NZ756328B2 - NUCLEIC ACIDS ENCODING HUMAN ANTIBODIES TO SIALYL-LEWISa - Google Patents

Abstract

The present invention provides compositions for the production of an antibody or functional fragment thereof directed against Sialyl-Lewisa (sLea). The compositions of the invention include polynucleotides encoding a heavy chain and/or a light chain variable domain that binds to sLea. The invention also provides an isolated antibody or functional fragment thereof and methods of treating or preventing a disease, such as cancer or tumor formation, wherein the antibody or functional fragment includes a variable heavy chain domain and a variable light chain domain that has an amino acid sequence provided herein. The invention further provides a conjugate of an antibody or functional fragment thereof conjugated or recombinantly fused to a diagnostic agent, detectable agent or therapeutic agent, and methods of treating, preventing or diagnosing a disease in a subject in need thereof. The invention further provides co-administering a chemotherapeutic agent with the antibody or functional fragment thereof. also provides an isolated antibody or functional fragment thereof and methods of treating or preventing a disease, such as cancer or tumor formation, wherein the antibody or functional fragment includes a variable heavy chain domain and a variable light chain domain that has an amino acid sequence provided herein. The invention further provides a conjugate of an antibody or functional fragment thereof conjugated or recombinantly fused to a diagnostic agent, detectable agent or therapeutic agent, and methods of treating, preventing or diagnosing a disease in a subject in need thereof. The invention further provides co-administering a chemotherapeutic agent with the antibody or functional fragment thereof.

Description

Nucleic Acids Encoding Human Antibodies To Sialyl-Lewis This application is a divisional application of New Zealand Patent Application No. 717428, which is the national phase entry of (published as ) dated 8 August 2014, and claims the benefit of priority of United States Provisional Application Serial No. 61/870,137, filed August 26, 2013, the entire contents of all of which are incorporated herein by reference.

This invention was made with government support under grant number CA-128362 awarded by the National Cancer Institute, NIH. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION The present invention relates generally to antibodies directed against Sialyl-Lewis (sLe ), and more specifically to polynucleotides encoding anti-sLe antibodies and the corresponding encoded antibodies or fragments thereof.

Passive administration of antibodies directed against tumor specific antigens may eliminate tumor cells and early metastases during cancer development. This treatment may also have a significant impact on cancer recurrence. Antibodies directed against tumor specific carbohydrates may be useful candidates in this cancer treatment. For example, many tumor-restricted monoclonal antibodies resulting from immunization of mice with human cancer cells have been shown to be directed against carbohydrate antigens expressed at the cell surface as glycolipids or glycoproteins. The carbohydrate sLe has been shown to be expressed on tumors of the gastrointestinal tract. Expression of sLe has also been shown to impact metastatic potential and correlates with increased metastatic potential in human colon cancer and pancreatic adenocarcinoma. However, carbohydrate chemistry has been rather challenging and the clinical development of antibodies that recognize such tumor specific carbohydrates has been slow.

Pancreatic carcinoma is one of the most aggressive adenocarcinomas and is often associated with a poor prognosis. Pancreatic carcinoma ranks as the fourth leading cause of cancer mortality. Despite advances in the screening for different carcinomas, the reliability of detecting malignant lesions stemming from the pancreas remains poor.

Positron emission tomography utilizing fluorodeoxyglucase (FDG-PET) has been indicated for the detection and staging of pancreatic cancer. However, FDG-PET is insensitive to differentiating pancreatitis from malignancy and remains problematic in staging small primary lesions (< 7mm) and liver metastases (<1 cm). One diagnostic screening method used to monitor the state of pancreatic ductal adenocarcinoma (PDAC) patients includes detecting elevated levels of circulating sLe antigen in sera. Patients with > 37 U/ml of circulating sLe antigen indicates cancer recurrence. However, development of alternative diagnostic tools that utilize such tumor specific carbohydrates has been slow.

Thus, there exists a need for identifying and generating antibodies that specifically recognize tumor specific carbohydrates, such as sLe , for the treatment of recurring cancers and for detecting malignant lesions and metastases. This invention satisfies this need and provides related advantages, or at least to provide the public with a useful choice.

