AU626007B2 - Dna sequences, recombinant dna molecules and processes for producing soluble t4 proteins - Google Patents

Description

CORRECTED

VERSION*

in WO 89/01940 IC07H 15/12] (No. 06/1989). under INID Number (81) "Dcsignated States". add "BIJ (OAPI patent), CF (OAPI patent). CG (OAPI patent). CM (OAPI patent). GA (OAPI patent). ML (OAPI patent); MR (OAPI patent). SN (OAPI patent). TD (OAPI patent). TG (OAPI patent)" "PC PTWORLD INTELLECTUAL PROPERTY ORGANIZATION 7'{ L ALICTION P Interna't onatN Bureau ~OTI Y INTERNATIONAL APPLICATION PUBLISHED.NER T P N|t)OP,'RATION TREATY (PCT) (51) International Patent Classification 4 C07H 15/12, C12Q 1/70, 1/02 C12Q 1/06, C12P 21/00, 19/34 C12P 1/04, C12N 15/00, 7/00 C07K 13/00,3/00, A61K 37/68 A61K 39/00, 45/02 (11) International Publication Number: (43) International Publication Date: WO 89/ 01940 9 March 1989 (09.03.89) (21) International Application Number: (22) International Filing Date: (31) Priority Application Numbers: (32) Priority Dates: PCT/US88/02940 1 September 1988 (01.09.88) 094,322 141,649 4 September 1987 (04.09.87) 7 January 1988 (07.01.88)

US

094,322 (CIP) 4 September 1987 (04.09.87) 141,649 (CIP) 7 January 1988 (07.01.88) (33) Priority Country: Parent Applications or Grants (63) Related by Continuation

US

Filed on

US

Filed on (72) Inventors; and Inventors/Applicants (for US only) FISHER, Richard, A. [US/ US]; 1079 Beacon Street, Brookline, MA 12146 GIL- BERT, Walter [US/US]; 107 Upland Road, Cambridge, MA 02140 SATO, Vicki, L. [US/US]; 43 Larch Road, Cambridge, MA 02138 FLAVELL, Richard, A. [GB/US]; 182 Reservoir Road, Killingworth, CT 06417 MAR- AGANORE, John, M. [US/US]; 84 Patrick Road, Tewksbury, MA 01876 LIU, Theresa, R. [US/US]; 102 Emerson Road, Milton, MA 02186 (US).

(74) Agents: HALEY, James, Jr. et al.; Fish Neave, 875 Third Avenue, New York, NY 10022-6250 (US).

(81) Designated States: AT (European patent), AU, BE (European patent), BJ (OAPI patent), CF (OAPI patent), CH (European patent), CG (OAPI patent), CM (OAPI patent), DE (European patent), DK, FR (European patent), GA (OAPI patent), GB (European patent), IT (European patent), JP, KR, LU (European patent), ML (OAPI patent), MR (OAPI patent), NL (European patent), NO, SE (European S patent), SN (OAPI patent), TD (OAPI patent), TG (OAPI patent), US.

Published With international search report.

Before the expiration of the time limit for amending the claims and to be republished in the event of the receipt of amendments.

(71) Applicant (for all designated States except US): BIOGEN, INC.

[US/US]; 14 Cambridge Center, Cambridge, MA 02142 (US).

(54) Title: DNA SEQUENCES, RECOMBINANT DNA MOLECULES AND PROCESSES FOR PRODUCING SOL- UBLE T4 PROTEINS (57) Abstract This invention relates to DNA sequences, recombinant DNA molecules and processes for producing soluble T4 protein. More particularly, this invention relates to DNA sequences that are characterized in that they code on expression in an appropriate unicellular host for soluble forms of T4, the receptor on the surface of T4+ lymphocytes, or derivatives thereof. In accordance with this invention, the DNA sequences, recombinant DNA molecules and processes of this invention may be employed to produce soluble T4 essentially free of other proteins of human origin. This soluble protein may then advantageously be used in the immunotherapeutic and diagnostic compositions and methods of this invention. The soluble T4-based immunotherapeutic compositions and methods of this invention are useful in treating immunodeficient patients suffering from diseases caused by infective agents whose primary targets are T4+ lymphocytes. According to a preferred embodiment, this invention relates to soluble T4-based compositions and methods which are useful in preventing, treating or detecting acquired immune deficiency syndrome, AIDS related complex and HIV infection.

I WO 89/01940 PCT/US88/02940 t1 DNA SEQUENCES, RECOMBINANT DNA MOLECULES AND PROCESSES FOR PRODUCING SOLUBLE T4 PROTEINS TECHNICAL FIELD OF INVENTION This invention relates to DNA sequences, recombinant DNA molecules and processes for producing soluble T4 proteins. More particularly, this invention relates to DNA sequences that are characterized in that they code on expression in an appropriate unicellular host for soluble forms of T4, the receptor on the surface of T4 lymphocytes, or derivatives thereof. In accordance with this invention, the DNA sequences, recombinant DNA molecules and processes of this invention may be employed to produce soluble T4 essentially free of other proteins of human origin.

This soluble protein may then advantageously be used in the immunotherapeutic, prophylactic, and diagnostic compositions and methods of this invention.

The soluble T4 protein-based immunotherapeutic compositions and methods of this invention are useful in treating immunodeficient patients suffering from diseases caused by infective agents whose primary targets are T4 lymphocytes. According to a preferred embodiment, this invention relates to soluble T4 protein-based compositions and methods which are useful in preventing, treating or detecting I.I. WO 89/01940 PCT/US88/02940 WO 89/01940 PCT/US88/02940 -2acquired immune deficiency syndrome, AIDS related complex and HIV infection.

BACKGROUND ART I The class of immune regulatory cells known as T cell lymphocytes can be divided into two broad functional classes, the first class comprising T 4 helper or inducer cells which mediate T cell pro- A liferation, lymphokine release and helper cell interi actions for Ig release, and the second class comprising T cytotoxic or suppressor cells which participate in T cell-mediated killing and immune response suppression. In general, these two classes of lymphocytes are distinguished by expression of one of two surface glycoproteins: T4 55,000-62,000 daltons) which is expressed on T helper or inducer cells, probably as a monomeric protein, or T8 (m.w.

32,000 daltons) which is expressed on T cytotoxic or suppressor cells as a dimeric protein.

SThe primary structures of T4 and T8 have been deduced from their respective cDNA sequences J. Maddon et al., "The Isolation and Nucleotide Sequence Of A cDNA Encoding The T Cell Surface Protein T4: A New Member Of The Immunoglobulin Gene Family", Cell, 42, pp. 93-104 (1985); D. R. Littman et al., "The Isolation And Sequence Of The Gene Encoding T8: A Molecule Defining Functional Classes Of T Lymphocytes", Cell, 40, pp. 237-46 (1985)]. Both predicted protein sequences define molecules with domains expected for surface antigens, including transmembrane and intracytoplasmic domains at the carboxyl end of the protein. In addition, both proteins contain an amino terminal region which shows striking homology to immunoglobulin and T cell receptor variable regions and which might function during target cell recognition [Maddon et al., supra].

WO 89/01940 PCT/US88/02940 -3- In immunocompetent individuals, T4 lymphocytes interact with other specialized cell types of the immune system to confer immunity to or defense against infection L. Reinhers and S. F.

Schlossman, "The Differentiation Function Of Human Lymp~oc-es T Cell, 19, pp. 821-27 (1980)]. More specifically, T4 lymphocytes stimulate production of growth factors which are critical to a functional immune system. For example, they act to stimulate B cells, the descendants of hemopoietic stem cells, which promote the production of defensive antibodies.

They also activate macrophages ("killer cells") to attack infected or otherwise abnormal host cells and they induce monocytes ("scavenger cells") to encompass and destroy invading microbes.

It has been found that the primary target of or receptor for certain infective agents is the T4 surface protein. These agents include, for example, viruses and retroviruses. When T4 lymphocytes are exposed to such agents, they are rendered nonfunctional. As a result, the host's complex immune defense system is destroyed and the host becomes susceptible to a wide range of opportunistic infections.

Such immunosuppression is seen in patients suffering from acquired immune deficiency syndrome AIDS is a disease characterized by severe or, typically, complete immunosuppression and attendant host susceptibility to a wide range of opportunistic infections and malignancies. In some cases, AIDS infection is accompanied by central nervous system disorders. Complete clinical manifestation of AIDS is usually preceded by AIDS related complex a syndrome accompanied by symptoms such as persistent generalized lymphadenopathy, fever and weight loss. The human immunodeficiency virus retrovirus is thought to be TO% WO 89/01940 PCT/US88/02940 -4the etiological agent responsible for AIDS infection' M. Poo/C_ and its precursor, ARC [1 C. Srndh--arn et al., "Detection, Isolation And Continuous Production Of Cytopathic Retroviruses (HTLV-III) From Patients With AIDS And Pre-AIDS", Science, 224, pp. 497-500 (1984)].* Between 85 and 100% of the AIDS/ARCS population test seropositive for HIV Shaw et al., "Molecular Characterization Of Human T-Cell Leukemia (Lymphotropic) Virus Type III In The Acquired Immune Deficiency Syndrome", Science, 226, pp. 1165-7) (1984)]. The number of adults in the United States infected with HIV has been estimated to be between 1 and 2.5 million Barnes, "Strategies For An AIDS Vaccine", Science, 233, pp. 1149-53 (1986); M. Rees, "The Sombre View Of AIDS", Nature, 326, pp. 343-45 (1987)]. These estimates include 64,900 individuals who do -not belong to an identified group at risk for AIDS L. Sivak and G. P. Wormser, "How Common Is HTLV-III Infection In The United States?", New Eng.

J. Med., 313, p. 1352 (1985)]. The apparent annual rate of diagnosis for those infected with HIV virus is between 1 and 2% a rate which may increase significantly in future years.

The genome of retroviruses, such as HIV, contains three regions encoding structural proteins.

The gag region encodes the core proteins of the virion. The pol region encodes the virion RNA-dependent DNA polymerase (reverse transcriptase). The 0 In this application, human immunodeficiency virus the generic term adopted by the human retrovirus subcommittee of the International Committee On Taxonomy Of Viruses to refer to independent isolates from AIDS patients, including human T cell lymphotropic virus type III ("HTLV-III"), lymphadenopathy-associated virus human immunodeficiency virus type 1 and AIDS-associated retrovirus will be used.

U I-i i i l o T O j S8 8/0 29 WO,9/01940 PCT/US8/02940 env region encodes the major glycoprotein found in the membrane envelope of the virus and in the cytoplasmic membrane of infected cells. The capacity of the virus to attach to target cell receptors and to cause fusion of cell membranes are two HIV properties controlled by the env gene. These properties are believed to play a fundamental role in the pathogenesis of the virus.

HIV env proteins arise from a precursor polypeptide that, in mature form, is cleaved into a large heavily glycosylated exterior membrane protein of about 481 amino acids gpl20 and a smaller transmembrane protein of about 345 amino acids which may be glycosylated gp41 Ratner et al., "Complete Nucleotide Sequence Of The AIDS Virus, HTLV-III", Nature, 313, pp. 277-84 (1985)].

The host range of the HIV virus is associated with cells which bear the surface glycoprotein T4. Such cells include T4 lymphocytes and brain cells J. Maddon et al., "The T4 Gene Encodes The AIDS Virus Receptor And Is Expressed In The Immune System And The Brain", Cell, 47, pp. 333-48 (1986)]. Upon infection of a host by HIV virus, the T4 lymphocytes are rendered non-functional. The progression of AIDS/ARCS syndromes can be correlated with the depletion of T4 lymphocytes, which display the T4 surface glycoprotein. This T cell depletion, with ensuing immunological compromise, may be attributable to both recurrent cycles of infection and lytic growth from cell-mediated spread of the virus.

In addition, clinical observations suggest that the HIV virus is directly responsible for the central nervous system disorders seen in many AIDS patients.

The tropism of the HIV virus for T4+ cells is believed to be attributed to the role of the T4 cell surface glycoprotein as the membrane-anchored virus receptor. Because T4 behaves as the HIV virus WO 89/01940 PCT/US88/02940 -6receptor, its extracellular sequence probably plays a direct role in binding HIV. More specifically, it is believed that HIV envelope selectively binds to the T4 epitope(s), using this interaction to initiate entry into the host cell G. Dalgelsts h et al., "The CD4 (T4) Antigen Is An. Essential Component Of The Receptor For The AIDS Retrovirus", Nature, 312, pp. 763-67 (1984); D. Klatzmann et al., "T-Lymphocyte T4 Molecule Behaves As The Receptor For Human Retrovirus LAV", Nature, 312, pp. 767-68 (1984)]. Accordingly, cellular expression of T4 is believed to be sufficient for HIV binding, with the T4 protein serving as a receptor for the HIV virus.

The T4 tropism of the HIV virus has been demonstrated in vitro. When HIV virus isolated from AIDS patients is cultured together with T helper lymphocytes preselected for surface T4, the lymphocytes are efficiently infected, display cytopathic effects, including multinuclear syncytia formation and are killed by lytic growth Klatzmann et al., "Selective Tropism Of Lymphadenopathy Associated Virus (LAV) For Helper-Inducer T Lymphocytes", Science, 225, pp. 59-63 (1984); F. Wong-Staal and R. C. Gallo, "Human T-Lymphotropic Retroviruses", Nature, 317, pp. 395-403 (1985)]. It has been demonstrated that a cloned cDNA version of human T4, when expressed on the surface of transfected cells from non-T cell lineages, including murine and fibroblastoid cells, endows those cells with the ability to bind HIV J. Maddon et al., "The T4 Gene Encodes The AIDS Virus Receptor And Is Expressed In The Immune System And The Brain", Cell, 47, pp. 333-48 (1986)].

During the course of HIV infection, the host mounts both a humoral and a cellular immune response to the virus. These responses include the appearance of antibodies which bind to a number of viral products and which exhibit neutralizing effect Tm WO 89/01940 PCT/US88/02940 -7or antibody dependent cellular cytotoxic functions [M..urff Rbrt et al., "HTLV-III-Neutralizing Antibodies In Patients With AIDS And AIDS-Related Complex", Nature, 316, pp. 72-74 (1985); F.

Barin et al., "Virus Envelope Protein Of HTLV-III Represents Major Target Antigen For Antibodies In AIDS Patients", Science, 228, pp. 1094-96 (1985); A. H. Rook et al., "Sera From HTLV-III/LAV Antibody Positive Individuals Mediate Antibody Dependent Cellular Cytotoxicity Against HTLV-III/LAV Infected T Cells", J. Immunol., 138, pp. 1064-67 (1987)].

Epitopes of the HIV envelope have been identified as important determinants in eliciting a neutralizing antibody response. And, determinants in antibody dependent cellular cytotoxicity ("ADCC") activity include HIV env and, possibly, gag epitopes.

In the absence to date of effective treatments for AIDS, many efforts have centered on prevention of the disease. Such preventative measures include HIV antibody screening for all blood, organ and semen donors and education of AIDS high-risk groups regarding transmission of the disease.

Experimental or early-stage clinical treatment of AIDS and ARCS conditions have included the administration of antiviral drugs, such as HPA-23, phosphonoformate, suramin, ribavirin, azidothymidine and dideoxycytidine, which apparently interfere with replication of the virus through reverse transcriptase inhibition. Although each of these drugs exhibits activity against HIV in vitro, only AZT has demonstrated potential benefits in clinical trials. AZT administration in effective amounts, however, has been accompanied by undesirable and debilitating side effects, such as bone marrow depression. It is likely, therefore, that hematologic toxicity will be a major rate limiting factor in the long term use of AZT.

4/ C11 UJ

WT_

WO 89/01940 PCT/US88/02940 -8- Other proposed methods for treating AIDS have focused on the development of agents having activity against steps in the viral replicative cycle other than reverse transcription. Such methods include the administration of interferons or the application of hybridoma technology. Most of these treatment strategies are expected to require the co-administration of immunomodulators, such as interleukin-2.

To date, the need exists for .the development of effective immunotherapeutic agents and methods for the treatment of AIDS, ARCS, HIV infection and other immunodeficiencies caused by T lymphocyte depletion or abnormalities.

DISCLOSURE OF THE INVENTION The present invention solves the problems referred to above by providing, in large amounts, soluble T4 and soluble derivatives thereof that act as receptors for infective agents whose primary target is the T4 surface protein of T4 lymphocytes. Advantageously, this invention also provides soluble T4 essentially free of other proteins of human origin and in a form that is not contaminated by viruses, such as HIV or hepatitis B virus.

As will be appreciated from the disclosure to follow, the DNA sequences and recombinant DNA molecules of this invention are capable of directing, in an appropriate host, the production of soluble T4 or derivatives thereof. The polypeptides of this invention are useful, either as produced in the host or after further derivatization or modification, in a variety of immunotherapeutic compositions and methods for treating immunodeficient patients suffering from diseases caused by infective agents whose primary targets are T4 lymphocytes. According to various embodiments of this invention, such compo- WO 89/01940 PCT/US88/02940 -9sitions and methods relate to a soluble receptor for HIV, soluble T4 proteins and polypeptides and antibodies thereto. The soluble T4 proteins and polypeptides of this invention include monovalent, as well as polyvalent forms.

The compositions and methods of this invention, which are based upon soluble T4 proteins, polypeptides or peptides and antibodies thereto, are particularly useful for the prevention, treatment or detection of the HIV-related infections AIDS and ARC. More specifically, the soluble T4-based compositions and methods of this invention employ soluble T4-like polypeptides polypeptides which advantageously interfere with the T4/HIV interaction by blocking or competitive binding mechanisms which inhibit HIV infection of cells expressing the T4 surface protein. These soluble T4-like polypeptides inhibit adhesion between T4+ lymphocytes and infective agents which target T4+ lymphocytes and inhibit interaction between T4 lymphocytes and antigen presenting cells and targets of T4+ lymphocytes mediated killing. By acting as soluble virus receptors, the compositions of this invention may be used as antiviral therapeutics to inhibit HIV binding to T4 cells and virally induced syncytium formation at the level of receptor binding.

This invention accomplishes these goals by providing DNA sequences coding on expression in an appropriate unicellular host for soluble T4 proteins* and soluble derivatives thereof.

As used in this application, "soluble T4 protein", "soluble T4" and "soluble T4-like polypeptides" include all proteins, polypeptides and peptides which are natural or recombinant soluble T4 proteins, or soluble derivatives thereof, and which are characterized by the immunotherapeutic (anti-retroviral) (footnote continued on following page) WO 89/01940 PCT/US88/02940 i i This invention also provides recombinant DNA molecules containing those DNA sequences and V unicellular hosts transformed with them. Those hosts Spermit the production of large quantities of the novel soluble T4 proteins, polypeptides, peptides I and derivatives of this invention for use in a wide variety of therapeutic, prophylactic and diagnostic compositions and methods.

The DNA sequences of this invention are selected from the group consisting of: the DNA inserts of p199-7, pBG377, pBG380, pBG381, p203-5, pBG391, pBG392, pBG393, pBG394, pBG395, pBG396, pBG397, p211-11, p214-10 S and p215-7; 15 DNA sequences which hybridize to one or more of the foregoing DNA inserts and which code i on expression for a soluble T4-like polypeptide; and DNA sequences which code on expression for a soluble T4-like polypeptide coded for on expression by any of the foregoing DNA inserts and sequences.

According to an alternate embodiment, this invention also relates to a DNA sequence comprising the DNA insert of p170-2, said sequence coding on expression for a T4-like polypeptide. And, this invention also relates to recombinant DNA molecules and processes for producing T4 protein using that DNA sequence.

(footnote continued from preceding page) or immunogenic activity of soluble T4 protein. They include soluble T4-like compounds from a variety of sources, such as soluble T4 protein derived from natural sources, recombinant soluble T4 protein and synthetic or semi-synthetic soluble T4 protein.

WO,89/01940 PCT/US88/02940 -11- BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is an autoradiograph depicting the purification of T4 protein from U937 cells by immunoaffinity chromatography.

Figure 2 depicts autoradiograph and Western blot data demonstrating that immunoaffinity-purified, solubilized native T4 protein binds to HIV envelope protein.

Figure 3 depicts the nucleotide sequence and the derived amino acid sequence of T4 cDNA obtained from PBL clone X203-4. In this figure, the amino acids are represented by single letter codes as follows: Phe: F Leu: L Ile: I Met: M Val: V Ser: S Pro: P Thr: T Ala: A Tyr: Y His: H Gln: Q Asn: N Lys: K Asp: D Glu: E Cys: C Trp: W Arg: R Gly: G position at which a stop codon is present.

In Figure 3, the T4 protein translation start (AA_ 23 is located at the methionine at nucleoides 201-203 and the mature N-terminus is located at the lysine (AA 3 at nucleotides 276-278.

Figure 4 is a schematic outline of the construction of cDNA clones pBG312.T4 (also called p171-1) and p170-2.

Figure 5 is a schematic outline of the construction of plasmid pEC100.

Figure 6 depicts amino acid comparisons at a positions 3, 64 and 231 of various T4 cDNA clones.

Figures 7A and 7B depict the protein domain structure of purified,solubilized T4 protein and recombinant soluble T4 mutants.

Figures 8A-8D are schematic outlines of constructions of various intermediate plasmids and other plasmids used to express recombinant soluble T4 ("rsT4") of this invention.

L

WOo 89/01940 PCT/US88/02940 -12- Figure 9A is a schematic outline of the construction of plasmid p199-7.

Figures 9B and 9C are schematic outlines of the construction of plasmid p203-5.

Figure 10 depicts the synthetic oligonucleotide linkers employed in various constructions i according to this invention.

Figure 11 depicts the nucleotide sequence ti of the entire plasmid defined by p199-7 (PLmutet.rsT 4 and its rsT4.2 insert and the amino acid sequence deduced from the rsT4 sequence. This includes the ClaI-ClaI cassette which defines the Met perfect rsT4.2 coding sequence.

Figure 12 depicts a protein blot analysis of an induction of rsT4.2 expression from i SG936/p199-7.

Figure 13 is a schematic outline of the construction of plasmid pBG368.

Figures 14A-14C are schematic outlines of constructions of various plasmids of this invention.

Figure 15 depicts the nucleotide sequence of plasmid pBG391.

Figure 16 depicts the nucleotide sequence of plasmid pBG392. In this figure, the T4 protein translation start (AA 2 3 is located at the methionine at nucleotides 1207-1209 and the mature N-terminus is located at the lysine (AA 3 at nucleotide 1281-84.

Figure 17 is a schematic outline of constructions of various plasmids of this invention.

Figure 18 depicts the synthetic oligonucleotide linkers employed in various constructions according to this invention.

Figure 19 depicts the nucleotide sequence of plasmid pBG394.

Figure 20 depicts the nucleotide sequence of plasmid pBG396.

WO 89/01940 PCT/US88/02940 -13- Figure 21 depicts the nucleotide sequence of plasmid pBG393.

Figure 22 depicts the nucleotide sequence of plasmid pBG395.

Figure 23 is a Coomassie stained gel of rsT4.2 purified from the conditioned medium of the pBG380 transfected CHO cell line BG380G of plasmid p196-10.

Figure 24 is a schematic outline of the construction of plasmid p196-10.

Figure 25 is a schematic outline of the construction of plasmid pBG394.

Figure 26 is a schematic outline of the construction of plasmid p211-11.

Figure 27 is a schematic outline of the construction of plasmid p215-7.

Figure 28 is a schematic outline of the construction of plasmid p218-8.

Figure 29A is a Coomassie stained gel of rsT4.113.1 purified from the conditioned medium of pBG211-11 transfected E.coli.

Figure 29B is an autoradiograph depicting a Western blot analysis of rsT4.113.1 expressed in E.coli.

Figure 30, panels depict the purification of rsT4.113.1 from E.coli transformants.

Figure 31, panels depict the refolding of purified rsT4.113.1.

Figure 32 is an autoradiograph depicting the immunoprecipitation of 35S-metabolically labelled CHO cell lines producing recombinant soluble T4.

Figure 33 depicts an immunoblot analysis of COS 7 cell lines producing recombinant soluble T4.

Figure 34 depicts in graphic form the results of a competition assay between rsT4.113.1 and rsT4.3 for binding to OKT4A or OKT4.

L I~ ~I1C~ i i WO 89/01940 PCT/US88/02940 -14- Figures 35-37 depict in graphic form the results of competition assays between rsT4.111 and rsT4.3 for binding to, respectively, OKT4A, Leu-3A and OKT4.

Figure 38 depicts in graphic form an ELISA assay for rsT4.113.1 from E.coli transformants.

Figure 39 depicts in graphic form the results of a p24 radioimmunoassay using recombinant soluble T4 according to this invention.

Figures 40 and 41 depict the results of syncytia inhibition assays using recombinant soluble T4 proteins according to this invention.

Figure 42 is a schematic outline of the construction of plasmid pBiv.l.

Figure 43 depicts the bivalent recombinant soluble T4 protein produced by pBiv.l.

DETAILED DESCRIPTION OF THE INVENTION We isolated the DNA sequences of this invention from two libraries: a Ngt cDNA library derived the T cell tumor line REX and a Xgtl0 cDNA library derived from peripheral blood lymphocytes.

However, we could also have employed libraries prepared from other cells that express T4. These include, for example, H9 and U937. We also used a human genomic bank to isolate various fragments of the T4 gene.

For screening these libraries, we used a series of chemically synthesized anti-sense oligonucleotide DNA probes based upon the T4 protein sequence set forth in Maddon et al. (1985), supra.

For screening, we hybridized our oligonucleotide probes to our cDNA libraries utilizing a plaque hybridization screening assay. We selected clones hybridizing to several of our probes. And, after isolating and subcloning the cDNA inserts of the selected clones into plasmids, we determined ii WO 89/01940 PCT/US88/02940 their nucleotide sequences and compared the amino acid sequences deduced from those nucleotide sequences to the amino acid sequences referred to in Maddon et al. (1985), supra. As a result of these comparisons, we determined that all of our selected clones were characterized by cDNA inserts coding for amino acid sequences of human T4.

We have depicted in Figure 3 the nucleotide sequence of full-length T4 cDNA obtained from deposited clone p170-2 and the amino acid sequence deduced therefrom. That cDNA sequence was subsequently subjected to in vitro site-directed mutagenesis and restriction fragment substitution so that its cDNA sequence was identical to that of Maddon et al.

After modifying our T4 cDNA sequence to be identical to that of Maddon et al., we truncated samples of it in various positions to remove the coding regions for the transmembrane and intracytoplasmic domains. The remaining cDNA sequences encoded a soluble T4 which retained the extracellular region believed to be responsible for HIV binding.

We then constructed various clones characterized by such cDNA inserts coding for human soluble T4. Those cDNA sequences may be used in a variety of ways in accordance with this invention. More particularly, those sequences or portions of them, or synthetic or semi-synthetic copies of them, may be used as DNA probes to screen other human or animal cDNA or genomic libraries to select by hybridization other DNA sequences that are related to soluble T4.

Typically, conventional hybridization conditions, about 200 to 27°C below Tm, are employed in such selections. However, less stringent conditions may be necessary when the library is being screened with a probe from a different species than that from I WO89/01940 PCT/US88/02940 -16which the library is derived, the screening of a mouse library with a human probe.

Such cDNA inserts, portions of them, or synthetic or semi-synthetic copies of them, may also be used as starting materials to prepare various mutations. Such mutations may be either degenerate, the mutation does not change the amino acid sequence encoded by the mutated codon, or nondegenerate, the mutation changes the amino acid sequence encoded by the mutated codon. Both types of mutations may be advantageous in producing or using soluble T4's according to this invention.

For example, these mutations may permit higher levels of production or easier purification of soluble T4 or higher T4 activity.

For all of these reasons, the DNA sequences of this invention are selected from the group consisting of: the DNA inserts of p199-7, pBG377, pBG380, pBG381, p203-5, pBG391, pBG392, pBG393, pBG394, pBG395, pBG396, pBG397, p211-11, p214-10 and p215-7; DNA sequences which hybridize to one or more of the foregoing DNA inserts and which code on expression for a soluble T4-like polypeptide; and DNA sequences which code on expression for a soluble T4-like polypeptide coded for on expression by any of the foregoing DNA inserts and sequences.

Preferably, the DNA sequences of this invention code for a polypeptide selected from the group consisting of a polypeptide of the formula AA_23-AA362 of Figure 3, a polypeptide of the formula AA1-362 of Figure 3, a polypeptide of the formula Met-AA -362 of Figure 3, a polypeptide of the formula AA1-374 of Figure 3, a polypeptide of the formula Met-AA-374 of Figure 3, a polypeptide of the formula AA1- 377 of Figure 3, a polypeptide of the formula

-J-

WO 8901940 PCT/US88/02940 -17- Met-AAI_377 of Figure 3, a polypeptide of the formula AA-23-AA 3 7 4 of Figure 3, a polypeptide of the formula AA 23-AA 3 7 7 of Figure 3, or portions thereof.

DNA sequences according to this invention also preferably code for a polypeptide selected from the group consisting of a polypeptide of the formula AA-23-AA182 of Figure 16, a polypeptide of the formula AAl-AA82 of Figure 16, a polypeptide of the formula Met-AA 1 _182 of Figure 16, a polypeptide of the formula AA 2 3

-AA

1 8 2 of Figure 16, followed by the amino acids asparagine-leucine-glutamine-histidine- I serine-leucine, a polypeptide of the formula AAI-AA182 of Figure 16, followed by the amino acids asparagine-leucine-glutamine-histidine-serine-leucine, a polypeptide of the formula Met-AA 1 82 of Figure 16, followed by the amino acids asparagine-leucineglutamine-histidine-serine-leucine, a polypeptide of the formula AA_-23AAl3 of Figure 16, a polypeptide of the formula AA 11-AA3 of Figure 16, a polypeptide of the formula Met-AA- 1 1 3 of Figure 16, a polypeptide of the formula AA 2 3

-AA

1 1 of Figure 16, a polypeptide of the formula AAI-AA 1 1 1 of Figure 16, a polypeptide of the formula MAAe-AAi311 of Figure 16, a polypeppetide of the formula AA_ -AA of Figure 16, a polyp peptide of the formula e AA -AA 3 1 f Figure 16, a poly Spolypeptide of the formula MA2-AA 14 of Figure 16, a polypeptide of the formula AA-AA14 of Figure 16, a polypeptide of the formula Met-AA 1 _145 of Figure 16, a polypeptide of the formula AA23AA66 of Figure 16, Sa polypeptide of the formula AAI-AA66 of Figure 16, a polypeptide of the formula Met-AA166 of Figure 16, a polypeptide of the formula Met-AA 1 6 of Figure 16, of mature T4 protein, a polypeptide of the formula WO89/01940 PCT/US88/02940 -18- AA1-362 of mature T4 protein, a polypeptide of the formula Met-AA 1 362 of mature T4 protein, a polypeptide of the formula AA 1 _374 of mature T4 protein, a polypeptide of the formula Met-AA 1 -374 of mature T4 protein, a polypeptide of the formula AA-37 of protein, a polypeptide of the formula AA of mature T4 protein, a polypeptide of the formula Met-AA_ 3 7 7 of mature T4 protein., a polypeptide of the formula AA_ 23 -AA374 of mature T4 protein, a polypeptide of the formula AA- 2

AA

3 7 7 of mature T4 protein, or portions thereof.

DNA sequences according to this invention also code for a polypeptide selected from the group consisting of a polypeptide of the formula AA -AA -23of mature T4 protein, a polypeptide of the formula of mature T4 protein, a polypeptide of the formula AA-AA82 of mature T4 protein, a polypeptide of the formula Met-AA 1 82 of mature T4 protein, a polypeptide of the formula AA- 23 -AA182 of mature T4 protein, followed by the amino acids asparagine-leucineglutamine-histidine-serine-leucine, a polypeptide of the formula AA 1

-AA

182 of mature T4 protein, followed by the amino acids asparagine-leucine-glutaminehistidine-serine-leucine, a polypeptide of the formula Met-AA 1 _182 of mature T4 protein, followed by the amino acids asparagine-leucine-glutaminehistidine-serine-leucine, a polypeptide of the formula AA-23-AA13 of mature T4 protein, a polypeptide of the formula AAl-AAl3 of mature T4 protein, a polypeptide of the formula Met-AA_-113 of mature T4 protein, a polypeptide of the formula AA -23AA ll of mature T4 protein, a polypeptide of the formula I AA AAI 11 of mature T4 protein, a polypeptide of the formula Met-AAl 111 of mature T4 protein, a polypeptide of the formula AA- 23

-AA

1 31 of mature T4 protein, a polypeptide of the formula AA -AA 131 of mature T4 protein, a polypeptide of the formula Met-AA -131 of mature T4 protein, a polypeptide of the formula AA-23-AA145 of mature T4 protein, a polypeptide of 1 1 1 1 WO89/01940 PCT/US88/02940 -19the formula AA -AA145 of mature T4 protein, a polypeptide of the formula Met-AA 1 -145 of mature T4 protein, a polypeptide of the formula AA_23-AA166 of mature T4 protein, a polypeptide of the formula AAl-AA166 of mature T4 protein, a polypeptide of the formula Met-AA 1 1 66 of mature T4 protein, or portions thereof.

