400 to 581 of SEQ ID NO:118, positions 400 to 581 of SEQ ID NO:116, positions 400 to 581 of SEQ ID NO: 114 or positions 400 to 581 of SEQ ID NO:112.
114. The use of Claim 98, wherein the HBV antigens consist of the amino acid sequence that is at least 90% cal to SEQ ID , SEQ ID NO:116, SEQ ID NO: 114 or SEQ ID NO:112.
115. The use of Claim 98, wherein the HBV antigens consist of the amino acid ce of SEQ ID NO:118, SEQ ID NO:116, SEQ ID NO: 114 or SEQ ID NO:112.
116. The use of Claim 98, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO:118, SEQ ID NO:116, SEQ ID NO: 114 or SEQ ID NO:112.
117. The use of Claim 98, wherein the fusion protein comprises the amino acid ce of SEQ ID NO:151.
118. The use of Claim 98, wherein the fusion protein consists of the amino acid sequence of SEQ ID NO:151.
119. The use of Claim 98, wherein the HBV antigens are arranged in the following order, from N- to C-terminus, in the fusion protein: HBV surface antigen, HBV core n.
120. The use of Claim 98, wherein the yeast vehicle is a whole heat-inactivated yeast from Saccharomyces siae.
121. Use of: a) a yeast comprising a fusion protein b) the fusion protein comprising an amino acid sequence that is at least 90% identical to SEQ ID , SEQ ID NO:116, SEQ ID NO: 114 or SEQ ID NO:112, in the cture of an immunotherapeutic composition for the treatment of hepatitis B virus (HBV) infection in a subject wherein the composition is formulated for administration to a subject with an anti-viral compound.
122. The use of Claim 121, wherein the fusion protein comprises an amino acid sequence that is at least 95% identical to SEQ ID , SEQ ID NO:116, SEQ ID NO: 114 or SEQ ID NO:112. 304153833 TIS510927NZPR
123. The use of Claim 121, wherein the fusion protein comprises an amino acid sequence that is at least 90% cal to SEQ ID NO:151.
124. The use of Claim 121, wherein the fusion protein comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:151.
125. The use of claim 121, wherein the anti-viral compound is a nucleotide ue reverse transcriptase inhibitor.
126. The use of claim 121, wherein the anti-viral compound is tenofovir disoproxil fumarate (TDF).
127. The use of Claim 121, wherein the immunotherapeutic ition is formulated for a subject that has ed an anti-viral compound prior to stration of the immunotherapeutic composition.
128. The use of Claim 127, wherein the therapeutic composition is formulated for a subject that has received an anti-viral nd at least one year prior to administration of the immunotherapeutic composition.
129. The use of Claim 121, wherein the immunotherapeutic composition is formulated for a subject wherein the subject is naive to any prior anti-viral treatment for HBV.
130. The use of Claim 121, n the immunotherapeutic composition is formulated for administration in a dose of 2 Y.U., 10 Y.U., 40 Y.U. or 80 Y.U.
131. The use of Claim 121, wherein the immunotherapeutic composition is formulated for administration monthly.
132. The use of Claim 121, wherein the immunotherapeutic composition is ated for administration at more than one site on the t.
133. The use of Claim 121, wherein the immunotherapeutic composition is formulated to result in a loss of detectable HBsAg proteins following administration of the immunotherapeutic composition and an anti-viral compound to the subject.
134. The use of Claim 121, wherein the immunotherapeutic composition is formulated to result in appearance of antibodies in serum of the subject against an HBV antigen following administration of the immunotherapeutic composition and an anti-viral nd to the subject.
135. The use of Claim 121, wherein the yeast vehicle is a whole heatinactivated yeast from Saccharomyces cerevisiae.
136. Use of: TIS510927NZPR a) a yeast comprising a fusion protein b) the fusion protein comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: SEQ ID NO:118, SEQ ID NO:116, SEQ ID NO: 114 or SEQ ID NO:112, in the manufacture of an immunotherapeutic composition for treating hepatitis B virus (HBV) infection in a subject receiving anti-viral treatment.
137. The use of Claim 136, wherein the fusion protein comprises an amino acid sequence is at least 95% cal to SEQ ID NO SEQ ID NO:118, SEQ ID NO:116, SEQ ID NO: 114 or SEQ ID NO:112.
138. The use of Claim 136, wherein the fusion n comprises an amino acid sequence is at least 90% identical to SEQ ID NO:151.
139. The use of Claim 136, wherein the fusion protein comprise an amino acid is at least 95% identical to SEQ ID NO:151.
140. The use of claim 136, wherein the anti-viral treatment ses a nucleotide analogue reverse transcriptase inhibitor.
141. The use of claim 136, n the anti-viral treatment comprises vir disoproxil fumarate (TDF).
142. The use of Claim 136, wherein the immunotherapeutic composition is formulated for administration to a subject that has been receiving anti-viral treatment at least one year prior to administration of the immunotherapeutic composition.
143. The use of Claim 136, n the immunotherapeutic composition is formulated for administration in a dose of 2 Y.U., 10 Y.U., 40 Y.U. or 80 Y.U.