SUMMARY OF INVENTION The present invention particularly relates to the treatment of cancer or tumor formations having cells expressing Sialyl-Lewis using an antibody or polypeptide comprising a functional fragment thereof, comprising a variable heavy chain (VH) domain and a variable light chain (VL) domain, where said VH domain and said VL domain respectively comprise CDR1, CDR2 and CDR3 of an amino acid sequence comprising residues 20-145 of SEQ ID NO: 14 and CDR1, CDR2 and CDR3 of an amino acid sequence comprising residues 23-130 of SEQ ID NO: 16, or a polynucleotide encoding said antibody or polypeptide, and an agent of chemotherapy.

In one particular embodiment of the above aspect, provided is the use of the antibody, polypeptide or polynucleotide, for the preparation of a pharmaceutical composition for the treatment of a cancer or tumor formation having cells expressing Sialyl-Lewis , said treatment comprising the administration of said composition and an agent of chemotherapy.

In another particular embodiment of the above aspect, provided is the use of an agent of chemotherapy, for the preparation of a pharmaceutical composition for the treatment of a cancer or tumor formation having cells expressing Sialyl-Lewis , said treatment comprising the administration of said composition and said antibody, polypeptide or polynucleotide.

In certain embodiments said antibody is a human antibody. In other embodiments the polypeptide is selected from the group consisting of a Fab, a Fab’, a F(ab’)2, a scFV, a diabody, a triabody, and a minibody, preferably diabody, still more preferably a diabody comprising the amino acid sequence of SEQ ID NO: 20.

In some embodiments the antibody is a monoclonal antibody. In other embodiments the antibody is an IgG or IgM isotype. Preferably the IgG antibody is an IgG1 subclass.

In certain embodiments the antibody or polypeptide is conjugated or recombinantly fused to a diagnostic agent, detectable agent or therapeutic agent. In certain embodiments the detectable agent is a radioactive material or a fluorescent material. Preferably the radioactive 89 131 125 124 123 121 14 11 material is zirconium ( Zr), iodine ( I, I, I, I, and I,), carbon ( C and C), sulfur 3 115 113 112 111 99 201 ( S), tritium ( H), indium ( In, In, In, and In,), technetium ( Tc), thallium ( Ti), 68 67 103 99 133 gallium ( Ga and Ga), palladium ( Pd), molybdenum ( Mo), xenon ( Xe), fluorine 18 15 13 64 94m 153 177 159 149 140 175 166 86 90 47 ( F), O, N, Cu, Tc, Sm, Lu, Gd, Pm, La, Yb, Ho, Y, Y, Sc, 186 188 142 105 97 68 57 65 85 32 153 169 51 54 75 Re, Re, Pr, Rh, Ru, Ge, Co, Zn, Sr, P, Gd, Yb, Cr, Mn, Se, 113 117 Sn, and Sn, or the fluorescent material is selected from the group consisting of umbelliferone, fluorescein, fluorescein isothiocynate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride and phycoerythrin. In certain embodiments the therapeutic agent is radioactive metal, for example an alpha-emitter, or an Auristatin molecule, for example auristatin PHE, bryostatin 1, solastatin 10, monomethyl auristatin E (MMAE) and monomethylauristatin F (MMAF).

In certain embodiments said cancer or tumor is selected from the group consisting of a tumor of the gastrointestinal tract, colon cancer, colorectal adenocarcinoma, metastatic colon cancer, colorectal cancer, pancreatic cancer, pancreatic adenocarcinoma, pancreatic ductal cancer, small cell carcinoma of the lung, bladder adenocarcinoma, signet ring ovarian cancer, ovarian cancer, metastatic carcinoma, adenocarcinoma of the stomach, adenocarcinoma of the esophagus, adenocarcinoma of the throat, adenocarcinoma of the urogenital tract, and adenocarcinoma of the breast.

In certain embodiments the treatment comprises concurrent or successive administration of the antibody or polypeptide, or the polynucleotide encoding said antibody or polypeptide, and the agent of chemotherapy.

In still further embodiments, the agent of chemotherapy is at least one agent selected from the group consisting of an anthracycline; a taxan; an antimetabolite; an alkylating agent, (followed by page 3A) lomustine, cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, cisdichlorodiamine platinum (II) (DDP), cisplatin; an antibiotic; an Auristatin molecule; a hormone; a nucleoside analoge, a DNA-repair enzyme inhibitor, a kinase inhibitor; a cytotoxic agent, a topoisomerase inhibitor; a DNA minor groove binder; and adenosine deaminase inhibitors; or pharmaceutically acceptable salts, solvates, clathrates, or prodrugs thereof. In particular embodiments the agent of chemotherapy is a taxan selected from paclitaxel (Taxol) and docetaxel (Taxotere) or is selected from the group consisting of doxorubicin, daunorubicin, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5- fluorouracil and decarbazine, mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BCNU), lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, actinomycin D, bleomycin, mithramycin, and anthramycin (AMC); auristatin PHE, bryostatin 1, solastatin 10, monomethyl auristatin E (MMAE) and monomethylauristatin F (MMAF), glucocorticoids, progestins, androgens, estrogens, gemcitabine, etoposide, topotecan, Gleevec, imatinib mesylate; maytansine, paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, 1- dehydrotestosterone, glucorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin and analogs or homologs thereof.