The amino terminal amino acid of mature T4 protein isolated from T cells begins at lysine, the third amino acid of the sequence depicted in Figure 16. Accordingly, soluble T4 proteins also include polypeptides of the formula AA 3 -AA377 of Figure 16, or portions thereof. Such polypeptides include polypeptides selected from the group consisting of a polypeptide of the formula AA 3 to AA 3 6 2 of Figure 16, a polypeptide of the formula AA 3 to AA 3 7 4 of Figure 16, a polypeptide of the formula AA3-AA182 of Figure 16, a polypeptide of the formula AA 3

AA

1 1 3 of Figure 16, a polypeptide of the formula AA 3

-AA

1 3 1 of Figure 16, a polypeptide of the formula AA 3

-AA

1 4 of Figure 16, a polypeptide of the formula AA 3

-AA

1 6 6 of Figure 16, and a polypeptide of the formula AA3-AA111 of Figure 16. Soluble T4 proteins also include the above-recited polypeptides preceded by an N-terminal methionine group.

Soluble T4 protein constructs according to this invention may also be produced by truncating the full length T4 protein sequence at various positions to remove the coding regions for the transmembrane and intracytoplasmic domains, while retaining the extracellular region believed to be responsible for HIV binding. More particularly, soluble T4 polypeptides may be produced by conventional techniques of oligonucleotide directed mutagenesis; restriction digestion, followed by insertion of linkers; or chewing back full length T4 protein with enzymes.

l 1 WO 89/01940 PCT/US88/02940 Alternatively, soluble T4 polypeptides may be chemically synthesized by conventional peptide synthesis techniques, such as solid phase synthesis B. Merrifield, "Solid Phase Peptide Synthesis.

I. The Synthesis Of A Tetrapeptide", J. Am. Chem.

Soc., 83, pp. 2149-54 (1963)].

The DNA sequences of this invention code for soluble proteins and derivatives that are believed to bind to Major Histocompatibility Complex antigens and envelope glycoprotein of certain retroviruses, such as HIV. Preferably, they also inhibit syncytium formation, believed to be the mode of intracellular HIV virus spread. And, they may inhibit interaction between T4+ lymphocytes and antigen-presenting cells and targets of T4 cell mediated killing. Most preferably, they also inhibit adhesion between T4 lymphocytes and infective agents, such as the HIV virus, whose primary targets are T4 lymphocytes.

The DNA sequences of this invention are also useful for producing soluble T4 or its derivatives coded for on expression by them in unicellular hosts transformed with those DNA sequences. As well known in the art, for expression of the DNA sequences of this invention, the DNA sequence should be operatively linked to an expression control sequence in an appropriate expression vector and employed in that expression vector to transform an appropriate unicellular host.

Such operative linking of a DNA sequence of this invention to an expression control sequence, of course, includes the provision of a translation start signal in the correct reading frame upstream of the DNA sequence. If the particular DNA sequence of this invention being expressed does not begin with a methionine, the start signal will result in an additional amino acid methionine being located at the N-terminus of the product. While 1 i WO3'9/01940 PCT/US88/02940 -21such methionyl-containing product may be employed directly in the compositions and methods of this invention, it is usually more desirable to remove the methionine before use. Methods are available in the art to remove such N-terminal methionines from polypeptides expressed with them. For example, certain hosts and fermentation conditions permit removal of substantially all of the N-terminal methionine in vivo. Other hosts require in vitro removal of the N-terminal methionine. However, such in vivo and in vitro methods are well known in the art.

A wide variety of host/expression vector combinations may be employed in expressing the DNA sequences of this invention. Useful expression vectors, for example, may consist of segments of chromosomal, non-chromosomal and synthetic DNA sequences, such as various known derivatives of and known bacterial plasmids, plasmids from E.coli including col El, pCR1, pBR322, pMB9 and their derivatives, wider host range plasmids, RP4, phage DNAs, the numerous derivatives of phage X, NM989, and other DNA phages, M13 and filamenteous single stranded DNA phages, yeast plasmids, such as the 2p plasmid or derivatives thereof, and vectors derived from combinations of plasmids and phage DNAs, such as plasmids which have been modified to employ phage DNA or other expression control sequences. For animal cell expression, we prefer to use plasmid pBG368, a derivative of pBG312 Cate et al., "Isolation Of The Bovine And Human Genes For Mullerian Inhibiting Substance And Expression Of The Human Gene In Animal Cells", Cell, pp. 685-98 (1986)] which contains the major late promoter of adenovirus 2.

In addition, any of a wide variety of expression control sequences sequences that con- WO: 89/01940 PCT/US88/02940 -22trol the expression of a DNA sequence when operatively linked to it may be used in these vectors to express the DNA sequence of this invention. Such useful expression control sequences, include, for example, the early and late promoters of SV40 or the adenovirus, the lac system, the trp system, the TAC or TRC system, the major operator and promoter regions of phage X, the control regions of fd coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, Pho5, the promoters of the yeast a-mating factors, the polyhedron promoter of the baculovirus system and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof. For animal cell expression, we prefer to use an expression control sequence derived from the major late promoter of adenovirus 2.

i A wide variety of unicellular host cells |1 20 are also useful in expressing the DNA sequences of this invention. These hosts may include well known eukaryotic and prokaryotic hosts, such as strains of E.coli, Pseudomonas, Bacillus, Streptomyces, fungi, such as yeasts, and animal cells, such as CHO and mouse cells, African green monkey cells, such as COS 1, COS 7, BSC 1, BSC 40, and BMT 10, insect cells, and human cells and plant cells in tissue culture.

SFor animal cell expression, we prefer CHO cells and COS 7 cells.

It should of course be understood that not all vectors and expression control sequences will function equally well to express the DNA sequences of this invention. Neither will all hosts function equally well with the same expression system. However, one of skill in the art may make a selection among these vectors, expression control sequences, and hosts without undue experimentation and without WO 89/0194..PCT/US88/02940 W089/0ssl1940 PCT/US88/02940 -23departing from the scope of this invention. For example, in selecting a vector, the host must be considered because the vector must replicate in it.

The vector's copy number, the ability to control that copy number, and the expression of any other proteins encoded by the vector, such as antibiotic markers, should also be considered.

In selecting an expression control sequence, a variety of factors should also be considered.

These include, for example, the relative strength of the system, its controllability, and its compatibility with the particular DNA sequence of this invention, particularly as regards potential secondary structures. Unicellular hosts should be selected by consideration of their compatibility with the chosen vector, the toxicity of the product coded for on expression by the DNA sequences of this invention to them, their secretion characteristics, their ability to fold proteins correctly, their fermentation requirements, and thle ease of purification of the products coded on expression by the DNA sequences of this invention.

Within these parameters, one of skill in the art may select various vector/expression control system/host combinations that will express the DNA sequences of this invention on fermentation or in large scale animal culture, CHO cells or COS 7 cells.

The polypeptides produced on expression of the DNA sequences of this invention may be isolated from the fermentation or animal cell cultures and purified using any of a variety of conventional methods. One of skill in the art may select the most appropriate isolation and purification techniques without departing from the scope of this invention.

The polypeptides produced on expression of the DNA sequences of this invention are essentially WO 89/01940 PCT/US88/02940 -24free of other proteins of human origin. Thus, they are different than T4 protein purified from human lymphocytes.

The polypeptides of this invention are useful in immunotherapeutic compositions and methods.

For example, the polypeptides of this invention are active in inhibiting infection by agents whose primary targets are T4 lymphocytes by interfering with their interaction with those target lymphocytes. More preferably, the polypeptides of this invention may be employed to saturate the T4 receptor sites of T4targeted infective agents. Thus, they exert antiviral activity by competitive binding with cell surface T4 receptor sites. This effect is plainly of great utility in diseases, such as AIDS, ARC and HIV infection. Accordingly, the polypeptides and methods of this invention may be used to treat humans having AIDS, ARC, HIV infection or antibodies to HIV. In addition, these polypeptides and methods may be used for treating AIDS-like diseases caused by retroviruses, such as simian immunodeficiency viruses, in mammals, including humans.

According to one embodiment of this inv.ention, antibodies to soluble T4 proteins and polypeptides may be used in the treatment, prevention, or diagnosis of AIDS, ARC and HIV infection.

The polypeptides of this invention may also be used in combination with other therapeutics used in the treatment of AIDS, ARC and HIV infection.

For example, soluble T4 polypeptides may be used in combination with anti-retroviral agents that block reverse transcriptase, such as AZT, HPA-23, phosphonoformate, suramin, ribavirin and dideoxycitidine. Additionally, these polypeptides may be used with anti-viral agents such as interferons, including alpha interferon, beta interferon and gamma interferon, or glucosidase inhibitors, such as ,WO89/01940 PCT/I!S88/02940 castanospermine. Such combination therapies advantageously utilize lower dosages of those agents, thus avoiding possible toxicity.

And, the polypeptides of this invention may be used in plasmapheresis techniques or in blood bags for selective removal of viral contaminants from blood. According to this embodiment of the invencion, soluble T4 polypeptides may be coupled to a solid support, comprising, for example, plastic or glass beads, or a filter, which is incorporated into a plasmapheresis unit.

Additionally, the compositions of this invention may be employed as immunosuppressants useful in preventing or treating graft-vs-host disease, i 15 autoimmune diseases and allograft rejection.

The compositions of this invention typically comprise an immunotherapeutic effective amount of a polypeptide of this invention and a pharmaceutically acceptable carrier. Therapeutic methods of this invention comprise the step of treating patients in a pharmaceutically acceptable manner with those compositions.

The compositions of this invention for use in these therapies may be in a variety of forms.

I 25 These include, for example, solid, semi-solid and liquid dosage forms, such as tablets, pills, powders, liquid solutions or suspensions, liposomes, suppositories, injectable and infusable solutions. The preferred form depends on the intended mode of administration and therapeutic application. The composi- B:l tions also preferably include conventional pharmaceutically acceptable carriers and adjuvants which are known to those of skill in the art.

Generally, the pharmaceutical compositions of the present invention may be formulated and administered using methods and compositions similar to those used for other pharmaceutically important poly- WO 89/01940 PCT/US88/02940 -26peptides alpha-interferon). Thus, the polypeptides may be stored in lyophilized form, reconstituted with sterile water just prior to administration, and administered by the usual routes of administration such as parenteral, subcutaneous, intravenous, intramuscular or intralesional routes. An effective dosage may be in the range of from 0.5 to 5.0 mg/kg body weight/day, it being recognized that lower and higher doses may also be useful.

This invention also relates to soluble receptors and their use in diagnosing or treating viral agents which target or bind to those receptors.

Such soluble receptors may be used as decoys to absorb viral agents and to halt the spread of viral infection. Alternatively, virus-killing agents may be attached to the soluble protein receptors, providing a direct mode of delivery of those agents to the virus.

More particularly, the polypeptides of this invention are useful in diagnostic compositions and methods to detect or monitor the course of HIV infection. Advantageously, these polypeptides are useful in diagnosing variants of the HIV virus, regardless of origin of the infecting HIV agent.

For example, soluble T4 proteins and polypeptides according to this invention, which have a high affinity for HIV, may be advantageously used to increase the sensitivity of HIV assay systems now based upon monoclonal or polyclonal antibodies.

More specifically, soluble T4 proteins and polypep- 1 tides may be used to pretreat test plasma to concentrate any HIV present, even in small amounts, so that it is more easily recognized by the antibody.

And soluble T4 proteins and polypeptides may be used to purify the HIV envelope protein Alternatively, the soluble T4 proteins and polypeptides of this invention may be used to replace L, 1 i i WO 89/01940 PCT/US88/02940 -27anti-HIV antibodies now used in various assays.

These soluble T4 proteins and polypeptides are be preferable to anti-HIV antibodies for two reasons.

First, soluble T4, exhibits an affinity for HIV of approximately 10 a level which exceeds the 10 to 10 values of anti-HIV antibodies. And, while anti-HIV antibodies are more likely to be specific for different HIV isolates, strain variations would not affect a soluble T4 protein-based assay, since all HIV isolates must be capable of interacting with the T4 receptor as a prerequisite to infectivity.

For example, a soluble T4 protein or polypeptide may be linked to an indicator, such as an enzyme, and used in an ELISA assay. Here, soluble T4 advantageously acts as a measure of both HIV in a test sample and any free HIV envelope gpl20 protein.

And, polyvalent forms of soluble T4 proteins or polypeptides may be produced, for example, by chemical coupling or genetic fusion techniques, thus increasing even further the avidity of soluble T4 for HIV.

In order that this invention may be better understood, the following examples are set forth.

These examples are for purposes of illustration only, and are not to be construed as limiting the scope of the invention in any manner.

EXAMPLES

Purification Of Native Solubilized T4 We purified native T4 from the T4 -promonocytic cell line U937 derived from a histocytic lymphoma to approximately 50% purity using immunoaffinity chromatography as follows.

We grew U937 cells [a gift from Dr. Scott Hammer, New England Deaconess Hospital] to 106 cells/ml in RPMI 1640, 10% FCS, harvested and washed them in 1X PBS. We then lysed the cell pellet WO 89/01940 PCT/US88/02940 in 20 mM Tris-HC1 (pH 0.5% NP-40 (a non-ionic detergent), 0.2% NaDOC, 0.2 mM EGTA, 0.2 mM PMSF and 7 pg/ml BPTI at 4 x 10 cells/ml. Because this purification was carried out in the presence of a non-ionic detergent, T4, which is normally membranebound via its hydrophobic transmembrane domain, was isolated as a solubilized protein. We spun the lysate in a GS 3 rotor for 10 min at 10,000 rpm and stored the supernatant at -70 0

C.

Subsequently, we preabsorbed the clarified cell extract with mouse IgG-Sepharose, followed by protein A Sepharose and then passed the flowthrough through an immunoaffinity column comprising immobilized 19Thy anti-T4 monoclonal antibody on [a gift from Dr. Ellis Reinherz, Dana Farber Cancer Institute, Boston, Massachusetts]. We washed the I column extensively and eluted the bound material with 50 mM glycine-HCl (pH 0.15 M NaCl, 5 pg/ml BPTI and 0.2 mM EGTA.

We then separated 10 pl aliquots of each elution fraction on a 10% SDS-PAGE under reducing conditions, with the bands being visualized by silver staining. As shown in Figure 1, a major silverstained band of 55 Kd was visible. We then carried out two assays on the 55 Kd protein and sequenced the amino terminus of the protein to confirm its identity as native solubilized T4.

Sequencing Of Native Solubilized T4 We determined the N-terminal amino acid sequence of our solubilized native T4 which we isolated from a detergent extract of U937 cells by immunoaffinity chromatography as described above.

Techniques for determining the amino acid sequences of various proteins and peptides derived from them are well known in the art. We chose automated Edman degradation to determine the amino WO 89/01940 PCT/US88/02940 -29terminus of our solubilized native T4. More specifically, we gel purified and electroeluted approximately 5 pg of the solubilized native T4 and then subjected it to automated Edman degradation using a gas phase sequencer (Applied Biosystems 470A). We then identified the PTH-amino acids produced at each cycle of the Edman chemistry by high pressure liquid chromatography, on-line with the sequencer, in a PTH-amino acid analyzer (Applied Biosystems 120A).

Direct analysis of the protein provided amino terminal sequence information which, when compared to the amino acid sequence deduced from the cDNA sequence of human T4 [Maddon et al. (1985), supra], identified the purified protein as human T4.

Radioimmunoassay Of Native Solubilized T4 To determine that our purification process enriched for T4, we assayed fractions from the immunoaffinity elution step in a T4-specific sandwich radioimmunoassay, based upon the ELISA assay of P. E.

Rao et al., in Cellular Immunology, 80, pp. 310-19 (1983). We coated each well of a Removawell strip (Dynatecn Labs, Alexandria, Virginia) with 50 1l of pl/ml OKT4 antibody (ATCC #CRL 8002) or MOPC195 (a background binding control) in 0.05 M sodium bicarbonate buffer (pH 9.4) at 4 0 C overnight. We washed the wells and then filled them with 1% FCS in PBS to saturate the protein binding capacity of the plastic. After removing the 1% FCS solution, we added test samples, in 50 pl aliquots, to the wells.

We then incubated the samples for 4 hours at room temperature. Subsequently, we removed the samples and washed the wells four times with 0.05% 125 in PBS. We then added I-labelled 19Thy antibody (50,000-100,000 cpm per well) and incubated the wells at 4 0 C overnight. We then washed the wells four

J

WO 89/01940 PCT/US88/02940 125 times and separated each well for bound 125I detection in a Beckman gamma detector.

As shown in Figure 1, in which values were plotted following subtraction for background, the peak fraction of solubilized native T4 protein detected by radioimmunoassay coincided with elution of the 55 Kd protein seen by silver staining.

Western Blot Assay For T4 Although many antibodies have been developed for detecting T4 antigen, none are useful for protein blot analysis (Dr. Ellis Reinherz, personal communication). In order to develop antibodies useful for Western blot detection of soluble T4 to follow the purification of T4 and recombinant soluble T4, we raised polyclonal, hyperimmune anti-T4 antisera in rabbits against three synthetic T4 oligopeptides.

follows: Oligopeptide Amino Acid Coordinates JB-1 44-63 JB-2 133-156 JB-3 325-343 We had previously synthesized these peptides using conventional phosphoamide DNA synthesis techniques.

See, Tetrahedron Letters, 22, pp. 1859-62 (1981). We synthesized the peptides on an Applied Biosystems 380A DNA Synthesizer and purified them by gel electrophoresis.

Coupling Of T4 Peptides To BTG We coupled each of these peptides to the carrier protein bovine thyrogobulin [Sigma, St. Louis, Missouri] according to a modification of procedures set forth in J. Rothbard et al., J. Exp.

Med., 160, pp. 208-21 (1984) and R. C. Kennedy et al., "Antiserum To A Synthetic Peptide Recognizes The xl- WO 89/01940 PCT/US88/02940 -31- HTLV-III Envelope Glycoprotein", Science, 231, pp. 1556-59 (1986).

More specifically, we mixed 10 mg of BTG diluted in 1 ml of PBS with 1.3 mg of m-maleimidobenzoyl-N-hydroxysuccinimide ester in 0.5 ml of dimethylformamide We mixed the reaction mixture well and reacted it for about 1 hour at 25 0

C.

Subsequently, we loaded the mixture onto a Sephadex gel filtration column (Pharmacia, Sweden) which had been pre-equilibrated with 0.1 M PBS (pH We then collected a total of thirty 2 ml aliquot elution fractions and read the absorbance of each fraction at 280 nm ("A 28 We then pooled the three peak fractions (15, 16 and 17) to create the activated carrier.

We dissolved 10 mg of NaBH 4 in 2.5 ml of 0.1 M sodium borate solution to produce a sodium borohydride solution. Subsequently, we diluted approximately 8 mg of each of synthetic T4 peptides JB-1, JB-2 and JB-3 with 1 ml of 0.1 M borate buffer and then mixed each solution with 200 pl of the sodium borohydride solution, incubating the mixture on ice for 5 minutes. We then warmed each peptide solution to 25 0 C, brought each solution to pH 1.0 with 1 N HCl (during which frothing occurred) and then brought each solution to pH 7.0 with 1 N NaOH (after the frothing had stopped).

We then coupled each peptide to BTG by adding 1.2 ml of the peptide solution to 6 ml of the activated carrier solution. We allowed the coupling reaction to proceed overnight by incubating the reaction mixture at room temperature.

(ii) Inoculation Of Test Animals We dissolved each of the BTG-coupled peptides prepared above in sterile Freund's complete adjuvant, to a final concentration of 1 pg/ml coupled 1 1 WO 89/01940 PCT/US88/02940 -32peptide in PBS. Subsequently, we inoculated each of three rabbits (New Zealand white) by intramuscular injection of 500 pg of one of the coupled peptides into each rabbit. We inoculated a fourth rabbit (New Zealand white) in the same manner with a mixture of the three coupled peptides. All rabbits were prebled prior to boosting to establish an average baseline for each response to be measured. The rabbits were boosted at 6 weeks with 500 pg coupled peptide in incomplete Freund's adjuvant.

Serum was collected from each rabbit monthly for 4 months after immunization. The serum was then assayed for antipeptide titer.

(iii) ELISA With Antipeptide Sera Against Peptide Coated Plates In this assay, we determined that antiserum raised in an animal by each of peptides JB-1, JB-2 and JB-3 binds to that peptide. Accordingly, those peptides are immunogenic and elicit a response in test animals.

To carry out the assay, we coated Immulon-2 (Dynatech Labs, Alexandria, Virginia) microtiter plates with 50 pl per well of 50 pg/ml uncoupled peptide in PBS and incubated the plates overnight at 4°C. Plates coated with peptide 46R*, which served as controls, were treated identically. We then washed the plates 4 times with PBS-Tween and 4 times with water. The plates were blotted dry by gentle tapping over paper towels. After blotting the plates, Peptide 46 corresponds to amino acids 728-751 of the env gene of the HIV genome. The amino acid numbering corresponds to that set forth for the env gene in L. Ratner et al., "Complete Nucleotide Sequence Of The AIDS Virus, HTLV-III", Nature, 313, Spp. 277-84 (1985). Peptide 46 has the sequence:

LPIPRGPDRPEGIEEEGGERDRDR.

-1 SWO 89/01940 PCT/US88/02940 -33we added 200 pl of a 5% FCS/PBS solution to each well and incubated the plates for 1 hour at room temperature.

We then assayed serum samples from the rabbits on the pre-coated plates prepared as described above. We assayed the antibody response to the immunogen peptide at an initial dilution of 1:100, followed by serial 10-fold dilutions in 5% FCS/PBS.

After a 2 hour incubation period at room temperature, we washed the plates and blotted them dry as described above. We then added 50 pl of a 1:1500 dilution of horseradish peroxidase conjugated goat anti-rabbit-IgG [Cooper Biomedical, Malvern, Pennsylvania] in 5% FCS/PBS to each well and incubated the plates at room temperature for 1 hour. We washed the plates with PBS-Tween We then added 50 pl of 0.42 mM TMB. We stopped the enzyme reactions with 50 pl of 2 M H 2

SO

4 We then analyzed the plates spectrophotometrically at 450 nm using a microtiter plate reader [Dynatech Labs, Alexandria, Virginia].

We observed that antiserum against each of peptides JB-1, J3-2 and JB-3 binds to the. corresponding peptide. We also observed that antiserum against a mixture of peptides JB-1, JB-2 and JB-3 binds to peptides JB-1 and JB-3 under the conditions set forth above. The titers of each of the four antisera tested against the peptides in the solidphase ELISA are shown below, where "ND" represents 30 values not determined: Approximate Titer Against: Peptide JB-1 JB-2 JB-3 JB-1 >1/50,000 0 ND JB-2 0 1/50,000 ND JB-3" 0 0 1/10,000 JB-1 JB-2 JB-3 1/4,000 ND 1/7,000 WO 89/01940 PCT/US88/02940 -34- Ig fractions from two of the three antipeptide sera raised against individual peptides, anti-JB-1 and anti-JB-2, recognized the 55 Kd T4 antigen band of native solubilized T4 in a Western blot analysis of protein eluted from the 19Thy (anti-T4) monoclonal antibody affinity column described above. As in the case of the radioimmunoassay of native solubilized T4, the detection of the Kd protein coincides with its apparent elution from the affinity column. This provides further evidence that our T4 purification procedure enriched for solubilized T4.

Thus, these polyclonal sera are useful in the detection of nanogram quantities of T4 (both native and recombinant forms) by Western analysis.

Binding of Cell-Free T4 To HIV Envelope We then tested our purified solubilized native T4 isolated from U937 cells for its ability to bind to the HIV envelope protein gpl60/gpl20. To carry out this direct binding assay, we incubated 35 S-labelled gpl60/gpl20 detergent cell extract derived from a recombinant cell line 7d2 (a gift from Drs. Mark Kowalski and William Haseltine, Dana- Farber Cancer Institute) with samples of solubilized native T4, each of which had been preincubated with one type of monoclonal antibody.

More specifically, we mixed 5 pl of solubilized T4 in a microfuge tube with 5 pg (about 3 pi) of OKT4 (ATCC #CRL 8002), a monoclonal antibody recognizing an epitope on T4 which does not interfere with HIV binding A. Hoxie et al., J. Immunol., 136, pp. 361-63 (1986)] or with 5 pg of OKT4A (Ortho Diagnostics #7142), a monoclonal antibody that inter-

S.

feres with HIV binding to T4 positive cells ~Seven McDougal et al., J. Immunol., 137, pp. 2937-2944 i (1986)]. Alternatively, we mixed 50 p1 of solubilized ;L _w y ^VTO~y r

I-..F-YI"P~

WO 89/01940 PCT/US88/02940 T4 with 5 pg of aHTLV III gpl20 (Dupont #NEN-9284).

We then incubated the mixtures on ice for 1 hour.

Subsequently, we added 150 pl of S- 35 labelled gpl60/gpl20 cell extract or S-labelled control cell extract (precleared with protein-A Sepharose) to the preincubated solubilized T4/monoclonal antibody mixtures and rocked the tubes overnight at 4 0 C. We then precipitated the T4/gpl60/gpl20 immune complexes by adding 30 p1 of protein-A Sepharose to each tube and rocking for 2 hours at 4°C to allow the protein-A Sepharose to bind to the antibody complexes. Subsequently, we spun down the beads in an Eppendorf microfuge and after extensive washings, we eluted with 40 pl SDS sample buffer at 65 0 C for 10 minutes. We then loaded 20 pl of the eluted material on a 7.5% SDS-PAGE gel which was run under reducing conditions.

Figure 2 depicts autoradiograph and Western blot results of the T4/gpl60/gpl20 coimmunoprecipitations. In Figure 2, lanes 1-5 were autoradiographed after treatment with 40% sodium salicylate and lanes 6-7 were developed on a Western blot with rabbit antisera JB-2.

As shown in Figure 2, gpl60/gpl20 protein was coimmunoprecipitated in the presence of T4 with OKT4 (lane 5) but not in the presence of T4 with OKT4A (lane Lane 3 shows the positive control for gpl60/gpl20 using aHTLV III gpl20 monoclonal antibody. Neither negative control with 35 S-labelled control extract (lane 1) or protein-A Sepharose alone (lane 2) showed bands migrating in the position of gpl60/gpl20. Based upon the bands that developed on the Western blot, the amount of T4 precipitated with either OKT4 (lane 6) or OKT4A (lane 7) appeared to be similar.

This demonstrates that purified, solubilized native T4, which is naturally membrane bound, can i WO 89/01940 PCT/US88/02940 -36still interact with the HIV glycoprotein in solution.

Accordingly, we believe that cell free soluble T4 is useful in preventing the binding interaction between HIV and the T4 receptor of T4 lymphocytes. By competing with cell surface T4 for binding to the HIV envelope protein gpl20, soluble T4 is useful in blocking HIV infection.

Synthesis Of Oligonucleotide DNA Probes The nucleotide sequence and a deduced amino acid sequence for a cDNA that purportedly encodes the entire human T4 protein have been reported [Maddon et al., (1985), supra]. The deduced primary structure of the T4 protein reveals that it can be divided into domains as demonstrated below: Amino Acid Structure/Proposed Location Coordinates Hydrophobic/Secretory Signal -23 to -1 Homology to V-Regions/ Extracellular +1 to +94 Homology to J-Regions/ Extracellular +95 to +109 Glycosylated Region/ Extracellular +110 to +374 Hydrophobic/Transmembrane Sequence +375 to +395 Very Hydrophilic/ Intracytoplasmic +396 to +435 Based on the sequence for the above-listed domains, we chemically synthesized antisense oligonucleotide DNA probes using conventional phosphoamide DNA synthesis techniques. See, e.g., Tetrahedron Letters, 22, pp. 1859-62 (1981). We synthesized the probes on an Applied Biosystems 380A DNA synthesizer and purified them by gel electrophoresis.

14 WO89/01940 PCT/US88/02940 -37- Furthermore, we synthesized the probes such that they were complementary to the DNA sequences which code for the amino acid sequence, the probes were antisense, to enable them to recognize and hybridize to the corresponding sequences in DNA, as well as in mRNA. The nucleotide sequences of the eleven selected regions of the T4 protein [corresponding to the nucleotide numbering set forth in Maddon et al., (1985), supra] were the following: Nucleotide Oligonucleotide Coordinates 1 145-171 2 742-765 3 1414-1440 6 427-453 7 1303-1329 8 1012-1038 9 97-118 10-36 11 1698-1724 12 397-423 14 261-287 Before using our DNA probes for screening, we 5' end-labelled each of the single-stranded DNA 32 32 probes with P using P]-ATP and T4 polynucleotide kinase, substantially as described by A. M. Maxam and W. Gilbert, "A New Method For Sequencing DNA", Proc. Natl. Acad. Sci. USA, 74, pp. 560-64 (1977).

Construction of Xgtl0 Peripheral Blood Lymphocytes cDNA Library To prepare our Peripheral Blood Lymphocytes (PBL) cDNA library, we processed PBL, from a single leukophoresis donor, through one round of absorption WO 89/01940 PCT/US88/02940 -38to remove monocytes. We then stimulated the nonadherent cells with IFN-y 1000 U/ml and 10 pg/ml PHA for 24 hours. We isolated RNA from these cells using phenol extraction [Maniatis et al., Molecular Cloning, p. 187 (Cold Spring Harbor Laboratory) (1982)] and prepared poly A mRNA by one round of oligo dT cellulose chromatography. We ethanol precipitated the RNA, dried it in a speed vac and resuspended the RNA in 10 pl H 2 0 (0.5 pg/pl). We treated the RNA for min at room temperature in CH3HgOH (5 mM final concentration) and p-mercaptoethanol (0.26 We then added the methyl mercury treated RNA to 0.1 M Tris-HCl (pH 8.3) at 43 0 C, 0.01 M Mg, 0.01 M DTT, 2 mM Vanadyl complex, 5 pg oligo dT 12 -18' 20 mM KC1, 1 mM dCTP, 32 dGTP, dTTP, 0.5 mM dATP, 2 pCi[a- P]dATP and 30 U pl AMV reverse transcriptase (Seikagaku America) in a total volume of 50 pl. We incubated the mixture for 3 minutes at room temperature and then for 3 hours at 44 0 C, after which time we stopped the reaction by the addition of 2.5 pl of 0.5 M EDTA.

We extracted the reaction mixture with an equal volume of phenol:chloroform and precipitated the aqueous layer two times with 0.2 volume of M NH 4 AC and 2.5 volumes EtOH and dried it under vacuum. The yield of cDNA was 1.5 pg.

We synthesized the second strand according to the methods of Okayama and Berg [Mol. Cell. Biol., 2, p. 161 (1982)] and Gubler and Hoffman [Gene, pp. 263-69 (1983)], except that we used the DNA polymerase I large fragment in the synthesis.

We blunt ended the double-stranded cDNA by resuspending the DNA in 80 pl TA buffer (0.033 M Tris Acetate (pH 0.066 M KAcetate; 0.01 M MgAcetate; 0.001M DTT; 50 pg/ml BSA), 5 pg RNase A, 4 units RNase H, 50 pM p NAD 8 units E.coli ligase, 0.3125 mM dATP, dCTP, dGTP, and dTTP, 12 units T 4 polymerase and incubated the reaction mixture for 90 min at WO 89/01940 PCT/US88/02940 -39- 37 0 C, added 1/20 volume of 0.5M EDTA, and extracted with phenol:chloroform. We chromatographed the aqueous layer on a G150 Sephadex column in 0.01M Tris-HC1 (pH 0.1 M NaCi, 0.001 M EDTA and collected the lead peak containing the double-stranded cDNA and ethanol precipitated it. Yield: 0.605 pg cDNA.