144. The use of Claim 136, wherein the immunotherapeutic composition is formulated for administration monthly.
145. The use of Claim 136, wherein the immunotherapeutic composition is formulated for administration at more than one site on the subject.
146. The use of Claim 136, wherein the immunotherapeutic composition is formulated to result in the appearance of antibodies in serum of the subject t an HBV antigen following stration of the immunotherapeutic composition and antiviral compound to the subject.
147. The use of Claim 136, wherein the yeast vehicle is a whole activated yeast from Saccharomyces cerevisiae.
148. Use of: 304153833 TIS510927NZPR a) a whole, heat-inactivated yeast from Saccharomyces siae comprising a fusion protein b) the fusion protein expressed by the yeast, and comprising SEQ ID NO:118, SEQ ID NO:116, SEQ ID NO: 114 or SEQ ID , in the manufacture of an immunotherapeutic composition for treating c hepatitis B virus (HBV) infection in a subject ing anti-viral treatment.
149. The use of claim 148, n the fusion protein comprises SEQ ID NO:151.
150. Use of: a) a whole, nactivated yeast from Saccharomyces cerevisiae comprising a fusion protein b) the fusion protein expressed by the yeast, and comprising SEQ ID NO:118, SEQ ID NO:116, SEQ ID NO: 114 or SEQ ID NO:112, in the manufacture of an immunotherapeutic composition for treating chronic hepatitis B virus (HBV) infection in a subject wherein the composition is formulated for administration with an anti-viral compound.
151. The use of claim 150, wherein the fusion protein ses SEQ ID
152. A recombinant nucleic acid molecule encoding a fusion protein comprising HBV antigens, wherein the HBV antigens consist of: i) an HBV surface antigen having an amino acid sequence that is at least 95% identical to positions 1 to 399 of SEQ ID NO:118, positions 1 to 399 of SEQ ID NO:116, positions 1 to 399 of SEQ ID NO: 114 or positions 1 to 399 of SEQ ID NO:112; and ii) an HBV core antigen having an amino acid sequence that is at least 95% identical to positions 400 to 581 of SEQ ID NO:118, positions 400 to 581 of SEQ ID NO:116, positions 400 to 581 of SEQ ID NO: 114 or positions 400 to 581 of SEQ ID NO:112.
153. The recombinant nucleic acid molecule of Claim 152, wherein the recombinant nucleic acid molecule comprises a nucleic acid ce of SEQ ID NO:117, SEQ ID NO:115, SEQ ID NO: 113 or SEQ ID NO:111.
154. An isolated cell transfected with the recombinant c acid molecule of Claim 152. 304153833 TIS510927NZPR
155. The isolated cell of Claim 154, n the cell is a yeast cell.
156. The recombinant nucleic acid molecule of Claim 152, wherein the fusion protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:118, SEQ ID NO:116, SEQ ID NO: 114 or SEQ ID NO:112.
157. The recombinant nucleic acid le of Claim 152, n the fusion protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:151.
158. The recombinant nucleic acid molecule of Claim 152, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO:118, SEQ ID NO:116, SEQ ID NO: 114 SEQ ID NO:112 or SEQ ID NO: 151.
159. The inant nucleic acid molecule of Claim 152, wherein the fusion protein consists of an amino acid sequence of SEQ ID NO:118, SEQ ID NO:116, SEQ ID NO: 114 or SEQ ID .
160. The inant nucleic acid molecule of Claim 152, n the fusion protein consists of an amino sequence of SEQ ID NO:151.
161. An isolated cell transfected with the recombinant nucleic acid molecule of Claim 159.
162. The isolated cell of Claim 162, wherein the cell is a yeast cell.
163. An isolated cell transfected with the recombinant nucleic acid molecule of Claim 160.
164. The isolated cell of Claim 163, wherein the cell is a yeast cell.
165. The recombinant nucleic acid molecule of Claim 152, wherein the amino acid sequence of HBV surface antigen consists of positions 1 to 399 of SEQ ID NO:118, positions 1 to 399 of SEQ ID NO:116, ons 1 to 399 of SEQ ID NO: 114 or positions 1 to 399 of SEQ ID NO:112.
166. The inant nucleic acid molecule of Claim 152, wherein the amino acid sequence of HBV core antigen consists of positions 400 to 581 of SEQ ID NO:118, positions 400 to 581 of SEQ ID NO:116, positions 400 to 581 of SEQ ID NO: 114 or positions 400 to 581 of SEQ ID NO:112.
167. An immunotherapeutic composition as claimed in claim 1, substantially as hereinbefore described with particular reference to any of the examples, figures and/or
NZ705184A 2011-02-12 2012-02-09 Yeast-based therapeutic for chronic hepatitis b infection NZ705184B2 (en)

Applications Claiming Priority (7)

Application Number Priority Date Filing Date Title
US201161442204P 2011-02-12 2011-02-12
US61/442,204 2011-02-12
US201161496945P 2011-06-14 2011-06-14
US61/496,945 2011-06-14
US201161507361P 2011-07-13 2011-07-13
US61/507,361 2011-07-13
NZ614733A NZ614733B2 (en) 2011-02-12 2012-02-09 Yeast-based therapeutic for chronic hepatitis b infection

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NZ705184B2 true NZ705184B2 (en) 2017-01-05

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