Further aspects and embodiments are described herein for completeness. Certain of these aspects form the subject of the parent application, NZ 717428.

Thus also provided are compositions for producing antibodies or functional fragments thereof that bind sLe . The compositions include an isolated polynucleotide encoding an antibody or a functional fragment thereof, wherein the antibody includes a variable heavy chain (VH) domain that has an amino acid sequence provided herein. The isolated polynucleotide of the invention can also include a nucleic acid sequence provided herein, wherein the nucleic acid sequence encodes the VH domain of the antibody or functional fragment thereof.

In another embodiment, the isolated polynucleotide can encode an antibody or a functional fragment thereof, wherein the antibody includes a variable light chain (VL) domain that has an amino acid sequence provided herein. The isolated polynucleotide can also include a nucleic acid sequence provided herein, wherein the nucleic acid sequence encodes the VL domain of the antibody or functional fragment thereof. (followed by page 3B) In a particular embodiment provided is an isolated polynucleotide encoding an antibody or a polypeptide comprising a functional fragment thereof that binds to Sialyl-Lewis , said antibody or polypeptide comprises a variable heavy chain (VH) domain and a variable light chain (VL) domain, where said VH domain and said VL domain respectively comprise CDR1, CDR2 and CDR3 of an amino acid sequence comprising residues 20-145 of SEQ ID NO: 14 and CDR1, CDR2 and CDR3 of an amino acid sequence comprising residues 23-130 of SEQ ID NO: 16.

The compositions also include an isolated antibody or functional fragment thereof, wherein the antibody binds to sLe . In some embodiments, the invention provides an isolated antibody or functional fragment thereof that binds to sLe , wherein the antibody or functional fragment thereof includes a VH domain having an amino acid sequence provided herein.

In some embodiments, provided is an isolated antibody or functional fragment thereof that binds to sLe , wherein the antibody or functional fragment thereof includes a VL domain having an amino acid sequence provided herein. In some embodiments, provided is an isolated antibody or functional fragment thereof that binds to sLe , wherein the antibody or functional fragment thereof includes both a VH domain and a VL domain, where the VH domain and the VL domain respectively include an amino acid sequence for the respective VH and VL domains of the clonal isolates provided herein.

In a particular embodiment provided is an isolated antibody or a polypeptide comprising a functional fragment thereof that binds to Sialyl-Lewis , said antibody or polypeptide comprising a variable heavy chain (VH) domain and a variable light chain (VL) domain, where said VH domain and said VL domain respectively comprise CDR1, CDR2 and CDR3 of an amino acid sequence comprising residues 20-145 of SEQ ID NO: 14 and CDR1, CDR2 and CDR3 of an amino acid sequence comprising residues 23-130 of SEQ ID NO: 16.

In some embodiments, provided is a conjugate having an antibody or functional fragment provided herein that is conjugated or recombinantly fused to a diagnostic agent, detectable agent or therapeutic agent. In some aspects, a conjugate of the invention that includes a detectable agent can be used in a method for detecting and/or diagnosing tumor formation is a subject. Such methods can include administering an effective amount of the conjugate to a subject in need thereof. In particular embodiments the invention provides the use of such a (followed by page 3C) conjugate in the manufacture of a medicament for the detection of a tumor which expresses sLe .

In particular embodiment provided is a fusion protein comprising a variable heavy chain (VH) domain or an antigen binding fragment thereof, a variable light chain (VL) domain or an antigen binding fragment thereof, and a heterologous protein, wherein said VH domain and said VL domain respectively comprise CDR1, CDR2 and CDR3 of an amino acid sequence of residues 20-145 of SEQ ID NO:14 and CDR1, CDR2 and CDR3 of an amino acid sequence of residues 23-130 of SEQ ID NO:16.

In some embodiments, provided are pharmaceutical compositions having one or more antibody or functional fragment of the invention and a pharmaceutically acceptable carrier.