We ligated the double-stranded cDNA to linker 35/36: 5'AATTCGAGCTCGAGCGCGGCCGC3' 3' using standard procedures. We then size selected the cDNA for 800 bp and longer fragments on a S500 Sephacryl column, and ligated it to EcoRI-digested bacteriophage lambda vector gtlO (a gift of Dr. Ellis Reinherz). We packaged aliquots of the ligation reaction in Gigapak (Strategene) according to the manufacturer's protocol. We used the packaged phage to infect E.coli BNN102 cells and plated the cells for amplification. The resulting library contained 1.125 x 106 independent recombinants.

We also screened a PBL cDNA library in the bacteriophage lambda vector gtlO (a gift of Dr. Ellis Reinherz), which was synthesized from mRNA from a T4 tumor cell line named REX, which expresses T4 protein at high levels Acuto et al., "The Human T Cell Receptor: Appearance In Ontogeny And Biochemical Relationship ofambda and Beta Subunits on IL-2 Dependent Clones And T Cell Tumors", Cell, 30 34, pp. 717-26 (1983)].

Screening Of The Libraries 32 We then used three of our P-labelled synthetic oligonucleotide antisense probes, probes 3, 6 and 9, to screen in parallel our two Xgtl0 cDNA libraries using the plaque hybridization screening technique described in R. Cate et al., "Isolation Of 4Ai TM,

J

WO 89/01940 PCT/US88/02940 The Bovine And Human Genes For Mullerian Inhibiting Substance And Expression Of The Human Gene In Animal Cells", Cell, 45, pp. 685-98 (1986), with minor modifications. We modified the Cate et al. procedure by hybridizing without tetramethyl ammonium chloride to accommodate our use of unique probes, rather than mixtures, to probe the plaque filters.

We used the three probes, which had been previously 5' end-labelled with P]-ATP according to the method of A. Maxam and W. Gilbert, Meth.

Enzymol., 68, pp. 499-&8 (1979) to screen in parallel the PBL cDNA library and the REX cDNA library discussed above.

From our screening of the PBL library, we 15 isolated a nearly full length soluble T4 cDNA clone X203-4 (or Xgtl0.PBL.T4) containing a 3.064 kb insert which could be cleaved from the Xgtl0 vector with EcoRI.

From our screening of the REX cell library, we isolated an incomplete T4 cDNA clone containing a 1,200 bp cDNA insert. We then further characterized S the DNA from these clones by DNA sequencing analysis.

We also screened a bacteriophage lambda human genomic library, constructed in the vector EMBL3 by Dr. Mark Pasek (Biogen Inc., Cambridge, Massachusetts) Murray in Lambda 2, eds. R. Hendrix, J. Roberts, F. Stahl, R. Weisberg, pp. @45-422 (1983)].

The library contains DNA fragments, created by partial restriction of chromosomal DNA from the human lymphoblastid cell line GM1416,48, XXXX (Human Genetic Mutant Cell Repository, Camden, New Jersey) with Sau3a, ligated onto EMBL3 arms which had been subjected to cleavage with BamHI according to the procedures outlined in Maniatis et al., (1982), supra.

Plating of the phage library, lysis, and transfer of the phage DNA onto nitrocellulose were performed as described by W. D. Benton and R. W. Pavid, "Screening V O WG~ 89/01940 PCT/US88/02940 -41of Lambda gt Recombinant Clones By Hybridization To Single Plaques In Situ", Science, 196, =j4 (1977) and Maniatis et al. (1982). Hybridization conditions were those described by Cate et al. (1986), supra, except that tetramethylammonium chloride (TMAC1) was omitted from the washing buffer.

Approximately 2 million plaques were screened in parallel hybridizations with probe 1 and probe 3 discussed above. One phage, called CM47, which hybridized with probe 3 in the primary screenings, was subjected to DNA sequence analysis to determine the existence and position of an intron between the coding sequences for the predicted extracellular and transmembrane domains. No phage clones containing T4 sequences were found screening with probe 1, probably because it includes a sequence interrupted by an intron R. Littman and S. N.

Gettner, Nature, 325, pp. 453-55 (1987); and our observations].

Partial sequence analysis of CM47 shows that an intron interrupts the sequence corresponding to the codon for valine (amino acid 363) of the deduced primary sequence for T4 (Figure 3 in which introns are indicated by a solid line). This intron defines a potential site for introducing a stop codon in order to express a soluble form of T4. Another intron found within the coding sequence for T4 interrupts the codon for arginine (amino acid 295) and a third intron in CM47 is found between the codons for arginine (amino acid 402) and P-qinine (amino acid 403) (Figure 3).

Sequencing Of cDNA Clones We then subcloned EcoRI digested DNA from clone X203-4 into animal expression vector pBG312 Cate et al., supra] to facilitate sequence analysis. More specifically, as depicted in Figure 4, 'm i i WO 89/01940 PCT/US88/02940 -42we then digested XgtlO.PBL.T4 with EcoRI to excise the 3.064 kbp EcoRI-EcoRI fragment containing the full length T4 cDNA. This cDNA sequence, including the entire coding region for soluble T4 and for full length T4 was deposited in p170-2. We used T4 ligase to ligate the fragment into animal expression vector pBG312 [supra] which had been previously cut with EcoRI, to form pBG312.T4 and p170-2 (Figure We then determined the nucleotide sequence of the EcoRI l 10 fragment of pBG312.T4 using Maxam Gilbert technology M. Maxam and W. Gilbert, "A New Method For Sequencing DNA", Proc. Natl. Acad. Sci. USA, 74, pp. 560-64 (1977)] (see Figure 3, which depicts the PBL cDNA sequence in comparison to that reported by Maddon et al., (1985), supra). This analysis showed that the 3.064 kbp PBL full length complementary DNA copy of T4 cDNA contained the coding sequence for T4, approximately 200 bp of 5' noncoding sequence and approximately 1500 bp of 3' noncoding sequence.

We then cut pBG312.T4 with PstI and removed the resulting 3' protruding ends with Klenow and isolated an approximately 2.5 kbp fragment. We then inserted the fragment into the polylinker of pBG312 (which had been previously restricted at the SmaI site) to form plasmid p170-2, which contains the full length PBL T4 cDNA sequence (see Figure 3).

As depicted in Figure 3, the PBL T4 cDNA contains a nucleotide sequence almost identical to the approximately 1,700 bp sequence reported by Maddon et al., (1985), supra. The PBL T4 cDNA, however, contains three nucleotide substitutions that, in the translation product of this cDNA, would produce a protein containing three amino acid substitutions compared to the sequence reported by Maddon et al. As shown in Figure 3, these differences are at amino acid position 3, where the asparagine of r Maddon et al. is replaced with lysine; position 64, WO 89/01940 PCT/US88/02940 -43where the tryptophan of Maddon et al. is replaced with arginine and at position 231, where the phenylalanine of Maddon et al. is replaced with serine.

The asparagine reported at position 3 of Maddon et al. instead of lysine was the result of a sequencing error (Dr. Richard Axel, personal communication).

The significance of the amino acid replacements at positions 64 and 231, which may represent allellic polymorphism C. Fuller et al., Human Immunology, 9, pp. 89-102 W. Stohl and H. G. Kunkel, Scand. J. Immunol., 20, pp. 273-78 (1984); N. Amino et al., Lancet, 2, pp. 94-95 (1984); and M. Sato et al., J. Immunol., 132, pp. 1071-73 (1984)], is not known.

DNA sequence analysis [Maxam and Gilbert, supra] of the insert in pEC100 of the REX clone suggests that it represents the product of a splicing error, because 5' noncoding sequence appears to have been spliced with coding sequence beginning with the GGT codon for glycine (amino acid 49) (see Figure 3 and Figure The T4 coding sequence in pECl00* from glycine (amino acid 49) to isoleucine (amino acid 435) is identical to the sequence of Maddon et al., (1985), supra.

In comparison, our earlier N-terminal protein sequence analysis of native T4 protein purified from U937 cells shows a T4 expression product with aspargine as amino acid 3. These differences are also set forth in Figure 6, which also depicts comparisons at corresponding positions of the partial clone from the REX cell line Xgtl0 library; our We constructed pEC100 by digesting the incomplete T4 cDNA clone from the REX library with EcoRI and isolating the 1,200 bp cDNA insert. We then ligated it to pUC12 (Boehringer Mannheim, Indianapolis, Indiana) which had been previously cut with EcoRI to RA- form pEClOO.

g U-f

W

WO 89/01940 PCT/US88/02940 -44genomic clone from a AEMBL3 library; mouse T4 sequences [Tourvieille et al., Science, 234, pp. 610-14- (1986)] and sheep T4 sequences [Classon et al., Immunogenetics, 23, pp. 129A(1 986 Construction of Soluble T4 Mutants We then employed the technique of in vitro site-directed mutagenesis and restriction fragment substitution to modify the T4 cDNA coding sequence of pl70-2 in sequential steps to be identical to that reported by Maddon et al., (1985), supra. We first used oligonucleotide-directed mutagenesis to modify the amino acids at positions 3 and 64. Next, we employed restriction fragment substitution with a fragment including the serine 231 codon of a partial T4 cDNA isolated from a T4 positive lymphocyte cell line Acuto et al., Cell, 34, pp. 717-26 (1983)] library in Xgtll (a gift from Dr. Ellis Reinherz), to modify the amino acid at position 231. We then truncated our modified T4 cDNA sequence to remove the coding regions for the transmembrane and intracytoplasmic domains. Subsequently, we constructed three different soluble T4 mutants from our full length T4 clone PBL T4 by linker insertion between restriction sites in order to increase the probability of empirically finding a stable, secretable T4 molecule. The structure of each of these mutants is depicted in Figure 7A.

Line A of Figure 7A represents a hydropathy analysis of our full length soluble T4 carried out using a computer program called Pepplot (University of Wisconsin Genetics Computer Group) according to J. Kyte and R. F. Doolittle, J. Mol. Biol., 157, pp. 105-32 (1982). Line B depicts the protein domain structure of full length T4 [Maddon et al., (1985) supra] in which represents the secretory signal sequence, represents the immunoglobulin-like

~RAC

g- I: WO 89/01940 PCT/US88/02940 variable region sequence, represents the immunoglobulin-like joining region seiquence, represents the unique, extracellular region sequence, "TM" represents the transmembrane sequence and represents the cytoplasmic region sequence. In line B, the transmembrane amino acid sequence and some flanking sequence is written below the TM domain. Line C depicts the protein domain structure of recombinant soluble T4 mutants rsT4.1 in pBG377, rsT4.2 in pBG380 and rsT4.3 in pBG381. Line D represents the protein domain structure of E.coli rsT4 gene (Met-perfect construct) (p199-7) which is deleted for the T4 N-terminal signal sequence We constructed the first three soluble T4 mutant gene fragments by truncating our full length soluble T4 cDNA at positions corresponding to either intron/exon boundaries or to protein domain boundaries defined by hydropathy analysis predictions. More specifically, we introduced synthetic linkers into the unique Aval site that is 5' to the transmembrane/ extracellular domain boundary to produce an in-frame translational stop codon, thus constructing T4 genes that lack the transmembrane and cytoplasmic domains of the full length T4 sequence.

For example, mutant rsT4.1 in pBG377 was truncated by the insertion of a stop codon following amino acid 362, lysine, which corresponds to the position of an intron separating the extracellular and transmembrane domain exons. The positions both of this intron and of the adjacent intron that splits I the transmembrane and cytoplasmic domains were determined by DNA sequence analysis of chromosomal T4 clones isolated from the XEMBL3 genomic library described above. Although the significance of the intron positions flanking the T4 transmembrane domain is not known, the determination of the genetic structure could provide important information for design- WO 89/01940 PCT/US88/02940 -46ing rsT4 mutants, since exons frequently define functional domains Gilbert, "Why Genes In Pieces?", Nature, 271, p. 501 (1978)].

We then constructed mutant rsT4.2 in pBG380 by truncating the T4 cDNA at the boundary of the transmembrane and extracellular domains at amino acid 374. And, we constructed mutant rsT4.3 in pBG381 by truncating the T4 cDNA at amino acid 377, three amino acids downstream from the transmembrane/ extracellular domain boundary and within the transmembrane domain.

We also employed the technique of oligonucleotide site directed mutagenesis, according to D. Straus et al., "Active Site Of Triosephosphate Isomerase: In Vitro Mutagenesis And Characterization Of An Altered Enzyme", Proc. Natl. Acad. Sci. USA, 82, pp. 2272-76 (1985), to construct a fourth soluble T4 mutant from our full length T4 clone PBL T4. The structure of this mutant is depicted in Figure 7A, line D, which represents the protein domain structure of E.coli rsT4 gene (Met-perfect rsT4.2) construct, deposited in p199-7, which is deleted for the T4 N-terminal signal sequence We also constructed various other soluble T4 deletion mutants to determine which smaller fragments of the T4 sequence provide a protein which binds to HIV. These constructions were based on our belief that only the amino terminal sequence of T4 is required for binding to HIV. This belief, in turn, ii 30 was based upon observations that the monoclonal antibody OKT4A blocks infection of T4 positive cells by HIV and it appears to recognize an epitope in the amino portion of T4 [Fuller et al., supra]. Such fragments of T4, which lack glycosylation and which are capable of binding HIV and blocking infection, may be produced in E.coli or chemically synthesized.

(y? 1 WO 89/01940 PCT/US88/02940 -47- The structure of each of these deletion mutants is depicted in Figure 7B. In that figure, line A depicts the protein domain structure of full length T4 [Maddon et al., (1985), supra; Figure 7A].

In line B, the protein structure of recombinant soluble T4 mutants are depicted as follows: rsT4.7 in p203-5, rsT4.7 in pBG392, rsT4.8 in pBG393, rsT4.9 in pBG394, rsT4.10 in pBG395, rsT4.11 in pBG397, rsT4.12 in pBG396, rsT4.111 in pBG215-7, rsT4.113.1 in pBG211-11 and rsT4.113.2 in pBG214-10.

We constructed soluble T4 derivatives p203-5, pBG392, pBG393, pBG394 and pBG396 by truncating our rsT4.2 gene after the StuI sites at amino acids 183 and 264 of rsT4.2. More specifically, we constructed derivative rsT4.7 in p203-5 and in pBG392 by truncating the rsT4.2 cDNA at amino acid 182.

And, we constructed each of derivatives rsT4.9 in pBG394 and rsT4.12 in pBG396 by truncating the rsT4.2 cDNA at amino acids 113, and 166, respectively. One may also construct each of derivatives rsT4.10 in pBG395 and rsT4.11 in pBG397 by truncating the rsT4.2 cDNA at amino acids 131 and 145, respectively.

Expression of T4 and Soluble T4 Polypeptides In Bacterial Cells The cDNA sequences of this invention can be used to transform eukaryotic and prokaryotic host cells by techniques well known in the art to produce recombinant soluble T4 polypeptides in clinically and commercially useful amounts.

For example, we constructed expression vector p199-7, as shown in Figure 9A, as follows.

We preceded the construction depicted in Figure 9A by the construction of various intermediate plasmids, as depicted in Figures 8A-8D. Those constructions were carried out using conventional SWO 89/01940 PCT/US88/02940 -48recombinant techniques. The linkers employed in those constructions are set forth in Figure As depicted in Figures 8A and 8B, starting with p170-2, which contains our full length T4 DNA sequence, coding for T4 characterized by three different amino acids than that of Maddon et al., (1985), supra, we produced various constructs which direct the expression of soluble T4. Some of these constructs are characterized in that one or more of those amino acid differences have been changed to correspond to the respective amino acids of Maddon et al. In this figure, as well as in the other figures, amino acid changes are reflected by an arrow.

Plasmid p192-6 contains the Met perfect rsT4.2 sequence derived by oligonucleotide sitedirected mutagenesis which removed the entire T4 N-terminal signal sequence as shown in Figure 8C.

And, to provide a convenient means of transferring the rsT4.2 Met perfect sequence into E.coli expression vectors, the steps described in Figure 8D were carried out to produce p195-8, a plasmid containing the Met perfect rsT4.2 sequence flanked by Clal restriction sites. The ClaI-Clal cassette of p195-8 optimizes the distance between the 5' Clal site and the initiating Met codon. In Figure 8D, ST8 rop is a tetracycline resistance encoding pAT153based plasmid containing the rop mutation that permits high plasmid copy number, a promoter and ribosome binding site from bacteriophage gene 32 and the gene 32 transcription termination sequence.

Cleavage of p195-8 with ClaI produced the fragment used to assemble p199-7, a construction which directs the expression of Met perfect rsT4.2 under the control of the PL promoter (Figure 9A).

As the first step, to construct a vector from which rsT4.2 expression is under control of the PL promoter, 89094 C/U8 24 WO 89/01940 PCT/US8/02940 -49we constructed the vector p197-12 from p1034 (plmuGCSF) (Figure 9A).

We then cut p1034 with EcoRI and BamHI to excise the GCSF cDNA insert and a portion of the phage mu ribosome binding site sequence which we subsequently reconstructed with oligonucleotides.

The synthetic linkers used were linkers 57-60 (Figure We then ligated the synthetic linker into the EcoRI/BamHI-cut p1034 to form p197-12. One could, instead, replace these steps by starting with any suitable E.coli expression vector containing a Clal site appropriately placed between the promoter and terminator sequences. We cut p197-12 with Clal and inserted a ClaI-ClaI cassette containing the cDNA sequence of rsT4.3 in pBG381 and phage transcription terminator derived from p1034. The sequence of this cassette is depicted in Figure 11. The resulting plasmid, p199-7, contains the rsT4.2 "Met perfect" gene in that vector.

Alternatively, one could derive the Met perfect rsT4;2 sequence from plasmid pBG380, deposited in connection with this application, and gap out the signal sequence to create p192-6.

We tested for expression of p199-7 as follows. SG936, an E.coli lon htpr double mutant A. Coldber c, [ATCC 39624] C-ff nd A. aCl dborg, "ATP-Dependent Protein Degradation In E.coli", in Maximizing Gene Expression, W. Reznikoff and L. Gold (eds.) (1986)], was transformed with p199-7 by conventional procedures [Maniatis et al. (1982)] to form SG936/p199-7, a transformant containing a plasmid with the Metperfect rsT4.2 gene behind the PL promoter. Trnsformants were selected on LB agar plates containing 10 mcg/ml tetracycline (tet). After streaking out several single colonies for single colony isolation, one was chosen at random for testing induction of WO8/14 CTU8/24 WG:89/1940 PCT/US88102940 rsT4.2 synthesis. We picked a single colony from an LB-agar tet plate into 20 ml Luria Broth (LB) and mcg/ml tet in a 125 ml shake flask and grew it overnight in a shaking air incubator (New Brunswick Scientific, New Jersey) at 30 0

C.

We then initiated an induction culture by adding 0.5 ml of the overnight culture to 50 ml LB and tet in a 500 ml flask which was grown at 30 0 C in a shaking air incubator. When the culture reached an OD(600) of 0.4, we transferred it to a 42 0 C waterbath and shook it gently for approximately 20 minutes.

After heat induction at 420C, the flask was transferred to a 39 0 C air incubator (New Brunswick Scientific, New Jersey) where it was shaken vigorously at 250 rpm. We withdrew samples just after the 42 0

C

heat shock, and at hourly time points for 4 hours, and then after overnight growth. The samples were measured for growth by OD(600) and analyzed following SDS-PAGE for the pattern of protein synthesis by Coomassie blue protein staining and by Western blot analysis with our rabbit antipeptide antibody probes (described above). Based on the relative molecular weight and protein blot analysis, the expression of rsT4.2 was induced from SG936/p199-7 following heat induction at 42°C (Figure 12).

We transformed p199-7 into a PLmu.tet expression vector, an E.coli expression vector, at the unique Clal site (see Figure 11). The nucleotide and amino acid sequences of p199-7 are shown in Figure 11.

The expression of soluble T4 from p199-7 in E.coli was measured by Western blot analysis of whole cell extracts following SDS-PAGE using the rabbit polyclonal anti-peptide JB-1 or anti-peptide JB-2 antibodies as probes (Figure 12).

We also constructed expression vector p203-5, as shown in Figure 9B, as follows.

t: ii _L Wi8t 9/01940 PCT/US88/02940 -51- We started with p197-7, which has the same sequence as the PLum vector p197-12 (see Figure 9A), except that there is a single nucleotide deletion in the 5' noncoding region following the PL promoter.

That deletion, which is a deletion of nucleotide adenine of p197-12 (see Figure 11), resulted from a deletion in the region that was constructed from linkers 57-60 (see Figure 10). p197-7 contains the rsT4.2 gene comprising 374 amino acids. Alternatively, one could also use p197-7 as a starting plasmid.

We cut p197-7 with Clal. We also cut p195-8 (see Figures 8D and 9A) with Clal to remove the Clal Clal cassette containing the cDNA sequence of rsT4.2. Subsequently, we inserted the ClaI-ClaI cassette into p197-7 to produce p198-2.

We then digested p198-2 with StuI to remove 80 amino acids (amino acid 185 to amino acid 264) of the mature T4 protein coding sequence. Unexpected methylation, however, prevented cutting at the second StuI site, so that only the StuI site at amino acid 184 was cleaved. Following ligation, the plasmid DNA was transformed into E.coli and we examined several plasmid clones for the deletion using standard procedures. None of those plasmids contained the expected StuI deletion.

Subsequent DNA sequence analysis of one of these plasmids, called p203-5, showed that two guanine residues (see amino acids 183 and 184; nucleotides 818 and 819 of Figure 3) of the StuI recognition sequence had been deleted following cleavage due to exonuclease digestion caused by the use of exonuclease-contaminated StuI enzyme. This dinucleotide deletion produced a translation frameshift following amino acid 182 (glutamine) and introduced a stop codon six amino acid codons downstream from the frameshift (Figure 9C). The unexpected f, WO 89/01940 PCT/US88/02940 -52methylation of the second StuI site together with the deletion that resulted in a new stop codon produced a gene encoding a shortened form of recombinant soluble T4, called rsT4.7. The rsT4.7 sequence encodes a 182 amino acid N-terminal segment of the mature T4 sequence followed by, at the C-terminus, six amino acids asparagine-leucine-glutaminehistidine-serine-leucine of non-T4 sequence and finally by a TAA stop codon.

The expression of soluble T4 from p203-5 in E.coli was measured by Western blot analysis as previously described.

Expression of T4 and Soluble T4 Polypeptides In Animal Cells We inserted both soluble T4 genes and the unmodified gene encoding membrane-bound T4 into animal expression vector pBG368. More specifically, we inserted each of the soluble gene constructs into pBG368 under the transcriptional control of the adenovirus late promoter, to give plasmids pBG377, pBG380 and pBG381. We also made two pBG312-based constructions, called pBG378 and pBG379, which direct the expression of recombinant full length T4 protein. pBG378 and pBG379 code for the same full length T4 protein but in pBG379, a portion of the 3' untranslated sequence has been removed. Subsequently, to test for expression of recombinant soluble T4 and recombinant full length T4, we cotransfected Chinese ti hamster ovary cells with one of each of those plasmids and with the plasmid pAdD26.

We first constructed pBG368 as follows.

As depicted in Figure 13, we cut animal cell expression vector pBG312 Cate et al., "Isolation Of The Bovine And Human Genes For Mullerian Inhibiting Substance And Expression Of The Human Gene In Animal Cells", Cell, 45, pp. 685-98 (1986)] with EcoRI and

-U

WO89/01940 PCT/US88/02940 -53- BglII to delete one of each of the two EcoRI and the two BglII restriction sites (the EcoRI site at position 0 and the BglII site located at approximately position 99). The resulting plasmid, pBG368, retained an EcoRI site in the cloning region and a BglII site after the cloning region. This left a single EcoRI site and a single BglII site in the polylinker for i cloning purposes.

iMore specifically, we deleted one EcoRI site and one BglII site by sequential partial diges- I tion of pBG312 with restriction enzymes EcoRI and BglII, respectively. We filled in with Klenow and 4 nucleotides then religated to produce pBG368, which contains unique restriction sites for EcoRI and BglII enzymes.

Once transient expression of soluble T4 was verified, we constructed stable cell lines that continuously expressed soluble T4. To do this, we employed the stable cell expression host, the dihydrofolate reductase deletion mutant (DHFR) Chinese hamster ovary cell line Kao et al., "Genetics Of Somatic Mammalian Cells X Complementation Analysis of Glycine-Requiring Mutants", Proc. Natl.

Acad. Sci., 64, pp. 1284-91 (1969); a- naa and f~ Ua l"Isolation Of Chinese Hamster Cell Mutants Deficient In Dihydrofolate Reductase Activity", Proc. Natl. Acad. Sci., 77, pp. &2-1 (1980)].

Using this system, we cotransfected each T4 gene construct with pAdD26 J. Kauvfman and P. A. Sharp, "Amplification And Expression Of SSequences Cotransfected With a Modular Dihydrofolate Reductase Complementary DNA Gene", J. Mol. Biol., 159, pp. 601-21 (1982j containing the mouse DHFR gene. Before carrying out the co-transfections, we linearized all plasmids by restriction enzyme cleavage and, prior to transfection, we mixed each plasmid with pAdD26 so that the molar ratio of pAdD26 to T4 MT.0 j WO 89/01940 PCT/US88/02940 -54was 1:10. This maximized the number of T4 gene copies per transfectant.

Within the cell, the plasmids were ligated together to form polymers that can become integrated into host chromosomal sequences by illegitimate recombination Haynes and C. Weissmann, "Constitutive, Long-Term Production Of Human Interferons By Hamster Cells Containing Multiple Copies Of a Cloned Interferon Gene", Nucl. Acids Res., 11, pp. 687-706 (1983); S. J. Scahill et al., "Expression And Characterization Of The Product Of A Human Immune Interferon cDNA Gene In Chinese Hamster Ovary Cells", Proc. Natl.

Acad. Sci. USA, 80, pp. 4654-58 (1983)]. We selected transfectants that express the mouse DHFR gene in culture medium lacking nucleotides. We then subjected these transfectants to a series of increasing concentrations of methotrexate, a toxic folate analogue that binds DHFR, to select for cells levels of DHFR.

Resistance to methotrexate by increased expression of DHFR is frequently the result of DHFR gene amplification, which can include the reiteration of large chromosomal segments, called amplified R. T. Sch'rnke units J. Kaufman and Sharp, "Amplification And Dxpra ion Of Loss Of Dihydrofolate Reductase Genes In A Chinese Hamster Ovary Cell Line", Molec.

Cell. Biol., 1, pp. 1069-76 (1981)]. Therefore, cointegration of DHFR and rsT4 sequences permitted the amplification of rsT4 genes. Stably transfected cell lines were isolated by cloning in selective growth medium, then screened for T4 expression with a T4 antigen (RIA) Klatzmann et al., Nature, 312, pp. 767-68 (1984)] and by immunoprecipitation 35 from conditioned medium after S] cysteine 35 S-Cys") metabolic labelling.

We also inserted the soluble T4 derivative rsT4.7 gene into an animal cell expression plasmid as follows.

i T0 WO 89/01940 PCT/US88/02940 As set forth in Figure 14C, we cut plasmid pBG381 (Figure 14A) with EcoRI and NheI. We then cut p186-6 with EcoRI and NheI to remove the 786 base pair fragment. We ligated that fragment into the digested pBG381 to form plasmid pBG391. The T4 sequence in pBG391 is identical to both that of Maddon et al. (1985) supra at positions 64 (tryptophan) and 231 (phenylalanine) and to tbiat of pBG381. However, at position 3, the asparagine reported by Maddon et al. and present in pBG381 is replaced with lysine. The nucleotide sequence of pBG391 is depicted in Figure We then digested p203-5 with Nhel and IOxanI to remove the 483 base pair fragment. We 15 inserted that fragment into NheI/OxanI-digested pBG391 to form plasmid pBG392, the animal cell expression construct of rsT4.7. The T4 sequence in rsT4.7 contains amino acids identical to that of Maddon et al.'s full length sequence at amino acid 20 positions 64 (tryptophan) and 231 (phenylalanine).

However, at position 3, the asparagine reported by Maddon et al. is replaced with lysine. The nucleotide sequence of pBG392 is depicted in Figure 16.

In Figure 14D, we have depicted the construction of other animal cell expression constructs containing sequences encoding the deletions rsT4.9 in pBG394, and rsT4.12 in pBG396. Those constructions were carried out using conventional recombinant techniques. The linkers employed in those constructions are set forth in Figure 18. The nucleotide sequences of pBG394 and pBG396 are shown in Figures 19 and Plasmid pBG393, shown in Figure 17, contains rsT4.8, the perfect form of rsT4.7. pBG393 contains 182 amino acids of the mature T4 sequence, without the additional non-T4 6 amino acids at the C-terminus following amino acid 182. The nucleotide sequence of BG393 is shown in Figure 2A.

t WO 89/01940 PCT/US88/02940 -56- Other animal cell expression plasmids according to this invention may be constructed as depicted in Figure 17. These include rsT4.10 in pBG395 and rsT4.11 in pBG397 (see Figure 18 for specific linkers).

The nucleotide sequence of BG395 is show in Figure 22.

Purification Of Recombinant Soluble T4 Recombinant soluble T4 construct pBG380 expressed in DHFR- CHO cells was grown to confluency in a e-Modified Eagles Medium (Gibco) supplemented with 10% fetal calf serum, 1 mM glutamine and the antibiotics penicillin and streptomycin (100 pg/ml of each). The cells were grown at 37 0 C in two 21 Cell Factory Systems (Nunc). We then washed the confluent i cells free of fetal calf serum with a-Modified Eagles IMedium without fetal calf serum and cultured the cells in a-Modified Eagles Medium at 37 0 C for 4 days.

Subsequently, we harvested the conditioned media, filtered it through a Millipore Millidisk 0.22p hydrophilic filter cartridge (Millipore #MCGL 305-01) and concentrated the secreted proteins on a fast-S ion exchange column (S-Sepharose Fast Flow, Pharmacia #17-0511-01) in 20 mM MES buffer (pH We then eluted the bound proteins with 20 mM Tris-HCl (pH 7.7) and 0.3 M NaCl. The elution pool was subsequently diluted with 2 volumes of 20 mM Tris-HCl (pH 7.7) and it was then loaded on a column Scomprising immobilized 19Thy anti-T4 monoclonal anti- J 30 body coupled to Affigel-10 [a gift of Dr. Ellis Reinherz, Dana Farber Cancer Institute, Boston, Massachusetts]. We washed the column extensively and eluted the bound material as 0.5 ml fractions with 50 mM glycine-HCl (pH 150 mM NaCl, 0.1 mM EGTA and 5 pg/ml bovine pancreatic trypsin inhibitor, Aprotinin (Sigma #A1153). We used Western blots f WO 89/01940 PCT/US88/02940 |j -57developed with rabbit antisera raised against peptide JB-2 to follow the purification. We employed silver stained gels to follow binding and elution of rsT4.2 during the chromatography. Figure 23 depicts a Coomassie stained gel of purified rsT4.2.

Gel sizing-column chromatography analysis of the purified rsT4.2 from the pBG380 transfected CHO cell line, BG380G, suggests that rsT4 is monmeric under physiologic pH and salt concentration.

Sequencing Of Recombinant Soluble T4 Protein We then determined the N-terminal amino acid sequence of a recombinant soluble T4, specifically rsT4.2, molecule purified from the conditioned medium of the pBG380 transfected CHO cell line as described above, by automated Edman degradation in an Applied Biosystems 470A gas phase sequenator B. Pepinsky et al., J. Biol Chem., 261, pp. 4239-46 (1986)].

The amino terminal sequence matched the sequence which we had previously determined for solubilized native T4 isolated from U937 cells, supra.

The amino terminal sequences of native solubilized T4 (sT4) and purified rsT4 protein are A2 proteins, as compared to the amino terminal sequence predicted by Maddon et al., (1985), supra, with the mature amino terminus located at position 3 of that sequence. The amino terminal sequences of solubilized native T4 i (sT4), recombinant soluble T4 (rsT4.2) secreted by CHO transfectant BG380G containing pBG380 and the protein sequence deduced by Maddon et al. (1985), supra are as follows: sT4: X-K-V-V-L-X-K-K-X-D-T-V-E-L-T-X-T-A-S-ErsT4.2: N-K-V-V-L-G-K-K-G-D-T-V-E-L-T-X-T-A-S-E- WO 89/01940 PCT/US88/02940 -58- Maddon et al. Q-G-N-K--V--L-G-K-K-G-D-T-V-E-L-T-C-T-A-S-E In the above sequences, the amino acids are represented by single letter codes as follows: Phe: F Leu: L Ile: I Met: M Val: V Ser: S Pro: P Thr: T Ala: A Tyr: Y His: H Gln: Q Asn: N Lys: K Asp: D Glu: E Cys: C Trp: W Arg: R Gly: G X: not determined or ambiguous.

We also constructed pBG211-11, a plasmid coding for the N-terminal 113 amino acids of soluble T4 protein. This construct, which codes for a protein characterized by a single disulfide bridge, between the cysteines at amino acid positions 18 and 86, is conveniently expressed in E.coli.

To construct p211-11, as depicted in Figure 24, we first cut p195-8 (see Figures 8D and 9A) with Clal to remove the ClaI-ClaI cassette containing the cDNA sequence of rsT4.2. We then digested pAT153y3SH16AAmp, the tryptophan operon promoter plasmid from the gamma interferon producing E.coli strain BN374 with Clal, and deleted the cDNA coding for gamma interferon. Subsequently, we inserted the Clal-Clal cassette into the Clal-cut E.coli plasmid in front of the tryptophan operon promoter and ligated to produce p196-10.

As shown in Figure 25, we then subjected pBG380 to oligonucleotide-directed mutagenesis to insert three tandem translational stop codons following the T4 cDNA sequence coding for amino acids -23 to 113 in pBG380, co produce pBG394.

We then constructed p211-11 from fragments of each of p196-10, pBG394 and p1034 as depicted in Figure 26. The first fragment including the vector sequences, was produced by restricting p196-10 with -u W89/01940 PCT/US88/02940 -59- HindIII and Clal to remove the T4 coding sequence from amino acids 61 through 374 of rsT4.2 and including vector sequence following the 3' end of the rsT4 gene. The second fragment, a HindIII BglII segment including the codons for T4 amino acids 61-113 of rsT4.9 immediately followed by a triplet of stop codons in tandem, was isolated by HindIII/BglII digestion of pBG394. The third fragment, a BamHI Clal fragment containing a bacteriophage T4 transcriptional termination signal N. Kirsch and B. Allet, S"Nucleotide Sequences Involved In Bacteriophage T4 Gene 32 Translational Self-Regulation", Proc. Natl.

Acad. Sci. USA, 79, pp. 4937-41 (1982)], was isolated by BamHI/ClaI digestion of p1034. We then ligated 15 these three fragments to produce p211-11, a T4 construct coding for a 113 amino acid soluble form of T4 protein, with asparagine at amino acid position 3 rsT4.113.1).

We then subjected p211-11 to oligonucleotide site-directed mutagenesis (Figure 27) to change the amino acid at position 3 from asparagine to lysine using the oligonucleotide T4-66:

I£

ATG CAG GGT AAA AAA GTA GTA CTG GGC 3'.

This produced plasmid p214-10, a fully corrected 113 amino acid soluble T4 vector coding i 30 for a 113 amino acid soluble form of T4 protein, with lysine at amino acid position 3 rsT4.113.2). As shown in Figure 27, we subjected p214-10 to oligonucleotide site-directed mutagenesis to delete glutamine and glycine at, respectively, S-_1 ~r L--~FC WO 89/01940 PCT/US88/02940 amino acid positions 1 and 2 of the T4 sequence using the oligonucleotide T4AID-87: I C GTA TCG ATT TGG ATG ATG AAA AAA GTA GTA 3'.

This produced p215-7, a 111 amino acid soluble T4 construct, including the trp promoter, which directs the expression of a 111 amino acid soluble form of T4 protein, with lysine at amino acid position 3 rsT4.111).

r We next constructed p218-8, a 111 amino acid construct which directs the expression of a 111 amino acid soluble form of T4 protein, with lysine i 15 at amino acid position 3 rsT4.111) under the control of the PL promoter, as depicted in Figure 28.

More specifically, we cut p197-12 (Figure 9A) with Clal to remove the 101 bp fragment containing linker and terminator sequences. We also cut p215-7 with Clal to remove the Clal Clal cassette containing the cDNA sequence of rsT4.111 and the OT4 transcriptional terminator sequence [Kirsch and Allet, supra]. Subsequently, we inserted the Clal Clal cassette into the Clal-cut p197-12 to produce p218-8.

In order to express rsT4.113.1, we transformed E.coli A89 with p211-11 by conventional techniques [Maniatis et al. (1982), supra] to form E.coli A89/p211-11. E.coli A89 is a tetracycline I sensitive derivative of E.coli SG936. We isolated E.coli A89 from E.coli SG936 according to the method of S. R. Maloy and W. D. Nunn, "Selection For Loss Of Tetracycline Resistance By Escherichia coli", J. Bact., 145, pp. 110-12 (1981), which is based upon the ability of the lipophilic chelating agent fusaric acid to selectively inhibit resistant strains.

WO89/01940 PCT/US88/02940 -61- More specifically, we plated E.coli SG936 on medium containing, per liter, 5 g tryptone, 5 g yeast extract, g NaCl, 10 g NaH 2

PO

4

H

2 0, 50 mg chlortetracycline- HC1, 12 mg fusaric acid, 0.1 mM ZnC12 and 15 g agar.

Colonies which grew at 30 0 C (putative tetracyclinesensitive strains) were retested for tetracycline sensitivity on L-agar plates containing 5 pg/ml tetracycline. One tetracycline-sensitive strain, designated A89, was then shown to be unable to grow on LB agar at 42 0 C, thus verifying the presence of the htpR mutation.

Transformants were selected by tetracycline resistance. We picked a single colony into 20 ml of minimal medium plus 0.2% casamino acids plus tryptophan (100 pg/ml) plus tetracycline (10 pg/ml) in a 100 ml shake flask placed in a shaking air incubator at 30 0 C and allowed the cells to grow up overnight.

The following morning, we inoculated 40 ml of minimal medium plus 0.2% casamino acids plus tryptophan (100 pg/ml) plus tetracycline (10 pg/ml) with the overnight culture at OD 600 0.05 in a 500 ml flask.

The cells were grown to midlog phase and then induced by pelleting, washing once in minimal medium and then resuspending in minimal medium plus 0.2% casamino acids plus tetracycline (10 pg/ml), in the absence of tryptophan. We removed 0.6 OD 600 of cells after 0, 1, 2, 3 and 4 hours incubation and after growth overnight.

The aliquots were centrifuged and cell pellets were subjected to lysis by boiling in Laemmli gel loading buffer. After centrifugation to remove cell debris, half of each sample was subjected to SDS-PAGE, followed by Western blot analysis with our rabbit antipeptide antibody probes or by Coomassie blue protein staining (Figures 29A and 29B).

WO 89O9f PCT/US 8/02940-- WO 89/01940D PCT/US88/02940 -62- Purification Of rsT4.113.1 We then purified rsT4.113.1 from the E.coli transformant by means of two essentially quantitative steps involving anion-exchange and gel-filtration chromatographies performed under reducing and denaturing conditions.

More specifically, we suspended 14 g of wet cells from a 4 L shake-flask fermentation in I 100 ml of a 20mM Tris (pH 7.5) buffer containing 20 pg/ml DNase, 20 pg/ml RNase and 1 mM phenylmethylsulfonylfluoride The suspension was applied to a French Press at 1000 psi in two passages and then centrifuged in an SA 600 rotor at 18,000 g for I 15 min at 4 0 C. The resulting pellet was solubilized i 15 in 20 ml of a 20 mM Tris (pH 7.5) buffer containing 7 M urea and 10 mM 2-mercaptoethanol. We then subjected the suspension to ultracentrifugation at 85,000 g for 90 min at 4 0 C. The supernatant was 1 diluted by the addition of 80 ml of 20 mM Tris i 20 (pH 7.5) buffer containing 7 M urea and 10 mM 2-mercaptoethanol and 40 ml of the sample was applied to a 3 x 4 cm Q-Sepharose fast-flow column (Sigma, St. Louis, Missouri) which had been preequilibrated in the same buffer. The column was developed with a gradient in 400 ml total volume of increasing NaCl from 0 to 0.3 M in the same Tris/urea/ 2-mercaptoethanol buffer. Column fractions were monitored for absorbance at 280 nm and for protein Scontent by SDS-PAGE (15% acrylamide). The fractions were also analyzed by Western blots. Figure panel is a chromatogram displaying the purification of rsT4.113.1 by ion-exchange chromatography.

In that figure, peaks containing rsT4.113.1 are identified. The rsT4.113.1 was found to elute early in the NaCI gradient and to be well-resolved from low-molecular weight contaminants.

-J

1;4 WO 89/01940 PCT/US88/02940 -63- In order to separate rsT4.113.1 from highmolecular weight contaminants, we carried out gelfiltration chromatography on an rsT4.113.1-containing pool for final purification of the protein to near homogeneity purity). More specifically, we prepared a pool containing 20 mg of protein in 50 ml and then concentrated to 10 ml in a stirred-cell ultrafiltration unit (Amicon, Danvers, MA.) using a membrane (Amicon). Subsequently, 5.0 ml of the concentrate was applied to a 1.5 x 95 cm S-300 column (Sigma) equilibrated and developed in the same Tris/urea/2-mercaptoethanol buffer. We monitored the column fractions for absorbance at 280 nm and for protein content by SDS-PAGE. The fractions were also analyzed by Western blots. A pool containing rsT4.113.1 (approximately 4 mg) in 15 ml was thus prepared. Figure 30, panel is a chromatogram displaying the purification of rsT4.113.1 by gel-filtration separation of the rsT4.113.1 pool.

In that figure, peaks containing rsT4.113.1 are identified.

Figure 30, panel is an SDS-PAGE analysis depicting the purification of the rsT4 derivative throughout the centrifugation and chromatography steps. In Figure 30, panel the lanes depicted are: lane A: molecular weight standards lane B: cell extracts lane C: cell pellet following solubilization 30 of cell extract in non-denaturing conditions lane D: supernatant following solubilization of cell extract in non-denaturing buffer lane E: supernatant following ultracentrifugation step

~'V

I

I

1* I i if

I,

'4 ii

I'

ill 1 c -I~ WO 89/01940 PCT/US88/02940 -64lane F: lane G: Q-Sepharose pool S-300 gel-filtration pool.

Refolding Of Purified rsT4.113.1 We refolded the purified rsT4.113.1 by 5 dilution and dialysis steps to non-denaturing and oxidized conditions. More specifically, refolding of the protein at a concentration of 0.5 OD (280)/ml was achieved by stepwise dialysis against 500 volumes of 3 M urea, 20 mM Tris (pH 500 volumes of 1 M urea, 0.1 M ammonium acetate (pH 6.8) and, finally, the same volume of a phosphate-buffered saline solution. Throughout the refolding procedure, samples of the protein were monitored for relative content by spectral analysis and by high-performance liquid 15 chromatography ("HPLC") performed on a 150A liquid chromatographic system (Applied Biosystems, Inc., Foster City, California). An octasilyl column (Aquapore RP-300, 0.46 x 3.0 cm) was equilibrated in 0.1% trifluoroacetic acid ("TFA")/water (solvent A) and 20% 0.085% TFA/70% acetonitrile (solvent B) and developed with a linear gradient of increasing acetonitrile concentration from 20% to 80% (solvent B) over 45 min at a flow rate of ml/min.

As shown in Figure 31, panel protein in 7 M urea, 10 mM 2-mercaptoethanol and 20 mM Tris(pH 7.5) eluted from the HPLC column at 49% acetonitrile in the gradient. In subsequent steps, from 1 M urea/l mM ammonium acetate (pH 6.8) [Figure 31, panel to phosphate buffered saline [Figure 31, panel an increasing percentage of rsT4.113.1 was found to elute earlier in the HPLC gradient at 47% acetonitrile. The identity of the earlier eluting peak as oxidized product was verified by reduction of rsT4.113.1 in non-chaotropic cl WO89/01940 PCT/US88/02940 solutions and application of sample thus treated to HPLC under the same conditions.

The elution of oxidized rsT4.113.1 prior to reduced protein on HPLC suggests that formation of the single disulfide bridge decreases relative hydrophobicity of the protein L. Browing et al., Anal.

Biochem., 155, pp. 123-28 (1986)]. Spectral analysis of rsT4.113.1 was performed throughout the course of refolding in order to monitor relative yield of soluble protein in the procedure. The refolding method allowed approximately 20% recovery of rsT4.113.1.

HPLC analysis indicated a less than 15% contaminant of reduced protein in the preparation (Figure panel lane G).

Sequencing Of Renatured rsT4.113 We then carried out amino acid analysis of rsT4.113.1 by automated Edman degradation in an Applied Biosystems 470A gas phase sequenator equipped with a 900 A data system. Phenylthiohyda;ition amino acids generated during the course of the degradative chemistry were analyzed on-line using an Applied Biosystems 120A PTH-analyzer equipped with a PTH-C18 2.1 x 220 mm column. Protein (10 pg) for sequence analysis was applied to SDS-PAGE (15% acrylamide) and electroblotted on an Immobilon membrane (Millipore Corp., Bedford. Massachusetts) as described by P. Matsudaira, J. Biol. Chem., 262, pp. 10035-38 (1987).

Amino acid analysis of protein samples was performed by hydrolysis of protein in 6 N HC1, in vacuo, for 24 h at 110 0 C. The hydrolysates were then applied to a Beckman 6300 Analyzer equipped with post-column detection by ninhydrin. Western blot analysis of the SDS-PAGE gels was carried out by standard techniques using rabbit antisera JB-1.

WO 89/01940 PCT/US88/02940 -66- Sequence analysis revealed an amino terminal sequence of: Met-Gln-Gly-Asn-Lys-Val-Val The purified rsT4.113.1 protein was found to contain stoichiometric quantities of the aminoterminal methionine placed in the protein construct for expression in E.coli and an intact polypeptide chain consistent with a sequence derived from the plasmid construction. Recovery of phenylthiohydani toinyl-methionine at the first cycle of the degradative chemistry was 60% consistent with routine initial yields obtained in the automated Edman. This observation excludes the possibiity that a significant percentage of the rsT4.113.1 lacked the initiation methionine, the NH -methionine was not removed 2 by expression of rsT4.113.1 in E.coli, or that sequence analysis was impaired by the presence of glutamine at the first cycle of the degradative chemistry.

Sequence analysis was performed for 40 cycles and no evidence of lysine carbamylation was observed. Amino acid analysis displayed a close correlation of actual and theoretical values for amino acids, thus indicating the marked absence of proteolytic degradation in the course of expression, or purification, or both.

Immunoprecipitation Of CHO Cell Lines Producing Soluble T4 We tested the conditioned media from 3 S-Cys metabolically labelled CHO cells transfected with one of the T4 mutant constructs pBG377, pBG380, pBG381, the full length recombinant T4 construct pBG379, of this invention or vector only, to determine whether any produced a molecule recognized by the anti-T4 monoclonal antibody 19 Thy. To carry out this test, we incubated about 107 CHO cells transfected with either pBG380, pBG381, pBG377, pBG379 or pBG312, for 5 hours at 37 0 C with 180 pCi/ml 3S-labelled cysteine 'i WO 89/01940 PCT/US88/02940 -67- [DuPont, New England Nuclear] in 4 ml RPMI cys- medium (Gibco). After labelling of the cells, 1 ml of filtered, conditioned media was made 0.5 mM with phenylmethyl-sulphonyl fluoride and immunoprecipitated with OKT4 and protein A Sepharose H. Sayre and E. L. Reinherz, Eur. J. Immunol., 15, pp. 291-95 (1985)]. Subsequently, we incubated media from the 35 S-labelled cells with OKT4 (ATCC #CRL 8002). We then immuno-precipitated with protein A Sepharose and subjected the immuno-precipitates to SDS-PAGE under reducing conditions on 10% polyacrylamide gels K. Laemmli, Nature, 227, pp. 680-85 (1980)].

Autoradiography was carried out with X-Omat X-ray film (Eastman Kodak).

As shown in lanes 3-5 of Figure 32, both pBG380 (rsT4.2) and pBG381 (rsT4.3) directed the 35 synthesis of a secreted, immune, S-labelled T4 protein that was recognized by the OKT4 anti-T4 antibody. The immunoprecipitated truncated molecules migrated as 49 Kd proteins, a result consistent with their predicted molecular weights. In contrast, no soluble T4 antigen could be detected in the conditioned media of cell lines stably transfected with pBG377 (rsT4.1) or pBG379 (rflT4).

Immunoprecipitation analysis of cellular extracts of cell lines transfected with pBG377 suggests that the rsT4.1 gene may be misfolded, which could account for a block in its secretion J. Gething et al., Cell, 46, pp. 939-50 (1986)].

In Figure 32, the lanes represent the following: Lane 1: immunoprecipitation from conditioned medium of CHO cells stably co-transfected with vectors pBG312 and pAdD26. Lane 2: blank.

Lanes 3 and 4: immunoprecipitation from conditioned medium of CHO cells stably co-transfected with pBG380 (rsT4.2) and pAdD26. Lanes 5 and 6: immunoprecipitation from conditioned medium of CHO cells stably WO 89/01940 PCT/US88/02940 H -68- Sco-transfected with pBG381 (rsT4.3) and pAdD26.

Lane 7: immunoprecipitation from conditioned medium of CHO cells stably co-transfected with recombinant full length T4 (pBG379) and pAdD26. In Figure 32, the arrow indicates the predicted position of the soluble T4 from pBG380 or pBG381 relative to the migration of standard molecular weight markers.

Immunoprecipitation Of COS 7 Cell Lines Producing Recombinant Soluble T4 We expressed recombinant soluble T4 derivatives pBG392, pBG393 and pBG394 in COS 7 cells by electroporation, essentially as described by G. Chu et al., "Electroporation For The Efficient Transfection Of Mammalian Cells With DNA", Nuc.

Acids Res., 15, pp. 1311-26 (1987). More specifically, we introduced 20 pg closed circular plasmid DNA and 380 pg of carrier (sonicated salmon sperm DNA) into 3 x 10 COS 7 cells. The cells were j electroporated using a Gene Pulser (Biorad) set at 300 volts. Subsequently, we incubated the COS 7 j cells in Dulbecco's Modified Eagle's Medium supplemented with 10% fetal calf serum for 24 hours. We then harvested the conditioned media, filtered it through a Millipore Millidisk 0.22 p hydrophilic filter cartridge (Millipore #MCGL 305-01) and concentrated the secreted proteins on a fast-S ion exchange column (S-Sepharose Fast Flow, Pharmacia #17-0511-01) in 20 mM MES buffer (pH We then eluted the bound proteins with "i 30 20 mM Tris-HCl (pH 7.7) and 0.3 M NaC1. The elution pool was subsequently diluted with 2 volumes of 20 mM Tris-HCl (pH 7.7) and it was then loaded on a column comprising either 19Thy anti-T4 monoclonal antibody and protein A Sepharose or OKT4A and protein A Sepharose. We washed the column extensively and eluted the bound material as 0.5 ml fractions with S WO9/01940 PCT/US88/02940 -69mM glycine-HCI (pH 150 mM NaC1, 0.1 mM EGTA and 5 pg/ml Bovine pancreatic trypsin inhibitor, Aprotinin (Sigma, #A1153). The immunoprecipitates were subjected to SDS PAGE (10% gel) followed by immunoblotting against rabbit antisera raised against peptide JB-1. We employed silver stained gels to follow binding and elution of rsT4 during chromatography.

Figure 33 depicts an immunoblot analysis of transiently expressed pBG392 (rsT4.7) [lanes 11]; pBG393 (rsT4.8) [lanes 4, 7, 8] and pBG394 (rsT4.9) [lane The standards are 50 ng purified rsT4.3 (lane 150 ng purified rsT4.3 (lane 2) and 250 ng purified rsT4.3 (lane The arrow indicates the expected position of migration of a protein with the relative molecular weight of rsT4.7: 21,000 daltons. The sample that was to be loaded into lane 4 was lost and lanes 6 and 9 are blank.

As shown in lanes 10 and 11 of Figure pBG392 (rsT4.7) directed the synthesis of a secreted, immune protein that was recognized by the anti-T4 antibodies OKT4A and 19Thy. Lanes 4, 7 and 8 also demonstrate that pBG393 (rsT4.8) directed the synthesis of a secreted, immune protein that was j 25 recognized by OKT4A and 19Thy. This analysis illustrates that rsT4.7 contains the OKT4A epitope.

It also suggests that the binding region for HIV envelope binding resides in the amino 182 terminal residues of T4.

In contrast, no soluble T4 could be detected in the media of cell lines transfected with pBG394 (rsT4.9) [see lane Immunoprecipitation analysis of cellular extracts of cell lines transfected with pBG397, however, showed that rsT4.9 was recognized by OKT4A. We believe that rsT4.9, a 113 amino acid construct, binds the HIV virus and that it represents a second generation soluble T4, one with only two t WO 89/01940 PCT/US88/02940 cysteines and one of three disulfide bridges.

Accordingly, rsT4.9 is easily produced in E.coli or yeast systems.

Similarly, although no soluble T4 could be detected in the media of cell lines transfected with pBG396 (rsT4.12), analysis of cellular extracts of those cell lines showed that rsT4.12 was recognized by OKT4A. Thus, rsT4.12 may also bind HIV virus.

Radioimmunoassay And Epitope Analysis Of rsT4.113 In order to determine if the 113 fragment of rsT4 contained structural determinants for binding to OKT4A, Leu-3A and OKT4, we then carried out radioimmunoassay and epitope analysis of rsT4.113 using a competitive inhibition radioimmunoassay J. Newby et al., "Solid-Phase Radioimmune Assays" in Handbook Of Experimental Immunology, D. M. Weir 1, pp. 34.1-34.8 (1986)]. As OKT4A and Leu-3A block infectivity of HIV in vitro [Dalgleish et al., supra] and binding of T4 to gp120/160 [McDougal et al., supra], this analysis served as a first approximation as to whether or not rsT4.113 contained structural elements for interaction with HIV.

We first coated U-bottom 96 well microtiter plates (Falcon) with 50 pl/well goat-anti-mouse IgG (Hyclone Typing Kit, Logan, Utah) in PBS (pH 7.0) to a concentration of 50 pg/ml and incubated the plates overnight at 4 0 C. We then rinsed the plates with 1X PBS and blotted them dry. The plates were then blocked by the addition of 100 pl/well of a iX PBS solution containing 5% bovine serum albumin for 1 hour at room temperature. We rinsed the plates with PBS, blotted dry and then spotted them with pl of one of three antibody solutions containing either OKT4 (10 pg/ml in block buffer); OKT4A (500 ng/ml in block buffer) or Leu-3A (Lecton- '4 WO 89/01940 PCT/US88/02940 -71- Dickinson) (500 ng/ml in block buffer). We let the plates stand for 2 hours at room temperature. We then washed the plates 3 times with a PBS/0.05% solution and 2 times with 1X PBS and blotted them dry.

In a separate plate, we titrated competitor samples of unlabeled rsT4.113.1 from 20 pg/ml and serially diluted twice (including no competitor control), with final volumes in each well of 25 pi.

1 10 The positive control for this assay was competition with unlabeled rsT4.3 (375 amino acids). We then added 25 pl of 125I-rsT4.3 containing 10,000 pl (prepared according to A. E. Bolton and W. M. Hunter, Radioimmunoassay And Related Methods, 15 Chapter. 2c). Subsequently, we spotted the entire pl content of each well onto the assay plate containing each of the three antibody solutions and incubated for 2 h at room temperature. We then Swashed the plates 3 times with a PBS/0.5% solution and 2 times with IX PBS, blotted them dry and then counted the wells in a Beckman gamma counter for radioactivity.

As shown in Figure 34, rsT4.113.1 competes with 1 I-rsT4.3 for absorption to an OKT4A solid phase in a dose-dependent manner. Additionally, 125 rsT4.113.1 competes with I-rsT4.3 for absorption to a Leu-3A solid phase in a dose-dependent manner.

By comparison to unlabeled rsT4.3, rsT4.113.1 exhibits a molar affinity for those antibodies within a factor of 3. In the 0.4 to 25 pg/ml concentration range tested, rsT4.113 did not compete with radiolabelled rsT4.3 for binding to OKT4. In a similar assay, we observed that rsT4.111 also competes with 125 I-rsT4.3 for binding to OKT4A and Leu-3A, but not to OKT4 [Figures 35-37].

Based on these results, we believe that the epitopes for OKT4A and Leu-3A are contained within WO89/01940 PCT/US88/02940 -72the amino-terminal 113 amino acids of T4. We also believe that the epitope for OKT4 binding is localized within the carboxy terminal of the T4 polypeptide.

Accordingly, we believe that the binding domain is localized within the amino terminal 113 or 111 amino acids of the T4 protein. Based on this belief, we synthesized various synthetic oligopeptides which contain sequence within that structural domain. These oligopeptides are represented in I 10 Figure 3 as follows: Oligopeptide Amino Acid Coordinates JB-1 44-63 rsT4 #6 18-29 rsT4 #7 5-56 rsT4 #8 84-97 rsT4 #9 30-63 We synthesized these peptides using conventional phosphoamide DNA synthesis techniques [Tetrahedron Letters, 22, pp. 1859-62 (1981)]. We synthesized the peptides on an Applied Biosystems 380A DNA Synthesizer and-purified them by gel electrophoresis.

ELISA Assay For rsT4.113 We also carried out an ELISA assay for rsT4.113.1 produced by p211-11-transformed E.coli.

Throughout this assay, dilutions were made in blocking solution and, between each step, we washed the plates with PBS/0.05% Tween-20. More specifically, we coated wells of Immulon 2 (Dynatech, Chantilly, Virginia) plates with .005 OD (280 nm)/ml of OKT4 30 (IgG2b) in 0.05 M bicarbonate buffer to a volume of pl/well and incubated the plates overnight at 4°C. We then blocked the plates with 5% bovine serum albumin in PBS, 200 pl/well, and incubated for minutes at room temperature.

Subsequently, we added 50 pl of 50 ng/ml rsT4.3 to each well, incubating overnight at 4 0

C.

WO 89/01940 PCT/US88/02940 -73- We then added 50 pl/well of a mixture containing rsT4.113.1 and 10 ng/ml of OKT4A and incubated for 2 1/2 hours at room temperature. Using a Hyclone Kit (Hyclone), we then carried out the following steps. First, we added 1 drop of rabbit anti-mouse IgG2a to each well and incubated the plates for i 1 hour at room temperature. We then added 100 pl of peroxidase-labeled anti-rabbit IgG, diluted 1:4000 i with blocking buffer to each well, and incubated for i 10 1 hour at room temperature.

We prepared a substrate reagent as follows.

We diluted substrate reagent 1:10 in distilled water and added two O-phenyl-ethylene-diamine ("OPD") chromophore tablets per 10 ml of substrate. We let i 15 the mixture dissolve thoroughly by mixing with a vortex. Alternatively, a TMB peroxidase substrate system (Kirkegaard Perry Catalogue #50-76-00) may be used. Subsequently, we added 100 pl of the chromophore solution to each well, incubated for 10-15 minutes at room temperature and then stopped the color development with 100 pl of 1N H 2

SO

4 We then measured OD at 490 nm, using an ELISA plate reader.

The results of the assay are demonstrated in Figure 38.

We then subjected the soluble T4 proteins produced by the T4 constructs of this invention to various functional assays.

Assays Of The Antiviral Activity Of Soluble T4 The antiviral activity of soluble T4 according to this invention was evaluated using modifications of various in vitro systems used to study antiviral agents and neutralizing antibodies D. Ho et al., "Recombinant Human Interferon Alpha Suppresses HTLV-III Replication In Vitro", Lancet, pp. 602-04 (1985); K. Hartshorn et al., WO 89/01940 PCT/US88/02940 -74- "Synergistic Inhibition Of HTLV-III Replication In Vitro By Phosphonoformate And Recombinant Interferon Alpha-A", Antimicrob Ag Chemoth, 30, pp. 189-91 (1986)].

For each of these assays, we prepared graded concentrations of soluble T4 and preincubated them with an H9 derived IIIB isolate of HIV [a gift from Drs. M. Popovic and R. Gallo, National Cancer Institute, Bethesda, Maryland]. The isolate was maintained as a chronically infected culture in H9 cells. Cell-free HIV stocks were obtained from supernatant fluids of HTLV-III infected H9 cultures (culture conditions: 1 x 10 cells/ml with 75% viable cells). We prepared serial 10 fold dilutions of recombinant soluble T4 ranging from 10 picograms/ml to 10 micrograms/ml and incubated them with fifty tissue culture infectious doses (TCID 50 of HIV for 1 hour at 37 0 C, in RPMI-1640 supplemented with heat inactivated fetal calf serum (FCS). We then added 150 pl of H9 cells to a final concentration of 0,5 x 106 cells/ml which were not HIV-infected to the wells containing aliquots of the recombinant soluble T4/HIV mixture.

We adjusted each virus inoculum to a concentration of 250 TCID 50 /ml. We preincubated 100 p1 of the virus inoculum with 200 p1 recombinant soluble T4 or 100 pi immunoglobulin prepared in triplicate serial 2-fold dilutions for 1 hour at 37 0

C

prior to inoculation onto 1.5 2 x 10 H9 cells in 30 5 ml RPMI 1640 supplemented fetal calf serum HEPES (10mM), penicillin (250 U/ml), streptomycin (250 pg/ml) and L-glutamine (2mM). On days 5, 6, 7, and 14, we examined each culture for characterisic cytopathic effects Neutralization was defined as the inhibition of syncytia formation comared with controls.

A

y WO 89/01940 PCT/US88/02940 The positive control used was HIV seropositive neutralizing serum, as described in D. D. Ho et al., "Human Immunodeficiency Virus Neutralizing Antibodies Recognize Several Conserved Domains On The Envelope Glycoproteins", J. Virol., 61, pp. 2024-28 (1987). The negative controls used were HIV seronegative serum only and buffer only.

Cytopathic Effect Assay (CPE) In this assay, following conventional protocols for cytopathic effect assays [Klatzmann et al. (1984), supra and Wong-Staal and Gallo (1985), supra], we microscopically examined the H9 cells for evidence of cytopathic effects of HIV.

The CPE was scored on a four point scale from 1+ to with 4+ representing the highest degree of CPE.

By day 14, wells containing recombinant soluble T4 according to this invention (rsT4.2, derived from the pBG380 transfected CHO cell line BG380) at 10 pg/ml showed no evidence of CPE, while the negative control showed 1+ to 3+ CPE.

p24 Radioimmunoassay We then tested soluble T4 as an inhibitor of viral replication in an HIV virus replication assay according to D. D. Ho et al., J. Virol., 61, pp. 2024-28 (1987) and J. Sodroski et al., Nature, 322, pp. 470-74 (1986). We carried out the assay essentially as described, except that the cultures were propagated in microtiter wells containing 200 pl. In this assay, we evaluated the ability of the soluble T4 polypeptides of this invention to block HIV replication, as measured by HIV p24 antigen production. We sampled supernatants twice weekly for HIV p24 antigen as described below.

L

i-

U-;

WO 89/01940 PCT/US88/02940

I

-76- We obtained an assay kit [HTLV-III p 24 Radioimmunoassay System, Catalogue No. NEK-040, NEK-040A, Biotechnology Systems, New Research Products, Dupont] which contains affinity purified 125I labelled HIV p24 antigen, a rabbit anti-p24 antibody and a second goat anti-rabbit antibody which is used to precipitate antigen-antibody complexes.

We carried out the assay according to the protocol included with the kit. Accordingly, we mixed a sample to be assayed or one of a series of amounts of unlabelled p24 antigen with a fixed amount of 125I labelled p24 and a fixed limited amount of rabbit anti-p24 antibody. We incubated the samples overnight at room temperature and then added a goat anti-rabbit immunoglobulin preparation for 5 minutes at 40 0 C. We centrifuged the samples in a microfuge 125 and aspirated the supernatant fluid. Pelletted I labelled p24 was quantitated for each sample by gamma 125 counting and a standard curve for the I p24 displaced by the known amounts of antigen added to standard tubes was constructed. We then calculated 125 the 1I labelled p24 displaced by the antigen present in the unknown samples by interpolation using the standard curve constructed from the known amounts of p24 antigen contained in the standard samples. The results are shown in the table below.