In some aspects, also provided is a method for treating or preventing a disease in a subject in need thereof, by administering a therapeutically effective amount of a pharmaceutical composition as provided herein. In other aspects provided herein is the use of the antibody or polypeptide in the manufacture of a medicament for treating or preventing a disease, wherein the disease is a cancer or a tumor formation having cells expressing sLe . In still another aspect, provided is the administering of a second therapeutic agent concurrently or successively with an antibody or functional fragment provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS shows the nucleotide sequence and the encoded amino acid sequence of the variable heavy (VH) chain domain of clone 5B1 and a leader sequence that can be used for recombinant expression. The top portion of the figure shows an alignment between the nucleotide sequence of SEQ ID NO: 1 and amino acid sequence of SEQ ID NO: 2. The three complementarity determining regions (CDR1, CDR2 and CDR3) are also identified. shows the nucleotide sequence and the encoded amino acid sequence of the variable light (VL) chain domain of clone 5B1 and a leader sequence that can be used for recombinant expression. The top portion of the figure shows an alignment between the (followed by page 4)

Claims (25)

CLAIMS 1.:

1. Use of a polynucleotide for the preparation of a pharmaceutical composition for the treatment of a cancer or tumor formation having cells expressing Sialyl-Lewis , said treatment comprising the administration of said composition and an agent of chemotherapy, the polynucleotide encoding an antibody or a polypeptide comprising a functional fragment thereof that binds to Sialyl-Lewis , said antibody or polypeptide comprises a variable heavy chain (VH) domain and a variable light chain (VL) domain, where said VH domain and said VL domain respectively comprise CDR1, CDR2 and CDR3 of an amino acid sequence comprising residues 20-145 of SEQ ID NO: 14 and CDR1, CDR2 and CDR3 of an amino acid sequence comprising residues 23-130 of SEQ ID NO: 16.

2. Use of an antibody or a polypeptide comprising a functional fragment thereof that binds to Sialyl-Lewis for the preparation of a pharmaceutical composition for the treatment of a cancer or tumor formation having cells expressing Sialyl-Lewis , said treatment comprising the administration of said composition and an agent of chemotherapy, said antibody or polypeptide comprising a variable heavy chain (VH) domain and a variable light chain (VL) domain, where said VH domain and said VL domain respectively comprise CDR1, CDR2 and CDR3 of an amino acid sequence comprising residues 20-145 of SEQ ID NO: 14 and CDR1, CDR2 and CDR3 of an amino acid sequence comprising residues 23-130 of SEQ ID NO: 16.

3. The use of an agent of chemotherapy, for the preparation of a pharmaceutical composition for the treatment of a cancer or tumor formation having cells expressing Sialyl- Lewis , said treatment comprising the administration of said composition and a polynucleotide encoding an antibody or a polypeptide comprising a functional fragment thereof that binds to Sialyl-Lewis said antibody or polypeptide comprising a variable heavy chain (VH) domain and a variable light chain (VL) domain, where said VH domain and said VL domain respectively comprise CDR1, CDR2 and CDR3 of an amino acid sequence comprising residues 20-145 of SEQ ID NO: 14 and CDR1, CDR2 and CDR3 of an amino acid sequence comprising residues 23-130 of SEQ ID NO: 16.

4. The use of an agent of chemotherapy, for the preparation of a pharmaceutical composition for the treatment of a cancer or tumor formation having cells expressing Sialyl- Lewis , said treatment comprising the administration of said composition and an antibody or a polypeptide comprising a functional fragment thereof that binds to Sialyl-Lewis , said antibody or polypeptide comprising a variable heavy chain (VH) domain and a variable light chain (VL) domain, where said VH domain and said VL domain respectively comprise CDR1, CDR2 and CDR3 of an amino acid sequence comprising residues 20-145 of SEQ ID NO: 14 and CDR1, CDR2 and CDR3 of an amino acid sequence comprising residues 23-130 of SEQ ID NO: 16.

5. The use of any one of claims 1 to 4, wherein said antibody is a human antibody.

6. The use of any one of claims 1 to 4, wherein said polypeptide is selected from the group consisting of a Fab, a Fab’, a F(ab’) , a scFV, a diabody, a triabody, and a minibody.