WO;89/01940 PCT/US88/02940 -77p24 ASSAY OF HIV REPLICATION INHIBITION rsT4.2 Patient Average Bound/ Day g/ml) Serum CPM Unbound 7 Negative 344 Positive 2,237 112.4 -551 19.9 1,766 86.6 Negative 230 2.2 Positive 2,459 124.6 0.5* 322 7.3 1,980 96.3 14 Negative 221 1.8 Positive 2,284 115.0 246 3.1 1,988 98.7 These results demonstrate that soluble T4 according to this invention at a concentration of pg/ml completely inhibits virus replication as measured in this standard 14 day assay. These results are also depicted in Figure 39 in graphic form. In Figure 39, values were calculated from a standard curve of p24 according to assay kit instructions.

This concentration was initially believed to be pg/ml, based upon our preliminary approximation that 1 unit of absorbance at 280 nm was equivalent to 1 mg of rsT4.2. Absorbanc at 280 nm is a commonly used first approximation of protein concentration. Upon amino acid analysis of the protein, however, we found that it had a higher extinction coefficient than originally approximated, with 1 A^ 0 unit of rsT4.2 being equivalent to 0.5 mg of the 2 rotein.

This concentration was initially believed to be 10 pg/ml, based upon our preliminary approximation that 1 unit of absorbance at 280 nm 0 was equivalent to 1 mg of rsT4.2. Absorbane at 280 nm is a commonly used first approximation of protein concentration. Upon amino acid analysis of the protein, however, we found that it had a higher extinction coefficient than originally approximated, with 1 A 0 unit of rsT4.2 being equivalent to 0.5 mg of the rotein.

WO 89/01940 PCT/US88/02940' -78- We then carried out a p24 replication assay as described above, except that the soluble T4 was added to the infected cultures during refeeding at days 3, 7 and 10, in order to maintain a constant rsT4 concentration throughout the infection period.

The results of this assay are shown in the table below.

INHIBITION OF HIV REPLICATION WITH CONSTANT CONCENTRATION OF rsT4 rsT4.2 p24 (pg/ml) (ng/ml) I 0.008 770 0.031 970 0.125 0.5 0 0 0 1120 uninfected 0 These results demonstrate that when soluble T4 protein according to this invention was maintained at a constant concentration throughout the infection period, as little as 0.125 pg/ml of the protein substantially blocked replication of 250

TCID

50 /ml of HIV-1.

Advantageously, soluble T4 protein according to this invention, at concentrations far exceeding those required to block viral replication, did not exert immunotoxic effects in vitro, as measured by three lymphocyte proliferation assays mixed lymphocyte response, phytohemagglutinin, and tetanus toxoid stimulated response.

Syncytia Inhibition Assay To further assess the effect of soluble T4 on HIV env-T4 binding, we evaluated the effect of two preparations of our soluble T4 protein on the syncytiagenic properties of HIV in the co-cultivation assay. We carried out a C8166 cell fusion assay SW 89/01940 PCT/US88/02940 -79as described in B. D. Walker et al., Proc. Natl.

Acad. Sci. USA, 84, pp. 8120-24 (1984).

We incubated 1 x 109 H9 cells chronically infected with HTLV-IIIB for 1 hour at 37 0 C in

CO

2 with various concentrations of one of two preparations of rsT4.2 in 150 p1 RPMI-1640 media supplemented with 20% fetal calf serum. We then added 3 x 104 C8166 cells in 50 pl media (a T4 transformed human umbilical cord blood lymphocyte line'[Sodroski et al., supra], to a final volume of 0.2 ml in each well. Final well concentrations of soluble T4 were 0.5 pg/ml* and 5.0 pg/ml* for preparation #1 and 1.25 pg/ml* and 12.5 pg/ml* for preparation We then counted total number of syncytia per well at 2 hours and 4 hours after adding the C8166 cells at 37 0 C in 5% CO 2 Parallel co-cultivations used buffer alone (negative control) or OKT4A at 25 pg/ml (positive control) as controls. We considered a positive result as a 50% reduction in syncytia compared to controls, at a time when at least 100 syncytia per 104 infected H9 cells were present in the control cultivations. The results of this assay are shown below and in Figure 40 (2 hour data).

These concentrations were initially believed to be, respectively, 1 pg/ml, 10 pg/ml, 2.5 pg/ml and 25 pg/ml, based upon our preliminary approximation that 1 unit of absorbance at 280 nm ("A 28 was equivalent to 1 mg of rsT4.2. Upon amino cid analysis of the protein, however, we found that it had a higher extinction coefficient than originally approximated, with 1 A^ unit of rsT4.2 being equivalent to 0.5 mg o "he protein.

l 1 11 11 1 1 1 1 J WO 89/01940 PCT/US88/02940' INHIBITION IN C8166 FUSION ASSAY Inhibition* Preparation [rsT4.2] (pg/ml) 2 Hrs 4 Hrs buffer 0 0 0 rsT4.2 30 42 rsT4.2 54 47 rsT4.2 1.25** 16 21 rsT4.2 12.5** 77 OKT4A (25 pg/ml) 0 100 100 As demonstrated in this table and in Eigure 40, soluble T4 according to this invention at pg/ml and 12.5 pg/ml inhibited syncytia formation at 2 hours, as compared to buffer alone. By 4 hours after the addition of C8166 cells, soluble T4 at 12.5 pg/ml continued to inhibit greater than syncytia formation, as compared to the negative control.

We also evaluated the effect of two preparations of our soluble T4 protein rsT4.7 on the syncytiagenic properties of HIV in a similar cocultivation assay. The results of this assay are shown below.

All assays were carried out in triplicate, and the number of syncytia counted per well was averaged to calculate inhibition. The inhibition represents the difference between the average number of syncytia in the negative control (without rsT4 or OKT4A) and the average number of syncytia counted when either rsT4 or OKT4A were present during the i assay, divided by the average syncytia count for the negative control and multiplied by 100.

These concentrations were initially believed to be, respectively, 1 pg/ml, 10 pg/ml, 2.5 pg/ml and 25 pg/ml, based upon our preliminary approximation that 1 unit of absorbance at 280 nm was equivalent to 1 mg of rsT4.2. Upon amino acid analysis of the protein however, we found that it had a higher extinction coefficient than originally approximated, with 1 A unit of rsT4.2 being equivalent to 0.5 mg oP he protein.

i i I WQ8901940 PCT/US88/02940 -81- INHIBITION IN C8166 FUSION ASSAY Average rsT4.7 Syncytia/50pl (pg/ml) aliquot Inhibition at 2 Hrs Preparation H9 cells (control) C8166 cells (control) HIV-infected H9 cells added to C8166 cells (control) OKT4A (control) Prep. 1 of rsT4.7 118

N/A

N/A

0 100 63.6 0 S5.0* 13 This concentration 'ras initially believed to be 10 pg/ml, based upon our preliminary approximation that 1 unit of absorbance at 280 nm ("A 80 was equivalent to 1 mg of rsT4.2. Upon amino acid analysis of the protein, however, we found that it had a higher extinction coefficient than originally approximated, with 1 A unit of rsT4.2 being equivalent to 0.5 mg of he protein.

1 i i I I WO 89/01940 PCT/US88/02940 -82- Assay daia ay1 Average rsT4.7 Syncytia/50pl (pg/ml) aliquot Inhibition at 2 Hrs Preparation H9 cells (control) C8166 cells (control) HIV-infected H9 cells added to C8166 cells (control) OKT4A (control) Prep. 2 of rsT4.7 0 1 141

N/A

N/A

0 100 80.9 0 S5.0* This concentration was initially believed to be 10 pg/ml, based upon our preliminary approximation that 1 unit of absorbance at 280 nm was equivalent to 1 mg of rsT4.2. Upon amino acid analysis of the protein, however, we found that it had a higher extinction coefficient than originally approximated, with 1 A unit of rsT4.2 being equivalent to 0.5 mg o 8 ihe protein.

i WO 89/01940 PCT/US88/02940 -83- Assay date: day 14 Average rsT4.7 Syncytia/50pl Inhibition Preparation (pg/ml) aliquot at 2 Hrs H9 cells 0 0 N/A (control) C8166 cells 0 0 N/A (control) HIV-infected 0 128 0 H9 cells added C8166 cells (control) OKT4A (control) 0 0 100 Prep. 1 of rsT4.7 5.0* 35 72.7 Prep. 2 of rsT4.7 z 5.0* 2 98.4 As demonstrated in these tables, soluble T4 protein rsT4.7 inhibited syncytia formation in HIV-infected H9 cells.

We also evaluated the effect of rsT4.113.1 and rsT4.1ll on the syncytiagenic properties of HIV in a co-cultivation assay. We carried out a C8166 cell fusion assay as described in Walker et al., supra.

4 We incubated 1 x 10 H9 cells chronically infected with HTLV-IIIB for 1 hour at 37 0 C in C0 2 with from 5 to 50 pg/ml rsT4.113.1 or rsT4.111 in 150 pl RPMI-1640 media supplemented with fetal calf serum in 96-well microtiter plates. We This concentration was initially believed to be 10 pg/ml, based upon our preliminary approximation that 1 unit of absorbance at 280 nm was equivalent to 1 mg of rsT4.2. Upon aming acid analysis of the protein, however, we found that it had a higher extinction coefficient than originally approximated, with 1 A 8 unit of rsT4.2 being equivalent to 0.5 mg o Phe protein.

WO 89/01940 PCT/US88/02940 -84then added 3 x 104 C8166 cells to the wells in 50 il aliquots. The plates were incubated for 2 hours at 37 0 C in 5% CO 2 and, following this incubation, the number of syncytia per well were counted.

Syncytia were defined as cells containing a ballooning cytoplasm greater than three cell diameters. All samples were counted twice. Parallel co-cultivation used OKT4A alone or rsT4.3 alone at a concentration of 25 pg/ml (positive controls) or H9 cells alone or C8166 cells alone (negative controls).

The results of this assay are shown below and in Figure 41.

INHIBITION IN C8166 FUSION ASSAY Preparation rsT4(pg/ml) Inhibition H9 cells (control) 0 0 C8166 cells (control) 0 0 rsT4.113.1 1.25 rsT4.113.1 2.5 63 rsT4.113.1 4.25 63 rsT4.113.1 6.25 82 rsT4.113.1 12.5 96 rsT4.3 12.5 100 OKT4A (25 pg/ml) 0 100 As demonstrated in this table and in Figure 41, rsT4.113.1 exhibited a dose-dependent i inhibition of HIV-induced syncytia formation. The molar specific inhibitory activity of rsT4.113.1 appeared to be reduced by an order of magnitude by comparison to anti-viral activity of longer forms of recombinant soluble T4. Thus, whereas rsT4.113.1 is effective toward neutralization of HIV-dependent cell fusion in vitro, its molar specific inhibitory WO 89/01940 PCT/US88/( activity is decreased by a factor of 10. It is undetermined whether this decreased potency is due to incomplete renaturation of the E.coli-derived protein, the presence of three additional amino acids at the N-terminus of rsT4.113.1 (Met-Gln-Gly) lacking in rsT4.2 or rsT4.3 produced in mammalian cells, or the absence of additional structure in rsT4.113.1 required for high-affinity binding to

HIV.

We also carried out a C8166 cell fusion assay with rsT4.111, as described for rsT4.113.1.

The results of this assay are shown below.

)2940 INHIBITION IN C8166 FUSION ASSAY Preparation H9 cell (control) C8166 cells (control) rsT4.111 rsT4.111 rsT4.111 rsT4.111 rsT4.111 rsT4.111 rsT4.3 rsT4.3 OKT4A (25 pg/ml) rsT4(p g/ml) 0 0 1.25 2.5 4.25 6.25 12.5 25.0 12.5 25.0 0 Inhibition 0 0 0 67 100 100 100 100 100 As demonstrated in this table, rsT4.111 exhibited a dose-dependent inhibition of HIV-induced syncytia formation. At a concentration of 12.5 pg/ml and 25.0 pg/ml, complete inhibition of cell fusion was achieved.

Kinetics Of Intramuscular Injection Of Soluble T4 We examined the kinetics of the appearance of a recombinant soluble T4 protein according to this invention (specifically, rsT4.3 from the pBG381transfected cell line BG381) in serum after intramuscular injection as follows.

q 1 WO 89/01940 PCT/US88/02940' -86- We obtained two cynomolgus monkeys (Macaca fascicularis) who were free of infectious disease and in good health. Each monkey had been subjected to a 6 week quarantine period prior to administration of the soluble T4 protein. Throughout the administration period, each monkey was maintained on a conventional diet of monkey chow supplemented with fresh fruit. A catheter and a vascular access port were surgically placed in a femoral vein of each animal prior to treatment in order to facilitate blood i collection.

Over a period of 28 days, each animal received recombinant soluble T4 protein twice daily by intramuscular injection to the large muscles of the thighs or buttocks. Injections were administered to each animal 8 hours apart and each injection contained a volume of 0.15 ml/kg (0.25 mg/kg) of rsT4.3 (from the pBG381-transformed cell line BG381), for a total dose of 0.5 mg/kg/day/monkey. Serum samples for clearance determination were collected on day 1 before the first treatment and at i, 2, 4 and 8 hours after the first injection, as well as 1, 2, 4, 14 and 16 hours after the second injection on days 7, 14 and 28.

We found that intramuscularly injected soluble T4 reached the maximum level in serum between 1 and 2 hours after injection, with the level falling off slowly and reaching half-maximum value at approximately 6 hours post-injection. According to data obtained for intravenous administration (not shown), the level of rsT4.3 in serum should drop below that attained via intramuscular injection aproximately 2 hours after intravenous injection. Thus, while the maximum rsT4.3 level in serum after intramuscular injection does not reach that attainable via intravenous injection, it is slowly released into the blood stream, remaining detectable in serum for a il- SW 89/01940 PCT/US88/02940

I

-87much longer time. This slow release mechanism associated with intramuscular routes of injection is advantageous because a higher level of soluble T4 protein is available over a longer period of time over a given concentration; thus remaining in a sustained level. Intramuscular administration of soluble T4 protein is particularly useful in treating early stage HIV-infected patients, to prevent the virus from disseminating, or in treating patients who have been exposed to the virus and who are not yet seropositive.

We determined serum levels of rsT4.3 using an ELISA assay. Throughout this assay, dilutions were made in blocking solution and, between each step, we washed the plates with PBS/0.05% More specifically, we coated wells of Immulon 2 plates with .01 OD (280 nm)/ml of OKT4 (IgG2b) in 0.05 M bicarbonate buffer to a volume of 50 pl/well and incubated the plates overnight at 4 0 C. We then blocked the plates with 5% bovine serum albumin in PBS, 200 pl/well, and incubated for 30 minutes at room temperature.

Subsequently, we added 50 pl of sample or standard to each well, incubating for 4 hours at room temperature. We then added 50 pl/well of OKT4A at 0.1 pg/ml and incubated overnight at 4°C. Using a Hyclone Kit (Hyclone) we then carried out the following steps. First, we added 1 drop of rabbit anti-mouse IgG2a to each well and incubated the plates for 1 hour at room temperature. We then added 4i 100 pl of peroxidase-labeled anti-rabbit IgG, diluted 1:4000 with 5% BSA/PBS'to each well, and incubated for 1 hour at room temperature.

We prepared a substrate reagent as follows.

We diluted substrate reagent 1:10 in distilled water and added two O-phenyl-ethylene-diamine ("OPD") chromophore tablets per 10 ml of substrate. We let L

J

WO 89/01940 PCT/US88/02940, -88the mixture dissolve thoroughly by mixing with a vortex. Alternatively, a TMB peroxidase substrate system (Kirkegaard Perry Catalogue #50-76-00) may be used. Subsequently, we added 100 p1 of the chromophore solution to each well, incubated for 10-15 minutes at room temperature and then stopped the color development with 100 pl of lN H 2

SO

4 We then measured OD at 490 nm, using an ELISA plate reader.

The results of the assay are demonstrated in the tables below.

I

i- V

I

;:ii WO8/I4 CTU8/2 W 8901940 PCT/US88/02940 -89- Monkey #7-91 Time(hr) 8 9** 12 22 24 Day 1 22.7* 278.8 281.8 214.9 72.3 246.2 259.6 136.0 23.8 13.4 rsT4 Level (ng/ml) Day 7 96.5 199.6 366.8 246.6 105.0 Day 14 158.0 360.7 306.4 363.9 199.4 Day 28 19.8 238.3 441.1 393.2 290.4 Monkey #7-92 Time(hr) Day 1 rsT4 Level (ng/ml)

A

ii ii 25 8 9** 12 22 24 6.7* 87.2 254.2 170.0 118.9 405.1 523.5 371.5 48.4 39.4 56.0 225.8 377.9 167.3 101.2 Day 14 106.3 178.0 253.2 308.2 176.5 Day 28 60.9 437.7 770.6 821.5 898.3 background second injection administered after the collection of the 8 hour sample.

Polyvalent Forms Of Recombinant Soluble T4 Receptors may be characterized by their affinity for specific ligands, such that, at equilibrium, the intrinsic affinity (K between monovalent receptor and monovalent ligand can be defined as where [RL] is the concentration of receptor bound to ligand and [Rf] and [Lf] are the concentrations of free receptor and ligand, WO 89014 PCT/US88/O294fJ I_ WO 89/01940 PcT/US88/02940 respectively A. Underwood, in Advances In Virus Research, ed. K. Maramorosch et al., 34, pp. 283-309 (1988)].

For a polyvalent receptor (with a valency of n) binding to a polyvalent ligand (with a valency of a functional affinity can be defined as n[Rb]/n[Rf]m[Lf], where [Rb] is the concentration of bound receptor sites, and n[Rf] and m[Lf] are, respectively, the concentrations of free receptor and ligand binding sites. The effect of increasing the valence (the number of binding sites) is to enhance the stability of ligand-receptor complexes. The affinity of a polyvalent receptor for a polyvalent ligand will depend on three factors: the intrinsic association constant of each binding site, the valency (number of binding sites) and the topicological relationship between the receptor and ligand binding sites. Under some circumstances, polyvalent binding interactions will lead to higher functional affinity. The decreased dissociation rate of polyvalent ligands with polyvalent receptors results in an increased functional affinity L. Hornick and F. Karush, Immunochemistry, 9, pp. 325-40 (1972); I. Otterness and F. Karush, "Principles Of Antibody Reactions", in Antibody As A Tool, ed. J. J.

Marchalonais and G.W. Warr, pp. 97-137 (1982)].

The simplest case for receptor polyvalency increasing functional affinity is represented by a bivalent soluble receptor, such as an antibody 30 molecule, which has two identical ligand binding sites, each capable of independently binding antigen with equal affinity. If the antigen is displayed polyvalently, for example, chemically coupled to a solid support such that the spacing between antigenic sites can be bridged by the antibody's two antigen binding arms, the functional affinity of the antibody for the antigen coupled to the solid support would be W0189/01940 PCT/US88/02940 -91greater than the intrinsic affinity of the antibody binding site for the monovalent antigen Crothers and H. Metzger, Immunochemistry, 9, pp. 341-57 (1972)]. Because virus particles represent polyvalent antigens, the greater functional affinity of antibodies for polyvalent antigens is an important factor for antibody-directed virus neutralization.

The association of recombinant soluble T4 and the HIV major envelope glycoprotein gpl20 is an example of monovalent receptor binding to monovalent ligand. The affinity of this interaction has been measured, and the association between T4 and _9 has a dissociation constant Kd 4 x 10 M Lasky et al., Cell, 50, pp. 975-88 (1987)].

Using the antibody analogy, we believe that polyvalent rsT4 will demonstrate a greater affinity for HIV-infected cells displaying than monovalent rsT4 and the topicological relationship between gpl20 on the virus particle or the infected cell surface, will determine the degree to which polyvalent rsT4 exhibits higher functional affinity than monovalent rsT4. One example of a polyvalent rsT4 is described below, with respect to the production of a recombinant bivalent rsT4 consisting of two tandem repeats of amino acids 3-178, followed by the C-terminal 199 amino acids of rsT4.3.

According to this invention, a "polyvalent" receptor possesses two or more binding sites for a given ligand. Furthermore, the intrinsic affinity of each 30 ligand binding site of a given polyvalent receptor need not be identical.

As shown in Figure 42, to construct bivalent rsT4, we digested pBG391 with NheI, which cleaves after the valine at position 178 in rsT4, and removed the NheI 5' overhang with mung bean nuclease. Next, we cleaved with BglII to remove the C-terminal half of the rsT4 coding sequence in pBG391. Finally, we WO 89/01940 PCT/US88/02940' -92ligated a DraI-BglII fragment containing the coding sequence for rsT4 amino acids 3 (lysine) through 377 (isoleucine) to the cleaved pBG391 to create pBiv.l, a plasmid coding for a fusion protein with a tandem duplication of the N-terminal 176 amino acids of rsT4, followed by the C-terminal 199 amino acids of rsT4.3. The protein produced by this plasmid, therefore, contains two adjacent N-terminal binding or OKT4A-binding domains (defined by amino acid residues 3 through 111 of rsT4.111), followed by one OKT4-binding C-terminal domain (Figure 43).

pBiv.l was transfected by electroporation into COS 7 cells to test expression of the bivalent rsT4 protein. Three days later, we tested the conditioned medium of the transfected cells for the presence of the rsT4 bivalent protein by immunoprecipitation, followed by Western blot analysis of the precipitated protein. Both OKT4A and OKT4 were used for immuno-precipitation to determine that the OKT4 epitope and at least one of the OKT4A epitopes had folded correctly. Both antibodies precipitated a protein of the predicted apparent molecular weight (60,000d) from the conditioned medium of the cells.

Bivalent rsT4 may be purified by immunoaffinity purification from an OKT4 column and the purified protein may then be used to perform quantitative competition assays with rsT4.3. We believe that the bivalent molecule would demonstrate equivalent competition against rsT4.3 for OKT4 binding, 30 but significantly greater competition against monovalent rsT4 for OKT4A binding. The ability of bivalent recombinant soluble T4 to block syncytium formation may also be demonstrated in the C8166 fusion assay. We also believe that bivalent recombinant soluble T4 would block syncytium formation at significantly lower concentrations than monovalent rsT4; based upon the higher

^J

WO 89/01940 PCT/US88/02940 -93functional affinity of bivalent recombinant soluble T4 for According to alternate embodiments of this invention, other methods for producing polyvalent rsT4 may be employed. For example, polyvalent rsT4 may be produced by chemically coupling rsT4 to any clinically acceptable carrier molecule, a polymer selected from the group consisting of Ficoll, polyethylene glycol or dextran, using conventional coupling techniques. Alternatively, rsT4 may be chemically coupled to biotin, and the biotin-rsT4 conjugate then allowed to bind to avidin, resulting in tetravalent avidin/biotin/rsT4 molecules. And rsT4 may be covalently coupled to dinitrophenol (DNP) or trinitrophenol (TNP) and the resulting conjugate precipitated with anti-DNP or anti-TNP- Igm, to form decameric conjugates with a valency of for rsT4 binding sites.

Alternatively, a recombinant chimeric antibody molecule with rsT4 sequences substituted for the variable domains of either or both of the immunoglobulin molecule heavy and light chains may be produced. Because recombinant soluble T4 possesses gpl20 binding activity, the construction of a chimeric antibody having two soluble T4 domains and having unmodified constant region domains could serve as a mediator of targeted killing of HIVinfected cells that express For example, chimeric rsT4/IgG 1 may be produced from two chimeric genes an rsT4/human kappa light chain chimera (rsT4/C and an kappa rsT4/human gamma 1 heavy chain chimera (rsT4/Cga Both C appa and C ama regions gamma-l kappa gamma-r have been isolated from human recombinant DNA libraries, and each has been subcloned into animal cell selection vectors containing either the bacterial neo resistance or bacterial gpt markers WO 89/01940 PCT/US88/02940 -94for selection in animal cell hosts against the antibiotic G418 or mycophenolic acid, respectively.

To construct rsT4/Cgamma-1 and rsT 4 /Ckappa chimeric genes, an rsT4 gene segment, including at least the secretory signal sequence and the N-terminal 110 amino acid residues of the mature rsT4 coding sequence and including a splice donor or portion thereof, is placed upstream of the gamma-1 and kappa constant domain exons. A suitable restriction 1-9 enzyme may be used to cut within the intron downstream of the desired rsT4 coding sequence, thus providing a donor splice site. Subsequently, a suitable restriction enzyme is used to cut within the introns upstream of the kappa and gamma-1 coding regions. The rsT4 sequence is then joined to the kappa or gamma-1 constant region sequence, such that the rsT4 intron sequence is contiguous with the gamma-1 and kappa introns. In this way, an acceptor splice site is provided by the kappa or gamma-i constant region intron. Alternatively, rsT4 chimeric genes may be constructed without the use of introns, by fusing a suitable rsT4 cDNA gene segment directly to the gamma-1 or kappa coding regions.

The rsT4/Cgamma-l and rsT 4 /Ckappa vectors may then be cotransfected, for example, by electroporation into lymphoid or non-lymphoid host cells.

Following transcription and translation of the two Schimeric genes, the gene products may assemble into chimeric antibody molecules.

Expression of the chimeric gene products may be measured by an enzyme-linked immunoadsorbant assay (ELISA) that utilizes monoclonal anti-T4 antibody OKT4A, as described infra, or in gpl20 competition assays and radioimmunoassays, as described infra.

Activity of the rsT4/IgG 1 chimeras may be measured by incubating them with HIV-infected cells in the presence of human complement, followed by quantitating 1 1 i WO 89/01940 PCT/US88/02940 subsequent complement-mediated lysis of these cells.

Alternatively, activity may be measured in HIV replication and HIV syncytium assays as described infra.

In order to determine if bivalent rsT4 has a greater potency than monovalent rsT4, we mixed OKT4, at various concentrations, together with a constant concentration of rsT4, so that the molar ratio of OKT4:rsT4 varied between 0.2 and 4. After preincubating the mixture overnight at 4 0 C, we added aliquots to the HIV syncytium assay described infra.

OKT4 has no observable effect in this assay when used alone. In addition, the concentration of recombinant soluble T4 chosen did not cause inhibition in this assay. Accordingly, we looked for indications that the OKT4/rsT4 mixture was more potent than rsT4 alone.

We observed that at ratios of OKT4:rsT4 greater than 0.2, partial to complete inhibition of syncytium formation occurred. We believe that under conditions where two rsT4 molecules are bound to 1 OKT4 molecule, the greatest inhibitory effect should be found.

Thus, polyvalent, as well as monovalent forms of recombinant soluble T4 are useful in the compositions and methods of this invention.

Microorganisms and recombinant DNA molecules prepared by the processes of this invention are exemplified by cultures deposited in the In Vitro International, Inc. culture collection, in Linthicum, Maryland, on September 2, 1987, and identified as: BG378: E.coli MC1061/pBG378 199-7: E.coli MC1061/pl99-7 170-2: E.coli JA221/p170-2 EC100: E.coli JM83/pEC100 BG377: E.coli MC1061/pBG377 BG380: E.coli MC1061/pBG380 BG381: E.coli MC1061/pBG381 These cultures were assigned accession numbers IVI 10143-10149, respectively.

WC

*1

E

i, d [1 ii )89/01940 PCT/US88/02940' -96- In addition, microorganisms and recombinant DNA molecules according to this invention are exemplified by cultures deposited in the In Vitro International, Inc. culture collection, in Linthicum, Maryland, on January 6, 1988, and identified as: BG-391: E.coli MC1061/pBG391 BG-392: E.coli MC1061/pBG392 BG-393: E.coli MC1061/pBG393 BG-394: E.coli MC1061/pBG394 BG-396: E.coli MC1061/pBG396 203-5 E.coli SG936/p203-5.

These cultures were assigned accession numbers IVI 10151-10156, respectively.

Microorganisms and recombinant DNA molecules according to this invention are also exemplified by cultures deposited in the In Vitro International, Inc. culture collection, in Linthicum, Maryland, on August 24, 1988 and identified as: 211-11: E.coli A89/pBG211-ll 214-10: E.coli A89/pBG214-10 215-7 E.coli A89/pBG215-7 These cultures were assigned accession numbers IVI 10183-10185 respectively.

While we have hereinbefore described a 25 number of embodiments of this invention, it is apparent that our basic constructions can be altered to provide othe embodiments which utilize the processes and compositions of this invention. Therefore, it will be appreciated that the scope of this invention is to be defined by the claims appended hereto rather than by the specific embodiments which have been presented hereinbefore by way of example.

1

Claims (39)

1. A DNA sequence selected from the group consisting of: the DNA inserts of p199-7, pBG377, pBG380, pBG381, p203-5, pBG391, pBG392, pBG393, pBG394, pBG395, pBG396, pBG397, p211-11, p21 4 -10 and p215-7; DNA sequences which hybridize to one or more of the foregoing DNA inserts and which encode a soluble T4-like polypeptide which inhibits adhesion between T4+ lymphocytes and infective agents which target T4+ lymphocytes and which inhibits interaction between T4+ lymphocytes and antigen presenting cells and targets of T4 lymphocyte mediated killing; and DNA sequences which encode a soluble T4-like polypeptide encoded by any of the foregoing DNA inserts and sequences.

2. A recombinant DNA molecule comprising a DNA sequence according to claim 1, 3aid DNA sequence being operatively linked to an expression control sequence in said recombinant DNA molecule.

3. The recombinant DNA molecule according to claim 2, wherein said expression control sequence is selected from the group consisting of the early or late promoters of SV40 or adenovirus, the lac system, the trp system, the TAC system, the TRC system, the major operator and promoter regions of phage A, the control regions of fd coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, the polyhedron 1 Lu 98 promoter of the baculovirus system and the promoters of the yeast a-mating factors.

4. A unicellular host transformed with a recombinant DNA molecule according to claim 2. The host according to claim 4, wherein said host is selected from the group consisting of strains of E.coli, Pseudomonas, Bacillus, Streptomyces, fungi, animal cells, plant cells, insect cells and human cells in tissue culture.

6. A polypeptide encoded by a DNA sequence according to claim 1, said polypeptide being essentially free of other proteins of human origin.

7. The polypeptide according to claim 6, wherein said polypeptide is selected from the group consisting of a polypeptide of the formula AA- 3 of Figure 3, a polypeptide of the formula AA,-AA, 6 of Figure 3, a polypeptide of the formula Met-AA, 3 of Figure 3, a polypeptide of the formula AA. 23 -AA 37 of Figure 3, a polypeptide of the formula AA,-AA 3 of Figure 3, a polypeptide of the formula Met-AA,.3, of Figure 3, a polypeptide of the formula AA 23 -AA 377 of Figure 3, a polypeptide of the formula AA,-AA 3 of Figure 3 and a polypeptide of the formula Met-AA, 3 7 of Figure 3.

8. The polypeptide according to claim 6, wherein said polypeptide is selected from the group consisting of a polypeptide of the formula AA_2 3 of Figure 16, a polypeptide of the formula AA,- of Figure 16, a polypeptide of the formula MAA-AAs, of Figure 16, a polypeptide of the formula AMet-AA,., of L 99 Figure 16, followed by the amino acids asparagine- leucine-glutamine-histidine-serine-leucine, a polypeptide of the formula of Figure 16, followed by the amino acids asparagine-leucine- glutam'ne-histidine-serine-leucine, a polypeptide of the formula of Figure 16, followed by the amino acids asparagine-leucine-glutamine-histidine- serine-leucine, a polypeptide of the formula AA-,-AA,, j of Figure 16, a polypeptide of the formula of Figure 16, a polypeptide of the formula of Figure 16, a polypeptide of the formula AA- 2 of Figure 16, a polypeptide of the formula AA,-AA,, of Figure 16, a polypeptide of the formula of Figure 16, a polypeptide of the formula AA- 2 of Figure 16, a polypeptide of the formula of Figure 16, a polypeptide of the formula Met-AA, 3 of Figure 16, a polypeptide of the formula AA 23 -AA, 4 of Figure 16, a polypeptide of the formula of Figure 16, a polypeptide of the formula Met-AA,,, of Figure 16, a polypeptide of the formula AA 23 -AA, 6 6 of Figure 16, a polypeptide of the formula of Figure 16, a polypeptide of the formula Met-AA,,, 66 of Figure 16 and portions thereof.

9. The polypeptide according to claim 6, wherein said polypeptide is selected from the group consisting of a polypeptide of the formula AA,-AA, 62 of mature T4 protein, a polypeptide of the formula Met- Sof mature T4 protein, a polypeptide of the formula AA,-_A 3 6 of mature T4 protein, a polypeptide of the formula :..formula Met-AA, of mature T4 protein, a polypeptide of the the formula Met-AA,_AA, of mature T4 protein, a polypeptide the formula AA,-AA 37 of mature T4 protein, a polypeptide of the formula Met-AA,_,, of mature T4 protein and portions thereof. 9 9 9* f S 2 Ir 100 The polypeptide according to claim 6, wherein said polypeptide is selected from the group consisting of a polypeptide of the formula of mature T4 protein, a polypeptide of the formula of mature T4 protein, a polypeptide of the formula of mature T4 protein, followed by the amino acids asparagine-leucine-glutamine-histidine- serine-leucine, a polypeptide of the formula Met-AA,_, of mature T4 protein, followed by the amino acids asparagine-leucine-glutamine-histidine-serine-leucine, a polypeptide of the formula AA,-AA,, 3 of mature T4 protein, a polypeptide of the formula Met-AA,,, of mature T4 protein, a polypeptide of the formula AA,- of mature T4 protein, a polypeptide of the formula Met-AA_,,, of mature T4 protein, a polypeptide of the formula AA,-AA,, of mature T4 protein, a polypeptide of the formula of mature T4 protein, a polypeptide of the formula of mature T4 protein, a polypeptide of the formula of mature T4 protein, a polypeptide of the formula AA,- AA,, of mature T4 protein, a polypeptide of the formula Met-AA,, 66 of mature T4 protein and portions thereof.

11. The polypeptide according to claim 6, wherein said polypeptide is selected from the group consisting of a polypeptide of the formula AA 3 -AA3,, of Figure 16, a polypeptide of the formula Met-AA 3 3 of Figure 16, a polypeptide of the formula AA,-AA,7 of 0 Figure 16, a polypeptide of the formula Met-AA3. 37 of Figure 16, a polypeptide of the formula AA 3 -AA 6 of Figure 16, a polypeptide of the formula Met-AA 3 1 62 of Figure 16, a polypeptide of the formula AA 3 of S *0 Figure 16, a polypeptide of the formula Met-AA,,, of Figure 16, a polypeptide of the formula AA3-AA,,, of :Figure 16, followed by the amino acids asparagine- 101 leucine-glutamine-histidine-serine-leucine, a polypeptide of the formula of Figure 16, followed by the amino acids asparagine- leucine-glutamine-histidine-serine-leucine, a polypeptide of the formula AA-AA,,,of Figure 16, a polypeptide of the formula Met-AA,_,, of Figure 16, a polypeptide of the formula AA-AA, of Figure 16, a polypeptide of the formula of Figure 16, a polypeptide of the formula AA 3 of Figure 16, a polypeptide of the formula of Figure 16, a polypeptide of the formula AA 3 of Figure 16, a polypeptide of the formula Met-AA_, 4 s of Figure 16, a polypeptide of the formula AA,-AA,, of Figure 16, a polypeptide of the formula Met-AA,., 1 of Figure 16, other A2 variants of the T4 polypeptide of Figure 16, and portions thereof.

12. A method for producing a polypeptide according to any one of claims 6 to 11 comprising the step of culturing a unicellular host transformed with a recombinant DNA molecule according to claim 2.

13. A pharmaceutical composition comprising an immunotherapeutic or immunosuppressive effective amount of a polypeptide according to any one of claims 6 to 11 and a pharmaceutically acceptable carrier.

14. A method for treating a patient comprising the step of treating them in a pharmaceutically acceptable manner with a composition according to claim 13. Luj J 0* 102 The method according to claim 14, wherein said patient is treated by intramuscular injection of the composition.

16. A diagnostic composition for detecting or for monitoring the course of HIV infection comprising a diagnostic effective amount of a polypeptide according to any one of claims 6 to 11.

17. A method for detecting or for monitoring the course of HIV infection comprising the step of employing as a diagnostic a composition according to claim 16.

18. A means for detecting or for monitoring the course of HIV infection comprising a diagnostic composition according to claim 16.

19. A pharmaceutical composition comprising an immunotherapeutic or immunosuppressive amount of an antibody to a polypeptide according to any one of claims 6 to 11 and a pharmaceutically acceptable carrier.

20. A method for treating a patient comprising the step of treating them in a pharmaceutically acceptable manner with a composition according to claim 19.

21. The use of a polypeptide according to any one of claims 6 to 11 to purify HIV virus.

22. The use according to claim 21, wherein the HIV virus is purified from a biological sample. 0 O ooo" 4 103

23. A method for purifying HIV virus from a sample comprising the step of exposing the sample to a polypeptide according to any one of claims 6 to 11.

24. The method according to claim 23, wherein the sample is a biological sample. A DNA sequence selected from the group consisting of: the DNA insert of pBiv.l; DNA sequences which hybridize to the DNA insert of pBiv.l and which encode a polyvalent soluble T4-like polypeptide; and DNA sequences which encode a polyvalent soluble T4-like polypeptide encoded by the DNA insert of pBiv.l.

26. A recombinant DNA molecule comprising a DNA sequence according to claim 25, said DNA sequence being operatively linked to an expression control sequence in said recombinant DNA molecule.

27. A unicellular host transformed with a recombinant DNA molecule according to claim 26. .e

28. A polypeptide encoded by a DNA sequence according to claim 25, said polypeptide being d essentially free of other proteins of human origin.

29.' The polypeptide according to any one of claims 6 to 11, wherein said polypeptide is polyvalent. .o

30. A method for producing a polyvalent polypeptide comprising the steps of: O S R 31 -£S 21 rj I II-~ -104 culturing a unicellular host transformed with a recombinant DNA molecule according to claim 2 to produce a polypeptide; and coupling said polypeptide to a carrier to form a polyvalent polypeptide.

31. A DNA sequence comprising: a first portion comprising a DNA sequence coding for the constant region of an immunoglobulin light chain; and a second portion comprising a DNA sequence according to claim 1, or portions thereof, said second portion being joined upstream of said first portion.

32. A DNA sequence comprising: a first portion comprising a DNA sequence coding for the constant region of an immunoglobulin heavy chain; and a second portion comprising a DNA sequence according to claim 1 or portions thereof, said second portion being joined upstream of said first portion.

33. An expression vector comprising the DNA sequence according to claim 31.

34. An expression vector comprising the DNA sequence according to claim 32.

35. An expression vector comprising the DNA sequence according to claim 31 and the DNA sequence according to claim 32. s RA4/ iI 0 105

36. A method for producing a chimeric rsT4/IgG, comprising the step of co-transfecting a host cell with the expression vector according to claim 33 and the expression vector according to claim 34.

37. A method for producing a chimeric rsT4/IgG, comprising the step of transfecting a host cell with the expression vector according to claim

38. A chimeric rsT4/IgG, produced by the method according to claim 36 or 37.

39. A pharmaceutical composition comprising an immunotherapeutic or immunosuppressive effective amount of a polypeptide according to claim 28 or 29. A method for treating a patient comprising the step of treating them in a pharmaceutically acceptable manner with a composition according to claim 39.

41. A diagnostic composition for detecting or for monitoring the course of HIV infection comprising a diagnostic effective amount of a polypeptide according to claim 28 or 29.

42. A pharmaceutical composition comprising an immunotherapeutic or immunosuppressive effective S' amount of a chimeric rsT4/IgG 1 according to claim 38. *43. A method for treating a patient comprising the step of treating them in a pharmaceutically acceptable manner with a composition according to claim 42. DATED this 24th day of February 1992 RA S: BIOGEN, INC. By their Patent Attorneys CULLEN CO. Tt T^ WO-89/01940 1/93 PCT/US88/02940 A 0 0 00 I 42000 I

3000. al VON -7282 113 W14fl r8 2000FIG jI-~ SUSIUT HE 1: A A I I I I If-sI A A A 4 I .WO089/01940 PCT/US88/02940 93 FIG2 9 9/ A IV f2 1207- 35 4567 It