7. The use of claim 6, wherein said polypeptide is a diabody.

8. The use of claim 7, wherein said diabody comprises the amino acid sequence of SEQ ID NO: 20.

9. The use of any one of claims 1 to 4, wherein said antibody is a monoclonal antibody.

10. The use of any one of claims 1-9, wherein said antibody is an IgG or IgM isotype.

11. The use of claim 10, wherein said IgG antibody is an IgG1 subclass.

12. The use of any one of claims 1 to 11, wherein the antibody or polypeptide is conjugated or recombinantly fused to a diagnostic agent, detectable agent or therapeutic agent.

13. The use of claim 12, wherein said detectable agent is a radioactive material or a fluorescent material. 89 131

14. The use of claim 13, wherein said radioactive material is zirconium ( Zr), iodine ( I, 125 124 123 121 14 11 35 3 115 113 I, I, I, and I,), carbon ( C and C), sulfur ( S), tritium ( H), indium ( In, In, 112 111 99 201 68 67 103 In, and In,), technetium ( Tc), thallium ( Ti), gallium ( Ga and Ga), palladium ( Pd), 99 133 18 15 13 64 94m 153 177 159 molybdenum ( Mo), xenon ( Xe), fluorine ( F), O, N, Cu, Tc, Sm, Lu, Gd, 149 140 175 166 86 90 47 186 188 142 105 97 68 57 65 85 Pm, La, Yb, Ho, Y, Y, Sc, Re, Re, Pr, Rh, Ru, Ge, Co, Zn, Sr, 32 153 169 51 54 75 113 117 P, Gd, Yb, Cr, Mn, Se, Sn, and Sn, or wherein said fluorescent material is selected from the group consisting of umbelliferone, fluorescein, fluorescein isothiocynate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride and phycoerythrin.

15. The use of claim 12, wherein said therapeutic agent is radioactive metal or an Auristatin molecule.

16. The use of claim 15, wherein said radioactive metal is an alpha-emitter, or wherein said Auristatin molecule is selected from the group consisting of auristatin PHE, bryostatin 1, solastatin 10, monomethyl auristatin E (MMAE) and monomethylauristatin F (MMAF).

17. The use of any one of claims 1 to 16, wherein said cancer or tumor is selected from the group consisting of a tumor of the gastrointestinal tract, colon cancer, colorectal adenocarcinoma, metastatic colon cancer, colorectal cancer, pancreatic cancer, pancreatic adenocarcinoma, pancreatic ductal cancer, small cell carcinoma of the lung, bladder adenocarcinoma, signet ring ovarian cancer, ovarian cancer, metastatic carcinoma, adenocarcinoma of the stomach, adenocarcinoma of the esophagus, adenocarcinoma of the throat, adenocarcinoma of the urogenital tract, and adenocarcinoma of the breast.

18. The use of any one of claims 1 to 17, wherein the treatment comprises concurrent or successive administration of the antibody or polypeptide, or the polynucleotide encoding said antibody or polypeptide, and the agent of chemotherapy.

19. The use of any one of claims 1 to 18, wherein the agent of chemotherapy is at least one agent selected from the group consisting of an anthracycline; a taxan; an antimetabolite; an alkylating agent, lomustine, cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, cisdichlorodiamine platinum (II) (DDP), cisplatin; an antibiotic; an Auristatin molecule; a hormone; a nucleoside analoge, a DNA-repair enzyme inhibitor, a kinase inhibitor; a cytotoxic agent, a topoisomerase inhibitor; a DNA minor groove binder; and adenosine deaminase inhibitors; or pharmaceutically acceptable salts, solvates, clathrates, or prodrugs thereof.

20. The use of claim 19, wherein the agent of chemotherapy is a taxan selected from paclitaxel (Taxol) and docetaxel (Taxotere).

21. The use of claim 19, wherein the agent of chemotherapy is selected from the group consisting of doxorubicin, daunorubicin, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil and decarbazine, mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BCNU), lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, actinomycin D, bleomycin, mithramycin, and anthramycin (AMC); auristatin PHE, bryostatin 1, solastatin 10, monomethyl auristatin E (MMAE) and monomethylauristatin F (MMAF), glucocorticoids, progestins, androgens, estrogens, gemcitabine, etoposide, topotecan, Gleevec, imatinib mesylate; maytansine, paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, 1-dehydrotestosterone, glucorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin and analogs or homologs thereof.

22. A use according to claim 1, substantially as herein described and exemplified.

23. A use according to claim 2, substantially as herein described and exemplified.

24. A use according to claim 3, substantially as herein described and exemplified.

25. A use according to claim 4, substantially as herein described and exemplified.