5415. 'Sr WW (ma) -200 -67 -43 SI I.. b~. 1~ SUBSTITUTE SHEET t it .W0189/01940 PCT/US88/02940 3/p3 FIG3 H BBE S j p M M NBsBCNC fl a n n laptosr f REX51 t 1 1 an1NRpF 1 3. 1 1 4221221 TTTTTTTTTTAAGCACGAcICTGCAGAAGGAACAAAGCACCCTCCCCACTGGGCTCCTGG &AAAAAAATTCGTGCTGAGACGTCTTCCTTGTTTCGTGGGAGGGGTGACCCGAGGACC F F L S T T LQ K E Q S T L P T G L LV- BH F N A BsgNSS M M nsa B BM 1lapisas n n u p b bn u nlApct 11 4 B v V1 1 221211 11 H 2 1 11 TTGCAGAGCTCCAAGTCCTCACACAGATACGCCTGTTTGAGAAGCAGCGGGCAAGAAAGA S 120 AACGTCTCGAGGTTCAGGAGTGTGTCTATGCGGACAAACTCTTCGTCGCCCGTTCTTTCT A E LQ0V L TQ I RL F E K Q R A R. K T- S m H HDHaP n g graum 1 a aae9s 1 1 12361 B B P S a DBsNAD~pPaS t da avrlusui X enipaaaMo9n 1 12222241161 S D DHNPa d ralsu e aeas9 1 23416 CGCAAGCCCAGA CCCTGCCATTTCTGTGGGCTCAGGTCCCTACTGGCTCAG-GCCCCTG 121 180 GCGTTCGGGTCT GGGACGGTAAAGACACCCGAGTCCAGGGATGACCGAGTCCGGGGAC Q A 0RP C H F C G L R S L L A Q A P A- REX S'PJice Dor S H F m mmHm HMNC i B B nl n nnan pacr n b b u 11lel apiF f v v 4 1. 1 131 Me+ 2111 1 1. 1 H CCTCCCTCGGCAAGGCCACTOACCGGGGAGTCCCTTTTAGGCACTTGCTTCTGGTGC 181 240 GGAGGGAGCCGTTCCGGTGTTACTTGGCCCCTC-AGGGAAAATCCGTGAACGAAGACCACG S L G KATM N R G VP F R H L L L V L- AA-, H F i HH rimm D B B n ha un n d b b P ae 41 1 e v V 1 12 Hl1 1 1 1 TGCAACTGGCGCTCCTCCCAGCAGCCACTCAGGGAAAGAAAGTGGTGCTGGGCAAAAA.G 241 300 ACGTTGACCGCGAGGAGGGTCGTCGGTGAGTCCCTTTCTTTCACCACGACCCGTTTTTTC Q L AL LP AAT 0QG K K V V L G K K G- R A~ M MM mm 1 1 AAL b bb b a u 0 00 0 1 1 2 22 2 GGGATACAGTGGAACTGACCTGTACAGCTTCCCAGAAGAAGAGCATACAATTCCACTGGA 301 360 CCCTATGTCACCTTGACTGGACATGTCGAAGGGTCTTCTTCTCGTATGTTAAGGTGACCT D T VE L TC T ASQ0K K S I Q F H WK- I. S12T!TTESHEET WO8/01940o PCT/US88/02940 F 3(cof 'd) H B S i NBaN F F AaS n laps o o vui f alip k k a9n 1 4222 1 1 261 AA6ACCTCCAACCAGATAAAGATTCTGGGAAATCAGGGCTCCTTCTTAACT GGTCCAT 361 420 TTTTGAGGTTGGTCTATTTCTAAGACCCTTTAGTCCCGAGGAAGAATTGATITCCAGGTA N SN QI K IL G N Q G SF L T KG PS S FH H S REX A MNDa niHT i M MANaSS Splice 1 bdpu unhh n b bvluit AcceptOr u oen3 DPaa f o oaa9ny 1 121A 2111 1 2 224611 ii// CCAAGCTGAATGATCGCGCTGACTCAAGAAGAAGCCTTCGGGACCAAGGAAACTTTCCCC 421 480 GGTTCGACTTACTAGCGCGACTGAGTTCTTCTTCGGAAGCCCTGGTTCCTTTGAAAGGGG K L N DR AD SR R S L RD Q G N F P L S H H S BMDNa i A i D M H m m MAMMa.M cbpdu n f n d b b n n nvnnun lone3 f 1 f e o 0 1 a1191 1112A 1 2 1 1 2 2 1 1 121161 TCATCATCAAGAATCTTAAGATAGAAGACTCAGATACTTACATCTGTGAAGTGGAGGACC 481 ACTAGTAGTTCTTAGAATTCTATCTTCTGAGTCTATGAATGTAGACACTTCACCTCCTGG I I K N L K I E D S D T T I C E V E D Q S H E i a c n e 0 1 1 B AGAAGGAGGAGGTGCAATTGCTAGTGTTCGGATTGACTGCCAACTCTGACACCCACCTG 541 600 TCTTCCTCCTCCACGTTAACGATCACAAGCCTAACTGACTTGAGACTGTGGGTGGACG K SE VQ L LVF GLTAN SD T H L L B B BBE S a 0 S BsscNc D p p! t aptoor d M y n1NRpF e 1 1 1 221221 1 TTCAGGGGCAGAGCCTGACCCTGACCTTGGAGAGCCCCCCTGTAGTACCCTCAGT 601 660 AAGTCCCCTCTCGGACTGGGACTGGAACCTCTCGGGGGGACTTGGATAG Q G Q S L T L T L E S: P P G S S P S V Q H N M M i S M M MDOM As n n n t b b n dn Ip 1 1 f y o o 1 el uB 11 1 1 2 2 1 11 12 AATGTAGGAGTCCAAGGGGTACATA aAGGGGGGGAAGACCCTCTCCGTGTCTCAG 720 TTPCATCCTCAGGTTCCCCATTTTGTATCTCCCCCCCTTCTGGAGAGGCACAGAGrG C R S P R G K N I Q G G K T L S V S Q L- L_ i i .Ir ,91,l~ cc~f~ W6 89/01940 PCT/US88/02940 FiG. 3(cont'd) BESH SB BES N P A BsscNScsS B NScc Ns M v 1 apt osarts a Itor Ip b u u n1NRApcFXt n aNRF aH 0 2 1 2212121111 1 4121 31 2 TGGAGCTCCAGGATAGTGGCACCTGGACATGCACTGTCTTGCAGAACCAGAAGAAGGTGG 780 ACCTCGAGGTCCTATCACCGTGGACCTGTACGTGACAGAACGTCTTGTCTTCTTCCACC E L Q D S G T W T C T V L Q N Q K K V SE H NM A HS MMmm b ha 1 at n nn n 0 ee u eu 1111 2 11 1 31 1 11 1 AGTTCAAAATAGACATCGTGGTGCTAGCTTTCCAGAAGGCCTCCAGCATAGTCTATAAGA 781 840 TCAAGTTTTATCTGTAGCACCACGATCGAAAGGTCTTCCGGAGGTCGTATCAGATATTCT F KID I VV LA F QK AS SI V Y K K A 1 1 AAGAGGGGGAACAGGTGGAGTTCTCCTTCCCACTCGCCTTTACAGTTGAAAAGCTGACGG 841 900 TTCTCCCCCTTGTCCACCTCAAGAGGAAGGGTGAGCGGAAATGTCAACTTTTCGACTGCC E GE Q VS F SF P LA F TV E K L T G- P S A M H H MiIMfMMNDa 1 nu n npnnlnbdpu u 11 lhllMloen3 1 1 1 1 111111121A II GCAGTGGCGAGCTGTGGTGGCAGGCGGAGAGGGCTTCCTCCTCCAAGTCTTGGATCACCT 960 CGTCACCGCTCGACACCACCGTCCGCCTCTCCCGAAGGAGGAGGTTCAGAACCTACTGGA S GE LWW Q A ERAS S S K SW ITS- B BES P S MM m am accADNpPDaS A n n b b ta tor vrlusdui 1 11 0 a Se NRLFaaaMse9n u 1 1 2 2 23 121 22411161 1 /I1/ CTGACCTGAAGAACAAGGAAGTGTCTGTAAAACGGGTTACCCAGGACCCTAAGCTCCAGA 961 1020 GACTGGACTTCTTGTTCCTTCACAGACATTTTGCCCAATGGGTCCTGGGATTCGAGGTCT DL KN KS VS VK RVT Q D P K LQ M- BE S AH H M scMc HS D M M H 1ip p n tonr at d n n p u h h 1NR1F eu e 11 h 1 1 1 1 1211 31 1 1 1 1 TGGGCAAGAAGCTCCCGCTCCACCTCACCCTGCCCCAGGCCTTGCCTCAGTATGCTGGCT 1021 1080 ACCCGTTCTTCGAGGGCGAGGTGGAGTGGGACGGGGTCCGGAACGGAGTCATACGACCGA G K K L P L H L T L P Q A L P Q Y A G S STITUT;.:E SHEET- WO: 9/0940PCT/US88/ 02940 H BES SFIG Xc'on/'d2 Hscc MMa f f 8CC p tor nanu a a tor h NRF 1e19 N N NRF 1 121 1316 1 1 121 CTGGAAACCTCACCCTGGCCCTTGAAGCGAAAACAGGAAAGTTGCATCAGGAAGTGAACC 1140 GACCTTTGGAGTGGGACCGGGAACTTCGCTTTTGTCCTTTCAACGTAGTCCTTCACTTGG G N L T L A L E A K T G K L H Q E V N L P S H H D A M M kDNNpPa p p d 1 nfl vrllusu h hea u 1 1 aaaa.Ms9 1 1 1 1 1 1 2244116 TGGTGGTGATGAGAGCCACTCAGCTCCAGAAAAATTTGACCTGTGAGGTGTGGGGACCCA -I i 1200 ACCACCACTAC TCGGTGAGTCGAGGTCTTTTTAAACTGGACACTCCACACCCCTGGGT V V M R A T Q L Q K N L T C E V W G PT- S S s f Df mm DE A HMH T i a da nin do 1 n n a n N eN lul cp u 1 1q 1 1 1111 111 CCTCCCCTAAGCTGATGCTGAGCTTGMACTGGAGAACAAGGAGGCAAAGGTCTCGAAGC 1201 1260 GGAGGGGATTCGACTACGACT CGAACTTTGACCTCTTGTTCCTCCGTTTCCAGAGCTTCG S P K L HL S L KL E NK E A KV S KR- H HMM DH F FO M i n nl do o od a n 1 1 at kc Ie e f 1l1 12 1 11 3 1 GGGAGAAGGCGGTGTGGGTGCTGAACCCTGAGGCGGGGATGTGG;CAGTGTCTGCTGAGTG 1261 12 CCCTCTTCCGCCACACCCACGACTTGGGACTCCGCCCTACACCGTCACAGACGACTCAC E K A V W V L N P E A G M W Q C L L S D- P PS H S S A AflpPaS iANaS HMNc v vrusui a vlui pscr a aaMs9n f aa9n apiF 1 221161 1 2361 2111 ACTCGGGACAGGTCCTGCTGGAATCCAACATCAAGGTTCTGCCC-ACATGGTCCACCCCGG 1321 1 13.80 TGAGCCCTGTCCAGGACGACCTTAGGTTGTAGTTCCAAGACGGGTGTACCAGGTGGGGCC S G Q V L L E S N I KV L P T W S T P V- F S H n aH B B H H A 9 HMNH HH MM M u ua b b g g hi. paaa n n b bba 4 9e v v a a aD ape. 1 1 o ooe H 63 1 1 1 1 21 2113 1 1 2 221 TGCAGCCAATGGCCTGATTGTGCAGGGGTCGCCGGCCTCCTGCTTTTCATTGGGC 1381 =mm ACGTCGGTTACCGGGACTAACACGACCCCCCGCAGCGGCCGGAGGACGAAAAGTAACCCG QP M A L I V L G G V A G L L L F I G L- SUBSTITUTE SHEET WO 89/01940 PCT/US88/O2940 F/G.3 (c on/'d) s S H H M f f B NHMBNN BAqHBHiNN D b a a a 1 psaa aluihbanal d 0 N N n a apnea naDaeePra e 2 1 1 1 4 21114 121112114 1 TAGGCATCTTCTTCTGTGTCAGGTGCCGGCACCGAAPGCCCAAGCACAGC GGATGTCTC 1441 1500 ATCCGTAGAACAAGACACAGTCCACGGCCGTGGCTTCCGCGGTGCTCCTACAGAG G I F F *C V R C R H R R R Q A E R M S Q- S H B BB MNDaF F i D m m MH MH Mo soN HM M M b~dpuo 0on d n n bp bbp bp pps ps n oen3k k f a 1 1 oh ooh oM iMp ap 1 1 121A1 1 1 1 1 1 21 221 21212 211 1 1501 1560 TCTAGTTCTCTGAGGAGTCACTCTCTTCTGGACGGTCACGGAGTCGTT I X R L L S E K K T C Q C P H R P Q KT NBE S s F BES mem o m4 m SCHc MeDaX nC a E M BM bnlnbp n n toar bdpuh u tor bn bn olaloH 1 1 NReF oen3o 4 NRF vi vl 213121 1 1 1231 121A2 H 121 11 11 Sfop REX Si-op CATGTAkGCCCCAT7IFGC-ACGAGGCCAGGCAGATCCACT GCGCCTCCCCAGGTGT 1620 GTACATCGCGGGTAAACTCCGTCTCCGCCGTCTAGGTGAA3CGGrTC C S P I *G T R P C R S H L Q pP 0 V S- Fs SBES B nT AaS MNDaeccx Hs H m uh uibdputorh at t an Da a9n oer3NRFo eu X e 1 21 261 121A1212 31 1 3 1 CTGCCCCGCGTTTCCTGCCTGCGACCAGATGAATGTAGCAGATCCCAGGC CTCTGGCCT 1680 CACGGGGCGCAAAGGACGGACGCCTGGTCTACTTACATCGTCTAGGGTCCGGAGACCGGA A P R F L P AD Q HMN V A D P R P L AS- BES 'K H mM m O4m mcc DHNPHMNac n n ni nl nn n tor raispacur 1 1 111NRF aea a i9F 1 1 1 1 1121 234121161 CCTGTTCGCCTCCTCTACAAI'TTGCCATTGTTTCTCCTGGTTAGGCCCCGGCTTC.ACTG 1740 GGACAACGAGGAGATGTTAACGTAACAAAlAGGACCCALTCCGGGGCCGAAGTGAC C S P P L Q P A I V S P G L G PCG F T G- 111 11 CTTCACTGTTGCTCTCTAGTTTCCAGAC<C TI'AATCACAC CCTC CTCCACCCCATTTC CT 1800 CAACTCACAACGAGAGATCAAMCGTCTCCGAATTACTGTGGCAGCAGGTGCGGTAAAGGA V VLL S S FQR L NH T VL HA I1SF- ____SUBSTITUTE SHEET a 1 WO 89/01940 PCTIUS88/02940 8/P3 F/G. 3(con/'d) H MM a nn a 11 1 11 TTTCCTTCAAGCCTAGCCCTTCTCTATTATTTCTCTACCCTC TCCCCACT 1860 AAAGG AGTTC GGATC GGGAAGAGAGTAATA AGAGAGAC TGGGAGAGGGG TGAC GAG TA SF K PS PS LII S L PS PH C SF B BE SS S BE S aMDNscNacX aH scMcM MM H H mbpdtolurh u a tofirn n n p p HoneNRa3Fo 9 a NR1F1 1 1 h h 1112124A12 6 3 12111 1 1 1 1 TTGGATCCCAGGGGAGTGTTCAGGGCCAGCTGGCGCTGGAGC- TGAGGCTGGGTGT 1920 AACCTAGGGTCCCCTCACAAGTCCCGGCGGGACCGACCGACCTCCCACTCCCCA G S Q G S VQG Q PWVL AG G G WV BE P SSS N N A.DNBcMpXAN.PaacSS S1 1 vrltonunvlsuurii a a aaaNRlMlaas99Fnn 3 3 2241211124166111 CTGGAAGCATGGAGCATGGGACTGTTCTTTTACA.AGACAGGACCCTGGGACCACAGAGGG 1980 GACCTTCGTACCTCGTACCCTGACAAGAAAATGTTCTGTCCTGGGACCCTGGTGTCTCCC G S H E H G T V L L Q D R T L G P Q R A- s S S f f M MMKNDFaXFF F a a n n bdpouhoo o N N 1 1 oenk3okk k 1 1 1 1 1211A211 1 I CAGGAACTTGCACAAAATCACACAGCCAAGCCAGTCAAGGATGGATGCAGATCCAGAGGT 1981 2040 GTCCTTGAACGTGTTTTAGTGTGTCGGTTCGGTCAGTTCCTACCTACGTCTAGGTCTCCA G T C T K S H S Q A S Q G W M Q 1 Q R F- F F n R B BB B M M HnN H AYNF u s b bb b n n pul p volo 4 a v vvv 1 1 h4a h akak H 1 1 11 1 1 1 1H3 1 2141 /1 TTCTGCAGCCAGTACCTCCTGCCCCATGCTCCCGCTTCTCCCCTATGTrGGTGGGAC 2041 2100 AAGAC C GTCGGTCATGGAGGAC GGGG TAC GACGGGC GAAGAGTGGGATACAC C CAC CC TG LAA ST SC P MLP A S H P MWVG P S H aS i MM NR ui n nn is 9n f 11 aa 61 1 11 31 CACAGACTCACATCCTGACCTTGCACAAACAGCCCCTCTGGACACAGCCCCATGTACACG 2160 GTGTCTGAGTGTAGGACTGGAACGTGTTTGTCGGGGAGACCTGT;TCGGGGTACATGTGC Q T H I L T L H K Q P L W T Q P H V H G- -t iTEu SHEZT WO89/01940 PCT/US88/02940 FIG 3(conrfd) H F F M M FF M D a o 0 nn 0 0 nin d e k 1 1 kk 11 e 3 1 1 11 1 1 11 1 GCCTCAAGGGATGTCTCACATCCTCTGTCTATTTGAGACTTAGAAAAATCCTACAAGGCT 2220 CGGAGTTCCCTACAGAGTGTAGGAGACAGATAAACTCTGAATCTTTTTAGGATGTTCCGA L K G C L T S S V Y L R L R K I L Q G W S BH A D BMDNa M M ADBsqNSS c d cbpdu n n idapisas c e lone3 1 1 uenlApct 1 1 1112A 1 1 11221211 GGCAGTAGACAGAACTAAGATGATCATCTCCAGTTTATAGACCAGAACCAGAGCTCAGAG 2221 2280 CCGTCATCTGTCTTGATTCTACTAGTAGAGGTCAAATATCTGGTCTTGGTCTCGAGTCTC T E L R S S P V Y R P E P E L R.- j H BES m HM H i scc a pa a n tor e ap a f NRF 1 21 1 1 121 AGGCTAGATGATTGATTACCAAGTGCCGGACTAGCAAGTGCTGGAGTCGGGACTAACCCA 2281 2340 TCCGATCTACTAACTAATGGTTCACGGCCTGATCGTTCACGACCTCAGCCCTGATTGGGT AR LIT K CR T SK C W SR D PR PS F ADNpPaS B B n B M MB H H vrlusui b b u s n ns p p aaaMs9n v v 4 m 1 1m h h 2241161 1 1 H 1 1 11 1 1 U //I GGTCCCTTGTCCCAAGTTCCACTGCTGCCTCTTGAATGCAGGGACAAATGCCACACGGCT 2400 CCAGGGAACAGGGTTCAAGGTGACGACGGAGAACTTACGTCCCTGTTTACGGTGTGCCGA S L V P S S T A A S M Q G Q M P H G S H R R a a a a a a 1 1 CT AGGCTAGTGGTGGTACTCAATGTCTT TTGGTTCACAGAAGCACAGCA 2401 2460 GAGTGTCACCGATCC,'.7- 4CCCATGA TTACACACAGTCTCGCT i Q W L V V G T Q C V L L G S Q K H S T SN N ANaS D HS F F MFM tc 1 vlui d at o o non yo a aa9n e eu k k lkl 11 3 2461 1 31 1 1 111 II CCCATGGGAAGGGTCCATCTCAGAGAATTTACGAGCAGGGATGAAGGCCTCCCTGTCTAA 2461 2520 GGGTACCCTTCCCAGGTAGAGTCTCTTAAATGCTCGTCCCTACTTCCGGAGGGACAGATT HGKGP SQ RI YEQG RP PCLK $UBSTTUTE $EE.T. WO89/01940 PCT/US88/02940 10/93 Jlo'd N H F M a iMMm M M o n n p n n na b b k II B f 1 le 0 0 1 11 2 1113 2 2 AATCCCTCCTTCATCCCCCGCTGGTGGCAGAATCTGTTACCAGAGGACAAAGCCTTTGGC Z521 2580 TTAGGGAGGAAGTAGGGGGCGACCACCGTCTTAGACAATGGTCTCCTGTTTCGGAAACCG S L L H P P L V A E S V T R G Q S L W L H BH iH A sqN P B BM nh 1 pis a a sa Pa u Ap t m me 11 1 212 1 1 1 3 TCTTCTAATCAGAGCGCAAGCTGGGAGCACAGGCACTGCAGGAGAGAATGCCCAGTGACC 2581---- 2640 AGAAGATTAGTCTCGCGTTCGACCCTCGTGTCCGTGACGTCCTCTCTTACGGGTCACTGG F S E R K L G A Q A L Q E R M P S D Q BES M occ M M AM M MAMMN a tor n n in n nlanh a NRF 1 1 ul 1 luele 3 121 1 1 11 1 11111 II AGTCACTGACCCTGTGCAGAACCTCCTGGAAGCGAGCTTTGCTGGGAGAGGGGTAGCTA 2641 2700 TCAGTGACTGGGACACGTCTTGGAGGACCTTCGCTCGAAACGACCCTCTCCCCCATCGAT S L T L CR T SW KR ALL GE G VA S- PS BBS H D N D ADMMNpPaS M M HsccH i H d 1 d vrnniuaui nn ptorp nh 6 a e aallaMs9n 1 1 hNRFh P a 1 4 1 221141161 1 1 11211 1 1 GCCTGAGAGGGAACCCTCTAAGGGACCTCAAAGGTGATTGTGCCAGGCTCTGCGCCTGCC 2701 2760 CGGACTCTCCCTTGGGAGATTCCCTGGAGTTTCCACTAACACGGTCCGAGACGCGGACGG L R G N P L R DL K G D C AR L C A C P- M M M MM H M MM n n nn n b ab ii liii 0 eo 11 1 111 2 32 CCACACCCTCCCTTACCCTCCTCCAGACCATTCAGGACACAGGGAAATCAGGGTTACAAA 2820 GGTGTGGGAGGGAATGGGAGGAGGTCTGGTAAGTCCTGTGTCCCTTTAGTCCCAATGTTT T PS L T L L Q TI Q D T G KS G L Q I- S B S MNDa D M aMDHNNaX HMHM M M D bdpu d b mbpbdluh p n pn nn d oen3 a o Honoea3o h 1 hi l 1e 121A 1 2 111224A2 1 1 11 1 1 1 TCTTCTTGATCCACTTCTCTCAGGACCCCTCTCTTCCTACCCTTCCTCACCACTTCCCT 2821 2880 AGAAGAACTAGGTGAAGAGAGTCCTAGGGGAGAGAAGGATGGGAAGGAGTGGTGAAGGGA FL I H F SQD P L S S Y P S SP LP SUBSTITUTE SHEE T A I IW 0,89/01940 PCT/US88/02940 11 /M. M M n n M4ADMfl nbhrnb loaa2.o 1.23122 BES 9CC tor NRF 1221 CAGTCCCAACTCCTTTTCCCTATTTCCTTCTCCTCCTGTCTTTAAAGCCTOCCTCTTCC-A 2940 GTCAGGGTTGAGGAAAAGGGATAAAGGAAGAGGAGGACAGAAATTTCGGACGGAGAAGGT V P T P F P Y F L L L L S L K P AS S R- F B B H MB B M M n NBsN SN n nb b b b u laps ps Ilv v o 0 4 anlp ip 2.11 2. 2 2 H 4222 22 GGAAGACCCCCCTATTGCTG~CTGGGGCTCCCCATTTGCTTACTTTGCATTTG.TG~ccCACT 3000 CCTTCTGGGGGGATAACGACGACCCCGAGGGGTAAACGAATGAAACGTAAACACGGGTGA K T P L L L L G L P IC L L C I C A H S- D A d 2. a U 1 1. CTCCACCCCTGCTCCCCTGAGCTGAAAT AATACAATAACTTACTATAGATAAA 3060 GAGGTGGGGACGAGGGGACTCGACTTTATTTTTATGTTATTTGAATGATATTTCTACTTT P P L L P E I K IQ*T Y Y K D E K- AAAA 3061. 3064 TTTT Enzymes that do cut: II U Ii ii I I Accl2 Banl BstE2 EcoR2 Hgi.Al Mae3 Nc il2 NspH. Sau3A Taq 1 Ah&2 Ban2 B stNl2 Eapi Hgi DI Mba 2. Nc ol2 Pf lM2 Sau96 Thal Ah&3 Bbe1l B stfl FnuD2 Hhal2 Mbo2 Nde2 Ppum2. ScrF. Xho2 Af 12 Bbvl2 Ddel2 Fnu4H Hint I Mnl 2. Nhe 1 Pal Sf aNl Alu 1 Bc 11 Dpn2. Fokl HinPi Map 1 Nla3 Psti Sinl Ava 1 Bami Dral2 Hae2 Hpa2 Mu t2 Nla4 Pvu 2 Suti Ava 2 Bspl2 Dra2 Hae 3 Hph 2 Nae 2 Nsp2 Rueal1 Stu I B amH 2 BSPM2. EcoB Hgal2 Mae 1 Nar. NspB2 Sacd Sty'. Enzymes that do not cut: Aat2 Bgl 2. EcoK Kpn 1 PaeR7 SnaBl Xma 2 Apa 2. Bgi 2 EcoK Hae2 Pvu 1 Spel Xma3 ApaLl BupM2 EcoRl Miul Rsr2 Sphl Xmn 1 Asp70 BuaH2 EcoRV Mti Sac2 Supi. Xor2 Asp7l Ct r2 Fspi Nd. 1 Saill Sst2 Au u2 Clal Hinc2 Not 1 Sca2. Tthl Avr2 Dra3 Hind 3 Nru 1 Sf il Xbal2 Ball Eagl, Hpal Nail Sma 1. Xho I 13" T T U T S4Z Sa1 T I. WO89/01940 PCT/US88/02940 WO"89/01940 PCT/US88/02940 1,2-/03 Ecq WVA FIG 4 gkbp) Psil ECORI 4 c~,iso/aie 3kba £Pragmeri pBG 312' Eco RI (.4-75 k bp) I ligqate wl/ T4 /igase 7-4: ps/I NEco A' Es/ k,/e now i /o /a te 2.5 kbp 7Tc{ymen~i I ISUBST!TUTSE SHEET W0789/01940 PCT/US88/02940 /3/93 (19) 104394 (12.3/d23) 604) erro 7cop *nrumrTse inpaernl 9 eses riefer *o PBL T4 cDt/A COOr-dialeS SUSSTlTU TE SH~ET__ FIG 6 AMM 4CID SEQ I/gA'CE COMPw4'/SON .4T POSI1T/Oh'S -3,6 4A/D 23/ f F 74 P05/1,0,7 A/b. Makddon el al c/one 2eox Clone Genorac "Ouse sheep K V 3 41c~ 6A ww w 64 P6 6 766 766 0 7- FF 231 [T TCT [[PT 7-7C 1. It ii II I A WO 89/01940 PCT/US88/02940 15/93 ~J U o K) i ii ii Vt Vt II 0 ~1) s u IR 8 Ir i T a 11 t 1 S- 19 tfll -203- (r57 T4 7 pB6392. 5 52iOp (r5T-4.7) 452 Slop jp 6O393. 5 PEFE /T pB6394 1 v VT J Y (rsT4.9) 113 p8,6395 5~ VJ (r T4 .1Y /31 e0B63Z7 SSV /09, (rsT4 1 "0 0I (rST4. ff) /6 p21-7 V /01 (rsT4. 1/1) J2/-1 5 v ,P21 4 (r5T4.113)ff FIG 78 W089/01940 PCT/US88/ 02940 I WO, 89/01940 PCT/US88/02940 17/193 direc/ed mulagelesls 1 76' -2 T4) co~r, Aval, I5o/a/e /256' khp lkers.4154/ 366' R~gl \I ase YhLoI(pa ria /3/ivL/z iSO50/0 kbp fragm wT llqa~e 1/42tH 7 /5 0/a le 4.5kb Praz~rnenL 4 "'UEG"I"ITUTE W089/01940 PCT/US88/02940 I WO 89/01940 PCT/US88/02940 18/ 3 F/G.88 1 ,7e,4 'fg T4) orq 9 r Ava I, co RI, Ts olie 1./2 kbA, frag me n Lrnker-s 4,6147, 48149 !X '5pBG 366-, CORAV g/z (9 ELco AYz/f r Z5 ola~ f, 3 kbF frag men rL n kers .50/51 p\B.o8G 36 8 -Eco Rfl _q iHE 1, -J IWO 89101"40 PCT/US88/02940 19/.93 /br eKD tess/on of rsT4 direcled RaIYQge/ esis (ga7/'-oatser/oy m FIG.G8C( Als*ff M.~tJI issf ff/A4fty T U TE ~E E T 1 PCT/US88/ 02940 /92-i ~/2 4 C/o I Bad/li Xh 02 to b/awL Xh oI b/indz 0 T4 C/al 19-omo er ,4195-8 (54 co//, expresslon T T Lract COr7S7Lrdr-t Lrac (4)la I SUSSTI'l"TT7 I WO089/01940 I 21/93 PCT/US88/02940 I I A I IL I Bel FIG. 9A n~ilI ,o/A 9 54 w C/o1 r:5T4.2 CiaX s uBs $T TIZ ,sH BSE 7 WO 89/01940 PCT/US88/02940 22/93 C/a T35~ C/aZ a le C/aT po/ 9 8 -2 (2 62) digestion, o -29 FIG 98 OUS'TITUTE -SME WO,189/01940 W~89/1940PCT/US88/ 02940 125/213 FIG 9C 180 Stul CTA OCT TTC CAG AAO C TCC ACC ATA OTO TAT deletion 180 182 1 CTA OCT TTC CAC AAC CTC CAC CAT ACT CTA TAA leu ala ph. gln jasn 3.eu gln his ser leu stop frame shift Ii IWa,89/01940 PCT/US88/02940 2li193 FIG /0 CTOM CCC.ACA TOG TCC ACC CCO CTO CAG CCA ATC TCA 3' OAT CTC ACA 'TO OCT GCA CCO G00 TCG ACC ATO T 3' TCG ACC CCO OTO CAG CCA ATO C0CC CTO ATT TGA .3' 3' 00 OOC CAC OTC OCT TAC COO GAC TMA ACT CTA G S' GAA GAA 0T TGT 000 ACC MCG 31 TCG GGA CAG GTC C'TG CTG GAA TCC AMC ATC AMO TOLA A 3' 3' CTG TCC AG ACC ACC TTA OCT TGT ACT TCA CTT CTA 0 S' CAG CCA CCC MOA GAM ACA M.G TOO 3' TCG OGA CGAG OTC CTO CTO GMA TCC MAC ATC MOG G= 3' 000 CAG MAC (,rr OAT OT OGA 7TC CAG CGO GAC CTO TC 3' W0 89/01940 m PCT/US88/02940 251/ 3 F/IG /O(con/'d) 54 S' ACC 2'TC GAC TCC ACC ATG CAG C-GA MAC AMA C7CG CG 3' JMTATGGATCAATAACGA=GAMATATCTAA =TGM AGGAA.%TCGATTC CATGG TCCTTACTAMGACCrACCCTAG ~7TITUTE SHEET WO089/01940 PCT/US88/02940 26 9 C S c D 0 f AT a d d a I ua, R N a en3q GluPheLeu~iaLeuValLysLouLuIleLauEndz1.'~brLysLau.A-gLyuSe-flOe r R .2 ACCTACIOCC ?GCC)CGACCCTCrrCCCTATGTCACCTTGACTGACA ~b A14 a 14a 1 22 2 130 ThzrAaS.z1mLyuLySrI1.Oltn~b.Bis?pLywanrAAnGlm!.Lys Ile- MUMW 7 AS A QIDA mix? i laps a 1n bdqu unh ani? k Aa 2 o~n3DPa& f 4223 3 261 1 1211JL2111 1 CATC/ Tr.I 1/CG 240 OACCACC CGGAAGAAW#.t1CCAGTA TC=TAC?AGCGC ToA LauGlyMmn1GlySrPh.L,uThrLyGlyPro$rLyaLar5.aWspArAgaL6P LA MA3IaSS SiCNa A 3 3. 2461 1112A 1 2 2I.41 300, SorAzgkzgerrourp~apolnolyksmPhProL~u ll! 1.Ly.AaLauLys Il1e- 24 MUMS K4 ud b m vuni a fe 1&91A 11 2 112611 4 360 TCT TGGTCTT ACAC TCATC CTO rC? CCCTCC.%CGTTMkC AT Gl1UpS*rAapnhrTyri 1.Cyoucl~VaiGlui~upclziLysc luGluValG lnLouLOu- SHEET WO 89/01940 27/93 PCT/US88f 02940 r /L7.ll(cuQ7[a/ C p (;GTCATTCACGCCAACTCGAC.CCCACCT~C rc G 'CTACCCTG 361 420 ValPhGlyLuThr1MaanSrsp2rHiLuLau1aGlYG~lSrL~u~ThrLu- BBC S H s scC3 i t aptoor da a t a3.NRpLF 1 t y 3. 221221 1 1 1. 3 ACCT'TGGAGAGCCCCCCGGTAGTAGCCCCTCAC TGC.ATGTAGGAGTCCAAGGGGTAAA 480 TG ACCTCTCGGGGGGACL:ATCATCGGGGAGTCACGTTACATCCTCAGGTTCCCCATrT ThrLeuuSer~roProGlySerSer~roSerVaGICys~rgSer-ProAzgGlyLys H 3BEX SB BES M4 34 34 AsP A 3ssecqScsS B Nscc b m d 3.pv I aptoisarts a Itor 0 1 a uBU u nlNRAc~cFXt m aNRF 2 1 3. 122 1 221.2121111 1. 4121 AACATACA G GCG AC CCTCTC CGTGTC TCAJGCTGGAGCTC CAGGATAG 'rGGC.AC C 4 ~~481 540 TTTATGTCCCCCCCTTCTGGGAGAGCGAGTCGA~CCTCGAGTCCTAT-ACCGTGG ni ileGlnGlyGlyLysThrLeuSerValSerGnLauGluLeuGlaAspSerG lyThr No lp bh ax a a 31 2 1 TOGACTGCACTGTCTrGCAGAACCAGAAGAAQGTOGAGTTAAAATAGACATCGTOGTG 541 600 ACCTGTACGTGACAGACGTCTGGTCZ'CTCCACCTCAAGTTTATCTGTAGCACCAC TrpThrCyuThrValLeuGlnAmnGlaLyLysValGluPeLysl~splIeValVaI m A HS 3434 a I at a a a u su 1 1 1 1 31 1 1 CTAGC??rCCAGAAOGCCTCCAGCATAGTCTATAAGAAAGACCCGiACirGTOGAGTI'C 601 660 GATCGAAGGTCTCCGCAGGTCGTATCAGATAT'rTCTcrf'CCCCC3.'TCCACCT.CAAG Leu~laPheG~nLyuA~laSerSerZ~eVa1TyrLysLyuGluGlyGluGlaVa1GluPhe A A 34 I I a TCCCCCACTCGCCTTACAGTAAAAGCTGACGGGCAGTGGCGACTGTGGGGCAG 661 720 AGGA.AGGGTGAGCGGAAATGTCAACcrr rACTGCCCGTCACCOCTCGACACC-ACCGTC SerPheProLeu3.aPhaThrValGluLysauThrGlySerGlyGluLouTrpTrpGln SUBSTITUTE SHEET A WU 89/01940 PCT/US88/02940 28193 F G 1c n d p S KE MIt MXDa H flp al bdpu b I h 1.4 oen3 0 11 11 12 1A 2 721 780 CGCCTCTCCCGAGAGGAGGTCAGAACCT.AG~r GAC ACG TCTGTCCCAkc ski see AD~qPDaS A A H ta tor vrlusdui I1 so kCRI aaa.MSe9n u u 23 121 22411161 11 TCTG'AAAC=?ACCCAGGACCCTAACCCAGAGGGCAAGAAGCTCCCGCTCCAC 781 840 AQACATT TCCC ATGGGTCCTGGGATCGAGGTCTACCCGTTC TTCGAGGGCGAGG SarValLysAzrgValThrGln.AspProLysLauGln~otalyLys Lys LeuProLeuHsis a~s ES S H ace HS DK H ace mIa m tor at d mn tor nau I NW *u a I NURI e9 1 121 31 1 1 1 121 136 CTCACCCTGCCCCAGGCI 1CTCCTCAGTATGCTGGCTCTGGAAACCTCACCCTGaCCCTT g00 LauThrLeuProGlaAlaLeuProGln'yrAaGlySerGlyAsnLuThrLuAlaLou S BES f ace H D A a tor d I 1 121 1 1 1 GAGCGAAAPICAGGAAAGTTGCATCAGGAAGTGAAACCTGGTGTGATGAGAGCCACTCA3 901 960 CT!TCGT TGTC C? CAAC GTAGTC CTTCACTTGGACCACCACTAC TCTC TGAG TC GluAlaLysThrGlyLyuLeuHisGlnGluVaIAsmLuValVal~et~rgA-IaThrUm K ADNNpPaS f D KA D n vrllusui a d al d 1aaaaiMa9u N 1IU 122441161 1 1 11 1 1020 rGGTCTTTAAACTGGAA TCCACACCCCTGGGTGGAGGGATCGACTACACTCA LeuGlnLysAsnL.uThrCySGluValTrGlyProTbrserProLyiLeu~etLeuSar KT H 1080 AACTTGACCCTTGTTCTCCGlTCAGAG~cCCc~GCCCCrACACCCACcAC LeuLysLauGluAnLysGIuAaLyVaSrLysArgGluLysAlaValTrpVa1Leu 1 1 WO089/01940 PCT/US88/02940 29/93 F/CG //(cont'd) H p S H DHF D m LiA ADpaS i ds 0 d a a y vrusUi n at k ae6 f a aaMo9m f1 12 1 1 3 1 1 221161 1 1140 TTGGG&CTCCG-CCCCTACACGTCACA=AGACTCACTGAGCCCTGTCCAGGAGACCTT AsaftoGluAlaGly~e tTrpGllcyuLeuL4uSerkspSerG lyG lnValLauLeu61u- H S F S F NSA±T lUQNc f BWNa.X i 2.acna pscr u bbpduh us aIccq api? 4 vome3o 4t 31121 2111 H 1112A2 H2. 2200 AGGTTGTAGTTCCAGACGGGGTACCAGCGGGGiCCACGTCGGTI'ACACTCTAGGACGT SerAsnhleLysVaLuProThrTrpSrThr~roVaG~nProMe apProAla P S P S S A MB ADNNpMPaS ADNpPaS loax 1 nmb vrlluasui vrlusui du u Iv aaaa.Meo9m aa&Ms9a oen3o 1 11 224411161 2241161 2.21A.2 GCCCGCTGACCCTAGGCCCCTTTTATTTGA~G~AGATCCCAATTCT 1201 2260 C GrGTCGAACC CCTGGG&ATCTC CAGCGGAAA AT) AAC ?TAACC CTCTAGGTTAAGA AlaGlmLeuGlyAspProAkrgGlyProLeuPheTy:PheGluLauGlykApProksnSer C/o I N A c AMN A I 1 1 lah II a u 1 a use U 3 1 1 111 1 CATGTTGACACTATCTCGATAAGCTACTTT'ATCGGTAGTTTATCACAGTzTAAA *2320 GTACAJACTGTCGAATAGTAGCTATTCGATCGAATTACG&CCATCAAAZAGTGTCAATTr HisVal~ndGlmLeuZlelleAspLysLaulaLuMetArgEmdPhell.ThrValLys H SN F i H HB at o a h pa nma k P a 1 14 1 11 211 TT~GCTAACGCAGTCAGGcGTATAAATcTAAcAATGcG.CTcATcC-TCATccTCow 2.3s0 A C ATTGCGTCAGTCC CTGGCACATACTTTAGATT ?TAC CC CAGTAGCAGTAGGACC LeuLauThGlmSr~lyValTyrGlulEdGlCysAaHis.rgHisProAzrg- S BaS S S N mOmf acc Fp !D R HNH I. ana tor 0 p. a pocuar a elN NR.? k ap a api9eF 4 311 121 1 21 1 211631 cCCCGTCCCCTGATGCTG&TAGCATAG=CTTOGTATGCCGTACTGCCCGG=C'CT 1381 1440 GTCGCrAGTGGGACCTACGACAXTCCGTATCCCAACCAATACCGCCATGACCGCCCGAG.AA HiskrgHisProGlyCysCy.AzgHinArgLeuCly*TyrAlaGlyThrAlaGlyProLau- ZUrBSTITUTE SHEET WEO89/01940 PCT/US88/02940 30/9 3 FIG//(confYd) M S MBfaNM i HHf a 0 aba u ha a haa IRevN 4so. &aON I V 311 3 11 L. 121. GCGGGATATCGTCCATTCCGCGCATCGCAGTCACTATGCGTGCTGCTAGCGCTATA 1441 cGCCcTATAGCAGGTAAGGCTGTCGTAGCGGTCAGTGATACCGCACGACGATCGCGATAT AlaGlyyArgPrOPhArg~nHiArgG~nSerLauTrpArgAlaAlaSerAlaI1. 3 BH F iPHM SqN C Hn aho .18 t au Ppat lAC r e4 1111212 1 3H TGCGTTGATGCAATTCTATGCGCACCCGTTCTCGAGCACTGTCCGACCGCT='GGC CC 1560 AC oC ACTAC OTT AAGATACC CTGGGCAAGACC CTC TGACAGGC TCG AAACCCCr CysVaI~spAlalleSerMetkrThrAgSerArgSrThrValArgProLauTrpPr:o N T nleQoaT H u 1 a ubdpuh 1 4 a qDoea3a a H 4 12121Al 3 CCGCCCAGTCCTCTCCTCCCTACTTGGACCGACTATC CACTACCCCATATGCGAC 1561 1620 CCC GGGTCAG C C GAAGC CATGAAC CTC GGTGATACCTGATCCGCTAC TAC C CTG ProProSerProAlaA;rPheA~aThr~rpSrHisTyAgLuArgAspHisClyAap KS H H SH CHH{HO~f iDQAqHEHiN a pa fgappsaa psahihbana f Alp raeahpeN apnaDaeaPr 1 21 11321111 2112111211 CJCACCCGTCCTGTGGATTCTCTACCCCGACGCATCGTGGCC CGCATCACCGCGCCAC 1680 GTGTGC' AC' 1CTAAGXATGCGCCTCTACCCGCCTAGTCCCGGTG HisThr~gProVaLpSrLaugrThHiArgGyrgHiHiArgrgHis S H H S Nf aBqH3Uifl H MYa a~ la ah~bana1 bdpu apbs am nafa..ftai oem3 alop 41 121112114 1 121A 2222 ACGTGC CO GCTGGCGCCTATATC CC ACATCAC C CATC'GGCATC CGGCTCC CA 1681 1740 TCZACG;CCACGACCCGATATAGCGCCTGTAOTGGCTACCCCTC"*TACCCGACGT AzgCysG lyCysTrpA~gL~uTyrAgArgHis Him rgTrpGlyArgSerGlySerPro a H S S Sam N i H DNa C HHMc aps 1 a ha ralau f apacr nip a P as aeaa9 r eapi? 222 3 1 12 23416 1 32111 COC TCATUACCCTTGrrT CCC TGGGTATOTOOCAGCCC COT C 0000 1800 C)A6GCCCGAGTACTCGCWCAGCCCACCCATACCACCTCCG00GACC0CCCCC LouA gk&iG~ga~ez~gl~yrlCl,-grA-~y~gl W089/1940PCT/US88/02940 EA93HN~FIG //(cont1~ MaDasepra. 4 4LAP e, 121112114 H H 212 3 81-- 1860 TGACACCCGCGTAGAGGAACGTGCGTGGTAAGGAACGCCG-CCGCCACGAGTTGCCGGA ThrValyArgHisLuLauAlaArghrZ leProCysGlyGlyGlyAlaGrLnArgPro H S a Ma H t b a ga v 1 4 f a b I 1 H 1 I CACCTACTACGG=GCTCCTAAGCAGGAGTcGCATAAGGAGAGCGTCGTCCGAT 1920 GTTGATGATGkCCC&ACGAAGGATACGTCCTCAGCGATCCCTCTCCGCAGCTA GlaProThr~hGyLuauPoAnAaGyVaAlaEfldGlyALrAlaSerSerAsp- HF A HK IaHlT N I p. a uhh I. u ap P oaa a 1 21 1 211 3 GC C CTAGACC? TCAAC CCAGTCAGCTC CTTCC rGTGG3C CGC GGCATGACTATC CT 1921 .980 CCGGAACTCTCGG?GGTCAGTCCAGGAACGCCACCCGCCCCCCTACTATAGCA AlaLauGluSerLeuClaProSerG~nLauL~uProVal~lyAlaGlyHisAspTyrArg- F~ FH u b a1 apaau aha 4 0 a n aape 4P aa H 2 3 1 4 211 H 1 12 4CCCCGCACTTATACTTCTrTCTC~TTA CCCCAATGCACGTGCGGrCCGCCA~cCCT 1981 2040 GCG C TCAATACTCACACAAGAMATAGCT GrAGCATC CTGTC CAC CC TC C A s H HF S BM AaS i aliT lelDa H bmVni a uhh bdpu a V1 a9m P Daa oea3 11 261 1 211 121A 3 CTtCGGTCATTTCGAGACCCT'ICCTG4ACCACATGATCCGCCTCTCGCT 2041 2100 GLACCCAGTAAAGCCCCTCCTCCCCACACCTCCCCCTGCTACTACC CGACACA Leu~lyHisPherAgGlyPoLuSerLauuArApAspAprgProVaLAla H S i K AaNS a a vuli f I1 a9an 1 1 3 2641 TrcGGTAZ'cGAgTrG rcccCTcGCTcAArccTc~rCGCrGGTCCCCCACCAA 2101 *26 ACGCCATAAGCCTTAGACTGCGGACATTCGAACAGTCACCAGGGCGGTGGTT CquClyIleArgAsaLauklaAxrgProArgSerSerLauArgHisTrpSerAxgHisG~a WO089/01940 PCT/US88/02940 FIG /4'cont'd) KM H HMN B ENCEnHX nR ita a p I gi auam unhh g a 4 ap 6 Ia rg4ea D~aa a e 2 3 211. 1 3 111H33 21.11 1 2 ACGTTCGGCGAG;CAGGCCATATCGCCGGCATGGCGGCCGACGCCTGIGCTACGT 2161 TGCAA.AGCCGCTCTTCGTCCGGTAATAGCGGC CGTACCCCCGCTGCGCCtACCCGATGCA ThrPherAgG.ukiaGlyHis~yrArgkHisGlyGlyArgArg.aGlyLuArg- M rxST aT H H FM i H n trh uh g o p I. Dua Oa a a ko t a 1 211 21 1 3 12 1. 2 CTTGCTGGC GZCGC CACGC GAGCGGATGGC CTTC CC CATTATCATTCTCTC GC?."C 2280 GCAAC GAC C OC)AGCCCTGC GCTC CGAC CTAC C GAAGCGGTAATAC TAAGAAGAGC GAAG LatuAaGyVaArgAspAaArgLeuAspGyLeuProHisTyrAspSerSer~rg~he- sip S F 3 BES M fu t nT F H aN sCC I au a ub o a p1 tor p N4 N Da k a Ma NRP 1 1H 1 21 1 '3 13 121 CCC GCATC GGGATGCCC C GC G?1CAGCCCATGCTGTC CAGGC.AGGTAGATCACGAC CA 2340 GCCG&CCGTAGCCCTACGG=CGC)..KCGTCCGGTACGAC'AGGTCCGTCCATCTACTCGCTGGT ArArgHi sAgJpA aAgVa JAI aGlyHi sA.1 aVaIG .nAl aG lyArgEndAr gPr o *1 s A Qfla nnT TMN~a AaS I. bdpua uuh abdpu vui u oen3 04a qoan3 a9m 1 121A 2HI 1121A 261 231TCAGGCACAGCTCAGGACGCTCGCGCCTCTTACCAGrCCTAACTTCGATCACTGGACC 24 2400 serG.yThrAaSerrgI.e2A.&AgGlySer~yrG2nProAanPbeAspHisTrpThr- PN S PFa p bdpua u 9 n pi.s 1 I a oen3e 4 Up i pa a 2 121.A3 H 1 1 212 3 3 GCTGATCG?.CACGGGA??I'ATGCCGCCTCGGCGAGCACATCAACGGGTGGCATGGAT 2460 CGACTAGCAGTGCCGCTAAATACGGCGGAGCCGCTCGTGTACCTTGCCCAACCGTACCTA AlaAspArgHisGlyAspLuCysArgLeuGlyGluHisMetGluArgValG.yMe.Asp H PH F F RHItLiNN H 1Hn N N MY ahi.hbuana. 9 uhn uh. I. I pac ma.Ga.4*Pra a Dal Da a a api 122111H2114 1. 211 21 3 4 2211 TG2?AGGCGCCGCCCTATACCTTGTCTGCCTCCCCGCGTTGCGTCGCGGTGCATGGAGCCG 2520 AACCGCGGCGGGATATGGAACAGACGGAGGGGCGCAACGCAGCGCCACGTACCTC=~ CyuAgA~gAgProePCyLuProPrArgVa.AlaSerArgCysMetGluPro A WO 89/01940 PCT/US88/02940 ,CG //(con'1d) sS 3F H *alc T m HV nsN H H I uar a apa UA Ia q I ape 4a a Itf 631 1 1 211 HI 4 1 1 1 G=CCACCTCGACCTGAATGAGCCGGCGGCACCTCGCTACGATCACCACTCCAAGA 2521 2580 CCGGTGGAG.CTGGACTTACCTTCG=CCGCCGTGGAGCGATrGCCTAAGTGGTGAGGTTCT GlyHisLau.AspLau.AaGlySrrArgxHiLaukPLksnflyPh1ThrThr~rokrg P H P f N iFSHM st H N a Ppm.at YM a A~rGAC)ATCATCTGCGGAACTGTATGCGCAAACCAACCCT1'GGCAGAAC 2581 2640 TAACCTCGGTTAGTTAAGAACrCCTCTAC.ACTCGcT*GTGGGAACCGTcT-rG Ii eG 1yAlaAsanGlmPheLaukzgArgThrVa L uiz.AaGlaThrAa nPOTrpGl n~ FF FTH S S nT a m aBiTH A 3 f BKONa A uhu u uubnhh v b cbpdu I Da4 4 D4v~aa a v N lone3 u 21 H H 2H1111 1 1 1 1112A 1 ATATCCATCQCGTCCGCCATCTCCAGCAGCCGCACGCGCGCATCCGWGGGATGATCAG 2700 TATAGGTAGCGCGACGCGGTAAGGCGTCGGCGTGCGrCCGCGTAGAGCc.CCCTACTAGTC IleSerIle~laSrAa1.SeS1SerAc'ThrAgAglSrGyApApGln FN YET FN s nsP? MiAETMT H H H NmsA HeC a upVO ~unhhah a lupi pocr b 43u DPAL1. a4Hu api? v H221 2121111 11 1 3HII 2111 1 CTGCCTCrrCGCGZTCGGTGATGACCGGAAAACCTCTGACACxGCrArCTCCCGGAGA6C 2760 GACGGAGCGCGCAGCCACTACTGCCACTTTTGItATGTGTACGTCGAAGGGCCTCTG LeuProrgAlaPh.Argznd~ndJrgndLyProL~uThrHisla&klaProGlyksp- S S H F H A f HMc F H i nHT a a 1 a pacr 0 g nuhh P a u N apir k a P Oaa B 3 1 1 2111 1 1 1 211 2 OGTCACAGCT'TGTCTGTAAGCGGATGCCGGGAGCA\GACAAGCCCGTC.AGGGCGCGT"CA-GC 2820 Gy~isaerLouSerVa1Ser~1yCysA~rgGluG1mhrSerProSerGy~rgVai r H I Ha N TBM H A a. hu I ba a c Pa&4 et hve a I IH 3 1.13 2 3 GGGTiG.TGGCGGGTCGCGCACCATACCCAGTCACGTAGCGATAGC GGAG'7GTA 2880 CCCACACCGCCCACArCCCCGCGTCGGTACTGGGTCAGTGCATCGrCTATCGcC: CACA\ g Gl~s ga~rlA-~ri~pr~!h~d-gnA-~ra Sl, 1 Z IfI T T ESl i 1-5 'r WO;89/01940 PCT/US88/ 02940 F/GI/(corif'd) p s A BH ua s d a pi.sd 44 a a L lApe H 1 1 1 2121 4 TACGCTAACTATGCGGCATcAGAGCAGA!'GTA-TGAGAGTGCACCATATGCGGTGT 2881 2940 ATGSACCGAATATACCCGTAGTCTCGTCTAACATACTCTCACGTG4GTATACrrCrCACA TyzrrpL~u.ASn'rAlaAlaSerGlIUl eValLau.ArgVal~isHisateArgCys s H S f H 5 a b n haa b N a P aeN v 1 2 1 121 1 GAAATACCGCACAGATGCGT 1 GGAATCGATAGGCCT rcTCC 3000 CTTTATIGGGGTCTACGCATTCCTCTTATGGCGTACCGCGAGACAAGGAGC GluZI.ProHisArqCyuValArg~gLys'ryrArql lArgArgSarSarAJlaSarSer- H PHR F H i a i H aaA aa u abh u u 1 I t 4 Pva 44 I I1 H111 H H1 CTACTACTCGCTGCGCTCGTCGTCGGCTCGGCGAGCGGTATCAGCTCACTCAAAc 3060 GAGTGACTGA=GACGCGA=cCACCAAOCCGACGCCGCTCGCcATAGcTGAGTGAGPfrC LeuThrAspS.?LeurqserVa1VaIAgLuArgArgAlaValSarAlaHisSerLys- H N i HS f aH J1 31 GCGGTAATACGGTTATCCCAGATCAGGGGATAACGCAGAAAGAAATGTGAGCAAAA 3 061 3120 AlaVa1ilegLuSa~hrG1I.S~rGyApAsflGyLysAaflm0aEfldAlaLys H scHc N Ha nT a toar 1au ub 3 NReF a e4 Da a *3 121,4, 4 3H 21 4 3180 CCGGTCG??rrCCGGTCCTTGGCATTT??rCCGCO"CAACGACCGCAAAAAGGTATCCGAG G1yGlnG1nLysAlaArgAa ArgLys LysAla.A1aLeuLeau.IPhePheHis~rgLau f T M H a a t N q 1 a CGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGCGAAACCCGACA 3240 GCGGCGGGACTGCT.CGTAGTGT7=TTAGCTGCGAAGTTCAGTCTCCACCGCTT"TGGGCT.GT )LrgPro~roApGuHiHiLyAsnrArgSrSrGmnrgrpAgAsnProThr ~2'I~I 'Ur"~4E WO89/01940 PCT/US88/02940 35/93 FG /crfd tor or 1 h NRF NUP u p al 121 121 1 1 11 GGCTATAAQATACC.AGGl:* CCCCCTGGACTCCCTCGTGCCTCTCCTGTTCCC 3241 3300 F H u ps m ha 4 ap P as H 21 1.312 ACCCTrCCCcTTrCCGGATA~CTGTCCGCCcLITCCCTTCGGAACGTGcGcTT~l CT 3301 3360 TGACGTGCCTATGGACAG=CCG" 1tA1G I A CCCZ'CGCACCGCGAAAGA ThrLauProLauThGly~yrLuSarAaPhL~uProSrGySerVaLA-LaLeuSer O A U T 1 CAT CT C CCTGTA=GTATC 'CAGTTC OGTGTAG GTC GTTC GCTC c AGCTGGC TGT 3420 GtACAGT4CGATCCATAGTCAAGCCACATCcAGCAA~cGAGGrrCGA~CCCGAc) 31nCyISerArCyAgIyrLauSerSerValEndValVa1AzgSarLysLauGlyCys A BH NY H H p 8911 3 on iH H204 a pisu b pu ah pea L U~p v 34 pa ape 3. 212 1 2H111 2131 GTCCAACCCCCCGTTZCAGCCCOACCGCTGCCCTATCCGGTAACTrATCGTCTTGAG 3480 CACGTGCTCCCCC 1 GTG=T=GACGCGAATAGCCATf-ATAGCAGAACTC ValHisGluProProVa1G~aProAspArgyA.aL~uSerGlyAsaTyrArgLeuGlu- S FF per u u ba b n api? 4 4 ve v 211.1 H H 13 1 1 TCCCCCCGLAACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATrAGC 3481 3540 H 3 AG&GCGAGGTATGTAGGCGGTGCTACAGAGTTC?.TGAAGTGGTGGCCTAACTACGGCTAC 3600 TC'TCGCTCe-ATACATCCGCCACG&ATGTCTCAGAACTTC-ACCACCGGATTGATGCrAT ArgAlag.ValyGyAaThrGuPhLeuLysTprrpProAsnyrGly~yr WO 89/01940 PCT/USF8/O294O ?423 FIG I/(corf'') m i H p a 3660 ThrArgArg'rhrValPh.GlylleCysAILaeuLeuLysProValThrPheGlyLysAzrg- 7s A bmfa~O4 I 3 bdpups p 4 ui con3ap a 1 12IA21 2 GTTGGTACTCTTGATCCGG>V CACCCCCG AGGT TTT TTTG 3720 CAACCATCGAGAACTAGGCCGTTGTTTGGTGCGACCATCrCCACCAAAAAAACAAACG ValGlySerSarEndSerGlyLyuGlnThrThrA.aGlyS~rGly G yPhePheValCya F pH S S sS n BteT 0Ta.X 1QfaX lMa u buhah bdpuh bdpuh bdpbu 4 v~apa oen3o oam3o oaao3 H 12111 12i2A2 121A2 1212A AAGCAGCAATGCGCAGAAI111 A &GATCCAAGAG TCC.GATC???TCTACG 4 3780 TTCGTCGTCTAATGCGCGTC r iCCTAGAGTTCTTAGGAAACrAGAAAAGATGC Ly.GlnG~nn1.ThrArgArgLysLysGlySerG~nGuAapProLauIlePheSerThr o H N d g a I. b 0 a 'a a o 2. 1 2 3 2 GGGTCTCACTCATGAACGAAAACTcACGTAAGGGA-~i. I iGGTCATGAGATATCA 3840 CCCAGACTGCGAGTCACCTTGCrrG AGTGCAATT'CCCTAAAACCAGTACTCTAATAGT GlySerAapAlaG~nTpAaGuaSerArg~ndG.yleLuVaI~etArgLauSer H XnIaX H MnfaI BMDNaX lOI~a A p bdpuh 0. t~dpuh lbpduh bdpui I h oem3o Q.oeii3o one3o oem3 u I 121AI 1 121A2 2112A2 121A 2 AAAGATCTCACCTAGATCCTTTCAGATCCCCGATCTTTA0CTGTCTTGGTTTGICC 3900 TTTCCTAGAAGTGGACTAGCAAAAGTCTAGAGGGCTAGAAATCGACAGAACCAAACGG LysAkrgtlaPheThrEnd!3.6LeuPheArgSrProAspLeuEn4LeuSerl.rpPhaAla H i H 0 M u h d na p a a la 3901 3960 G?&.TCGCGTAACGTATTAGAAGCCC)ATACGCACAAGGTATGTTGGAGGAATCATGT O3nSerAlaLeuHisAmnLeuSer01yLuCysVa.Va1PrTyrArnLeuLeuSer~hr- sUBSTITUTE SH11T WO089/01940 I N NsH &Hh 1 3962. 'GC.AACCA' I ACGTTGGT PCT/US88/02940 FI/G //l(con/'d) TTATCACCGCCAGAGTAAATAGTCAACACGACGGTAG2ATATTTATC 4020 kATAGTGGCGrGTCTCCATTTTATCAGTTGTGCGTGCC-ACJATCTATAAATAG CyuAn~is?yzisiAgGrgndASSrGlMiAlaArgCyuEnlI6Phole BM F pisa n u I IAp 1 4 u 1.2 212 1 H I C C? GGATAGATTTAAC GTATGAGCACA AAAGAAC CXTTAACACAAGACCAGC 4080 GGAAC GC CACTATCTA AZGCATACTC GTGT C=TTGGTAATTGTGTT C-TCGCTCG ProCyaGlyAmaprgPhesmValEndAlaG2nLy AgksaHi aEndHisLys SerSer bg AGWZG CA GTC GeC AGC)A? rTATCAAAAAAAAMAAATCGAAC TTCGC TT.AT 4140 AACTCCTGCGTCACGGAAZTCGTAATAC?~rTTrcTTTTACTGAACCAATA LauArg~rhrHisvaLA~aLauLysG2.nPhaMetLyuLyuArgLysxeatanLeuAklaTyr 3ES H $cc i F tor a 2.21 2.1 C CCAGG ATCTGTC GC.AGAC AGATGGG TGCGGCAGTCAGGC GTTGG TCCTA"I 4200 GGGTCTAGACAGCGTCTCTACCCCTACCCCGTCAGTCCGCACCACGAAATAAAT ProArgAmLauSerGlThrArgTrpGlyTrpGlySerG~nAklaLouValLauTyrLeu HI. 4 ATGGCATCATGCATTAATGCTTATAACCCGCATTGCTTACAAAAATCTCAAAGTTA 4260 TAC C TAGTTAC CTAATTTAC C ATATTCC GTAAC CAATG TTTTTAAGAGTT.TCAAT Met.A.aSerMetHisEndMetLau 3.eThr~roHisCysLeuGInLysPheSerLyeLeu m b 2 GC GTTGAAGAATTrTAGC C CTCAATC CCCAGAGAA.ATCTACrGAGATGTATGAAACC-GTTA 4320 CGCAAC" CTAAATCGGMGTTAGCGTCTCI.'=AGATGCTCTACATACT.CCCAAT AJlaLauLysAaLu2.LeLuGInSarProG1uLysSe.ThrArgCysMetLysAxgLau J~3~TU ~SHEET WO 89/01940 PCT/US88/02940 38/93 FIG //(con/'d n H D a R u a d b 4 *a a a H 3 11 1 3 CALTACTCGGCJGTGAATC=CATCATACTCGCA 1 'AA&&1XAGTACAA GTCCG-TC Va1CysSerAg~iaL.uGuV3SorMtS~reuh*UPh hSeu~tPorglu H DP± AD f pdon Id a ekd ut 1 113 1111 4381 4440 C CTAC AAGTGGATTCGAATCTTGA ATG TC CACTACDC CTCTC TAC CCATTCG f am 1 11 3 4441 4500 G~nProLysLysProVal1I.Lau~iuSerGlyLurgLauLyIVallla~roEnfldro- NN a duc a 4 3 1 2 1 CACAAGTCACAG""CTAC~ATTATCCTGCCGG V 4501 4560 HiaG~nGlnAlaProSarGlnAlaPheLauThrGluCysEfldPhaerLauThrLeuSer- tor pp 121 1 1 AGGTGTGACCAGGTGATTTCTGCATAGCAGACTTGG,'GGTG4ATGAGTT.TACCT.TCA 4620 rCcGCACTCGGTCCACTAAAGACTATCGGTCTGAACCCC-ACTACTCAAATrGGAA&T ArgLtuLauSarflVa16SerAadProAspLeuGyVaMatSerLeuProSer- a 4680 TCTf"TGATTAATCCCTATCGCCAGTCCACAA=kk GT GGTGATTTGGGTGTCAT GGT ArgsnEnLuGlylleAlaVaJ~CyheTyrAaHisfldThrHiSerThrG.la TESrt WO 89/01940 PCT/US88/02940 WOS9/01940 PCT/US88/02940 391/93 FIG /I(coru/'d) S mDa, N m H bdpu I a a oen3 a 0 12 LA 3 2. 3 TGATCCCATGCAATGAGAGTTGTTCCGT'TGTGGGCLAAAGTTATCGCTAGTCAGTGCCCTG 4740 ACTA=GTACGTTACTCTCAACAAGGCAACACC :Z"-.,C.ATAGCATAGTACCGGAC !ndSrHiAaMet.ArgValVa1ProL~uTrpGlyLysLeuS~rLeuVaI.Ser, 31yLau S larDA bdpu oon3 1.2 IA H i H a AAaAGACGTTTGGCTGATCGGCAAGGTGTCTGGTCGGCGCATAGCTGATAACAATTGAG 4800 TTCTCTCAAACCGACTAGCCGTTCCACAAGACCAGCCGCGTATCGACTATG?1'TAACTC Lys~AzgAuaAlspAgGlnGlyVaLuVaGy.1a~ndLou IieThrI leGlu H PS S is at nMP BM R KNax MzaU uge ba pe pbdpuh fv 4N1 4&t V1 ap hoonz3b 11 HI K11 11 21 11221A2 CAAAATCATCGGGGCTGCAGCCCACATGCGTCCGGCGTAGAGGATCTCTCACCTA 4860 GTCTTAGAMGTACCCCGAcCGGG=TGCTAcCAGGCCGCATCTCCTAGACUAGTGGAT GiaGluS.:S~rSeGlyLuGProThe.ArgProAaEndALzgIeSerHisLou C CAAACAATGCCC C CCTGCAAAAAkATAAATTCATATAAAAAACATACAGATAAC CATC TG 4920 G~rTAGGGAGTTTTTATT~-rvGAG'..A~.GAA ProAsnAsmAaProLeuGlnLyalleAsnSerTyrLyLysHisThrApAs~n~iuLeu H SH Hag pp 3. 1 1 121 4980 GC CTATTTAATA AGAC C C CACAACTGTATT'rATGGTGAC C CCAC TATGACTC GTG AgEnd~ndleZleSerGlyG2.yVa2ApIesaThrrGyyApThrG.uHis N R NH m H a 1 La gp pa a.*al 2 1 33 22 ATCAGCAOGGAC GCCTrGAC CAC CAT ,AGGTGAAC 'CTCT A AATTAAG CC C GAAGAAG 5040 TAGTCGT"CCTGCGTGACGGTGTACTTCCACTGCGAGIr.AATTCGGACT"T.rC -WSTVTUTS SHE~ET WO 89/01940 WO 8901940PCT/US88/02940 F IG //(cont'd) u ob b 3 12 1 5092 C CGTC GTAAGTC GTC?"C CGAAAC CCCACACACTATGCTTTCTTC GTAA GIySrZ..GlnSerArgrgLupGlyVlEdTyGluThrLyu~is??7 Enzymes that do cut: Accl Aha2 Af12 Alul ApaL. Aval Ava2 Beni Ban2 Bbel Bbvl BC11 3ql1 Bq12 Semi 3sp12 BSpt41 BstZ2 3stNl BstXl Cfrl V~al Odel Dpni Dra2 Zaq. ZooS Ecol Eceal ZcoR2 EcoRV FaUD2 Fau4H FOIkl FPp Ha*2 Hae3 aqal HgiAl HgiDl Khal Hinc2 Hind3 Hin~l Hin~l Hpa2 Hphl 'aol Ma*2 Mae3 Mbal Mbo2 Mnl1 Hopi Msat Kst2 Heel Narl Mcii Ndel Nde2 Hhol Nla3 Nla4 Nrul H;il Nap2 NspB2 NspHI PfU4L PpuH1 Pool Pstl Pvu2 Real Sacl Sall Sau3A Sau96 ScrFl SfaXI Sinl Sotl Stul Styl Taql Thai ?thl U0o2 Xm&aZ Enz~ymes that do not cut: Aat2 Aha3 Apal A~Ap70 Aap7l Asu2 Av?2 Ball Sam.Hl 3spm.2 BaaH2 Oral Dra 3 Eupl Hpal Kpfll Miul Mcol Hotl PaGR7 PVUJ. Rsr2 Sad2 Scal Stil SnaLI Sflaal Spel Sphl SUPI Sst2 Xbal X-.ol Xmal Xmnl Xor2 Z1L103STITUTE. SHEEFT 4 WO 890194PCT/US88/02940 WO 89/01940 FIG/12 o roL~ 2e00,000 97,400 6'd 0'o0 700 Id 400 lan-e Pre-InduLced lane 4 Jn1*nducect lane 5= 3/hr. posi flducht'on Ianl 1Overllh pos'- indaci-or C(-1/Ghr) Mojeculair tf. mrr~kers "3UBSTITUTE SHEET I WO- 89/01940 WO89/1940PCT/US88/02940 A co Sut TITUTZ SHEIFT WO. 89/0 1940 PT S8 24 PCT/US88/02940 L/3/93 FIG. 14A P70-2-15J7/heo Iso/ate c,3kblo ,oSG6377 p S7.38 -CO13 91p £frag/9en 1 p185-23 ifr11s/,/6q/f, Tsci/ateP L t 5 as 4 ljs SUBSTITUTE SHEET W'O 89/01940 W'O 8901940PCT/US88/02940 FIG. 14B obG6 3 77 Elo Z/ 5f 15o /a te 7.31Kbp fragrnenL PB6 36 8 -EcoRrl// 1Z #Ind=L H S WO 89/01940 WO 8901940PCTIUS88/ 02940 q.51/93 G./14 I U WO 89/01940 PCT/US88/ 02940 %/93 pBG39I :BG368 backbone zsoluble T4#3 :AA #3 LYS bg38l.seq Length: 6151 FIG 1 GAATTAATTC CAGCTTGCTG TGGAATGTGT GTCAGTTAGG GTGTGGAAAG 51 101 151 201 251 301 351 401 1 501 551 601 651 701 751 801 851 901 951 1001 1051 1101 1151 1201 1251 1301 1351 14U 1 TCCCCAGGCT GTCA GCA AC C TGCAAAGCAT CCGCCCATCC TGGCTGACTA CT GA GCT ATT GTATA GA AA C C GA AGGA GGC ACTCGCTCCA GGTGATTGGT GGCTATAAAA CT GT CTGC GA C GGT CT TTC C CT CC GCCAC C CT CTCGA GA A CGTGGCGGGC TGCTGATGAT GTGAGGTGTG TGACATCCAC TGGATCCAAG CC CACT GGGC GTTT GAGAA G TCTGTGGGCT GCCACAATGA ACTGGCGCTC AAAAAGGGGA ATACAATTCC GGGCTCCTTC CCCCAGCAGG AGGTGTGGAA GAT CTCA AT CGC CCC TAA C ATTTTTTTTA CCA GA AGT AG TC GGA CCA CT TAAGTGGGAG GGGTGTGAAG TTATAGGTGT GGGGGTGGGG GGGCCAGCTG A GT ACT CTTG GAGGGACCTG A GGC GTCT AA GGCAGCGGGT GTAATTAAAG GCAGGCTTGA TTTGCCTTTC CTT CGACT CG TCCTGGTTGC CA GCGGG CA A CA GGT C CCTA ACCGGGGAGT CTCCCAGCAG TA CAGTGGA A ACTGGAAAAA TTAACTAAAG :AGAAGTATG kGTCCCCAGG TAGTCAGCAA TCCGCCCAGT TTTATGCAGA TGAGGAGGCT CTGAGACGAA GGGTAGCGGT A CA CA TGT C A GGC CA CGTG GCGCGTTCGT TTGGGCTCGC OAT C CGAAA C AOC GA GT CCG CCAGTCACAG GGCGGTCGG T AGGC GOTC T GATCGATCTG T CT CCACA GO A GGA ATT CCC A GA GC TC CAA GA AAGA C CA C TGG C TCA 06 CC CTTTTA GO CCA CTCAGGG CTGACCTGTA CTCCAACCAG GTCCATCCAA :AAAGCATGC CTCCCCAGCA CCAT AGT C CC rCCGCCCATT GGC CGA GGC C TTTTTGGAGG GGCTCGCGTC CGTT GTC CA C CC CT CTTC GG AC CGGGT OTT C CTCA CTCT C GGTTGAGGAC C CGT CGGC CT C AT CGA C CGG TC GC AA GGT A GTTGTTTCTG TGAOACGGCG GCATA CA CT T GT CCA CTC C C GA AGGA ACA GTCCT C ACAC AGCCCAGAGG CCCCTGCCTC CA CTTC TT C AAAGAAAGTG CA GCTTC CCA ATAAAGATTC GCTGAATGAT ATCTCAATTA GOCAGAAGTA GCCCCTAACT CTC CGC C CCA GC CT CGGC CT GGTCCTCCTC CAGOCCAGCA TA GGGGGTC C CATCAAGGAA C CT GA A 006 TTCCGCATCG A AACT CTT CG CCOAACGGTA AT CGGAA AA C GGCTGAGCAC GCGGAGGTGC GATGGTCGAG TGAGTGACAA C AGGT CCAA C AAOC A CCCT C A GATA CGC CT C CCT GC C ATT C CT C GCAA G TOOT OCTGC A GTGCTGGGCA GAAGAAGAGC TGGGAAATCA C OC OC TGACT SHE ET WO 89/01940 PCT/US88/02940 q7/ 93 1451 CAAGAAGAAG 1501 CTTAAGATAG 1551 GGAGGAGGTG 1601 ACCTGCTTCA( 1651 AGTAGCCCCT 1701 GGGGAAGACC 1751 GGACATGCAC 1801 ATCGTGGTGC 1851 GGGGGAACAG 1901 TGACGGGCAG 195) AAGTCTTGGA 2001 GGTTACCCAG 2051 TCACCCTGCC 2101 CTGGCCCTTG 2151 GGTGATGAGA 2201 GACCCACCTC 2251 GCAAAGGTCT 2301 GGGGATGTGG 2351 CCAACATCAA 2401 CTGATTTGAG 2451 TGGACAAACT 2501 TTAAGTGTAT 2551 GATTCCAACC 2601 ATGAGGAAAA 2651, GCTACTGCTG 2701 AGAAGACCCC 2751 CTGTGTTTAG 2801 GAAAAAGCTG 2851 CTTTATAAGT 2901 CTCCACACAG 2951 TGTACCTTTA 3001' GTATAGTGCC FIG. /5(con/'d) CTTGTGGGAC CAAGGAAACT TTCCCCTGAT AAGACTCAGA 1 CAATTGCTAG I GGGGCAGAGC CAGTGCAATG- CTCTCCGTGT TGTCTTGCAG TAGCTTTCCA GTGGAGTTCT TGGCGAGCTG TCACCTTTGA GACCCTAAGC C CA GGC CTTG AAGCGAAAAC GC CA CT CAGC CCCTAAGCTG C GA AGC GGGA CAGTGTCTGC GGTTCTGCCC ATCTTTGTGA AC CTA CA GAG AATGTGTTAA TAT GGA A C G CCTGTTTTGC A CT CTCAA CA AAGGACTTTC TA ATA GA ACT CACTGCTAT" A GGC A TA ACA GCATAGAGTG GCTTTTTAAT TTGA CTA GAG rAcTTAcATC 1 ~GTTCGGATTC TGACCCTGA rAGGAGTCCA :TCAGCTGGA AACCAGAAGA AAGGC CTC C CTT C CCA CT TGGTGGCAGG C CT GAA GAA C TCCAGATGGG CCTCAGTATG A GGA A AGTT G TC C AGAAAAA ATGCTGAGTT GAAGGCGGTG TGAGTGACTC A CAT GGT CGA AGGAACCTTA ATTTAAAGCT A CTA CTGA TT ATGAATGGGA T CA GA AGA AA TTCTACTCCT CTTCAGAATT CTTGCTTGCT CA AGA AA ATT GTTATAATCA TCTGCTATTA TTGTAAAGGG ATCA TAAT CA rGTGAAGTGG ICTGC CAA C :CTTGGAGAG kGGGGTAAAA ICTCCAGGAT AGGTGGAGTT AG CA TAGT CT CGCCTTTACA CC.GAGAGGGC AAGGAAGTGT CAA GA AGCT C CTGGCTCTGG CAT CAGGAAG TTTGACCTGT T GA AA C TGGA TGGGTGCTGA GGGACAGGTC CCC CGGTGCA CTTCTGTGGT CTAAGGTAAA CTAATTGTTT GCAGTGGTGG TGC CAT CTAG C CAAA AA AGA GCTAAGTTTT TTGCTATTTA A TGGA AA AA T TAACATACTG A TAA CTA TGC GTTAATAAGG GCATA CCA C CATCAAGAAT AGGACCAGAA TCTGA CACC C CCC CC CTGGT ACATACAGGG A GTGGC A CCT CAAAATAGAC A TA AGA AA GA GTTGAAAAGC TTCCTCCTCC C TGT A AAACG CCGCTCCACC AAACCTCACC T GA AC C TGGT GAGGTGTGGG GAACAAGGAG A CC CTGACYGC CTGCTGGAAT GCCAATGGCC GTGACATAAT TAT AA A ATTT GTGTATTTTA AATGCCTTTA T GA TGA TGAG A GA GA AAGGT TT GA GTCA TG CACCACAAAG A TTCT GTAA C TTTTTTCTTA TCAAAAATTG AATATTTGAT ATTTGTAGAG 1 3051 GTTTTACTTG CTTTAAAAAA CCTCCCACAC CTCCCCCTGA ACCTGAAACA ~SUS T I T U t t 4,4 'T~ WO 89/01940 W089/194oPCT/US88/ 02940 18/ 93 3101 TAAAATGAAT FIG GCAATTGTTG /5 (cant 'd) TTGTTAACTT GTTTATTGCA 3151 3201 3251 3301 3351 3401 3451 3501 3551 3601 3651 3701 3751 3801 3851 3901 3951 4001 4051 4101 4151 4201 4251 4301 4351 4401 4451 'i501 4551 4601 4651 GTTACAAATA TCACTGCATT TGTCTGGATC CAGGTGCGGT C GGGC T CGC C GGCAGGCCCG ATTCCTTGCG TCCTAATGCA GCCTTCAACC CGCCGCACTT CGGCAGCGCT ACGATGATCG TCAAGCCTTC CCATTATCGC TTCr3CGACGC C GGC GGC A TC ATGA CGA CCA CTAACTTCGA GGC GA GCAC A TTGTCTGCCT ACCT GAAT GG ATTGGAGCCA TT GG CAGA AC GCATCTCGGG C GA GCAT CA C GACTATAAAG C C TGTT C CGA GGGAAGCGTC TGTAGGTCG7 CCCGACCGC7 AAGACACGAC AAGCAATAGC CTAGTTGTGG CT CTA CGC CG TGCTGGCGCC A CTT CGGGCT TGGCCGGGGG GCGGCGGTGC GGAGTCGCAT CA GTCA OCT C A TGA CTGT CT CTGGGTCATT GCCTGTCGCT GTCACTGGTC C GG CATGGC G GAGGCTGOAT GGGATGCCCG TCAGGOACAG TCACTOGACC T GG AA CGGGT CCC CGC GT TO AAGC CGGCG AT CAATT CTT AT AT C CATCG CCGCGTTGCT AA A AAT CGA C IATACCAGGCG CCCTGCCGCT iGCGCTTTCTC T C CT C C A A GCGCCTTATC TTAT COC CA C AT CA CAAATT TTTGTCCAAA OACGCATCGT TATATCGCCG C ATGA GCOCT A CTGTTGGGC TCAACGGCCT AAGGGAGAGC CTTCCGGTGG TCTTTAT CAT TTC OG CGA GG TGCGGTATTC CCGCCACCAA GCCOACGCOC GC CTTC CC C C OTT GCAGGC C TTC A AGGA T G CT GATCGT C TGGCATOGAT CGTCGCGGTG CACCTCGCTA GCGOAGAACT C GT C COCCAT GGCGTTTtTC OCTC A AGTC A TTTCCCCCTG TACCOGATAC AATGCTCACG ICTGGGCTGTG CGGTAACTAT TGGCAGCAGC TCACAAATAA CTCATCAATG GGCCGGCATC A CA TCAC CGA TGT T'TCOGC G G CC ATCTC CT CAACCTACTA GT CGA CCGA T GCOCGGGGCA GCAACTCGTA ACCGCTTTCG GOAATCTTGC A CGTTTC GOC TOOOCTACGT ATTATOATTC C ATOC TGTC C CGCTCOCGOC A CGGC GA TTT TGTAGGCGCC CATOGAGCCG A C GOATTCA C GTGAATGCGC CTCCAGCAGC CA TAGGC TC C GA GOTGG CGA GAA GCT C CCT CTGT C C GCCT CTGTAGGTAT TGCA CGA AC C CGTCTTGAGT CA CTGGTAA C OCTTATAATO AGCATTTTTT TAT CTTA TC A A C CGGC GCCA TGGGGAAGAT TO GGT A TGOT TO CA TGCAC C CT GGG CTG CT GC C CTTGA GA TGACTATCGT GGACAGGTGC CTGGAGCGCG A CGC CCT CGC GA GA AOC AGG CTTGCTGGCG TT CT COCTT C AGOCAGOTAG T CTTA CCA GC A TOC COCCT C 0CC CTA TA CC GGC CA CCT CG CA CT CCAA GA AAA CCAA CC C CGCACGCGGC GCCCCCCTGA AACCCOACAG CGTOCOCTCT TT CT C CCTT C CTCA OTT CGG C CCC OTT CAG C CA A CCCGGT A OGATT AOC A 4701 OAOCGAGGTA TGTAGOCGGT GCTACAGAGT TCTTGAAGTO GTGGCCTAAC u T.1 E'S r WO 89/01940 WO 8901940PCT/US88/02940 19/93 FI/G. 475,1 480'1 485 1 490.1 495-1 5001 5051 5101 15 F1 52.01 5251 5301 5351 5401 5451 5501 5551 5601 5651 5701 5751 5801 5851 5901 5951 6001 6051 6101 61511 TA CGGC TA CA A GTTA C CTT C CCGCTGGTAG AAAAAAGGAT TCAGTGGAAC AAAGGATCTT AT CTA A AGTA CA GTGA GG CA CCTGACTCCC GGCCCCAGTG TTTATCAGCA CTGCAACTTT A GA G TAAGTA TGCAGGCATC C CGGTTC CCA AAAGCGGTTA CGCAGTGTTA TCATGCCATC TCATTCTGAG AACACGGGAT TTGGAAAACG A GA TC CAGTT TTTTACTTTC CC GCAA AA AA TTCCTTTTTC C GGA TACA TA GCACATTTCC A TGA CATTAA C TA GA AGGA C GGAAAA AGAG CGGTGGTTTT CTCAA GA AGA GAAAACTCAC CACCTAGATC TA TAT GA TA C CTAT CT CAG CGTCGTGTAG C TGCA A TGAT ATAAACCAGC ATCCGCCTCC GTTCGCCAGT GT GGT GT CA C ACGATCAAGG G CT CCT TC GG TCACTCATGG CGTAAGATGC AATAGTGTAT AA TA C CGCGC TTCTTCGGGG C GA TGTAAC C ACCAGCGTTT GGGAATAAGG AATATTATTG TTT GAA TGT A C CGAA A AGTG C CTA TAA AA A AGTATTTGGT TTGGTAGCTC TTTGTTTGCA TCCTTTGATC GTTAAGGGAT CTTTTAAATT AACTTGGTCT/ C GA TC TGTC T A TA AC TA CGA A CCGC GAGA C C AG CCGGA.A G ATCCAGTCTA TAATAGTTTG GCTCGTCGTT C GA GTTA CA T TC CT C CGATO TTATGGCAGC TTTTCTGTGA GCGGCGACCG CA CA TA GCAG C GAA A ACT CT CACTCGTGCA CTGGGTGAGC GC GA CA CGGA AAGCATTTAT TTTAGAAAAA C CA C CTGA CG TAGGCGTATC ATCTG, GCTC TTGATCCGGC AG CAGC AGA T T TT TCTA CGG TTTGGTCATG AAAA A TGAAG 'GACAGTTACC A TTT CGTTC A TACGGGAGGG C CA CGCT CA C GGCCGAGCGC TTAATTGTTG CGCAACGTTG TGGTATGGCT GA TCC C CCAT G TT GTCAGA A ACTGCATAAT CTGGTGAGTA AGTTGCTCTT AA CTTTA AA A CAAGGATCTT CCCAACTGAT AAAAACAGGA AATGTTGAAT CAGGGTTATT TAAACAAATA T CT A AGAAA C A CGA GGCC CT TGC TGA AGC C AAACAAACCA TA CGC GCA GA GGTCTGACGC A GA TTA TCA A TTTTAAATCA AATGCTTAAT TCCATAGTTG CTTACCATCT CGGCTC CA GA A GA AGT GGT C CCGGGAAGCT TTGCCATTGC TCATTCAGCT GTTGTGCAAA GTAAGTTGGC T CT CTTA CTG CT CA A CCAAG GCCCGGCGTC G TGCT CA TCA A CCG CTGTTG CTTCAGCATC AGGCAAAATG A CTCA TA CTC GTCTCATGAG GGGGTTCCGC CATTATTATC TTCGTCTTCA ~U~3STUTE SHEET I. 7-u WO 8901940PCT/US88/ 02940 WO 89/01940 50/23 p3G392 :5G368 backbone :soluble T4#7 :AA #3 LYS 18 2AA+6AA :from 203-5 bg392.seq Length: 6149 FfG.16 1 GAATTAATTC CAGCTTGCTG TGGAATGTGT GTCAGTTAGG GTGTGGAAAG 51 101 151 201 251 301 351 401 451 501 551 601 651 701 751 801 851 901 951 1001 1051 1101 1151 1201 1251 1301 TCCCCAGGCT CCCCAGCAGG CAGAAGTATG GTCAGCAACC AGGTGTGGAA AGTCCCCAGG TGCAAAGCAT GCATCTCAAT TAGTCAGCAA CCGCCCATCC CGCCCCTAAC TCCGCCCAGT TGGCTGACTA ATTTTTTTTA TTTATGC.AGA CTGAGCTATT CCAGAAGTAG TGAGGAGGCT GTATAGAAAC TCGGACCACT CTGAGACGAA CGAAGGAGGC TAAGTGGGAG GGGTAGCGGT ACTCGCTCCA GGGTGTGAAG ACACATGTCG GGTGATTGGT TTATAGGTGT AGGCCACGTG GGCTATAAAA GGGGGTGGGG GCGCGTTCGT CtGTCTGCGA GGGCCAGCTG TTGGGCTCGC CGGTCTTTCC AGTACTCTTG GATCGGAAAC CTCCGCCACC GAGGGACCTG AGCGAGTCCG CTCTCGAGAA AGGCGTCTAA CCAGTCACAG CGTGGCGGGC GGCAGCGGGT GGCGGTCGGG TGCTGATGAT GTAATTAAAG TAGGCGGTCT GTGAGGTGTG GCAGGCTTGA GATCGATCTG TGACATCCAC TTTGCCTTTC TCTCCACAGG TGGATCCAAG CTTCGACTCG AGGAATTCCC CCCACTGGGC TCCTGGTTGC AGAGCTCCAA GTTTGAGAAG CAGCGGGCAA GAAAGACGCA TCTGTGGGCT CAGGTCCCTA CTGGCTCAGG MET GCCACAT-!-A ACCGGGGAGT CCCTTTTAGG ACTGGCGCTC CTCCCAGCAG CCACTCG AAAAAGGGGA TACAGTGGAA CTGACCTGTA CAAAGCATGC CTCCCCAGCA C CA TAGTCCC TCCGCCCATT GGCCGAGGCC TTTTTGGAGG GGCTCGCGTC CGTTGTCCAC CC CT CTT CGG ACCGGGTGTT C CTCA CTCTC GGTTGAGGA C C CGT CGGC CT C ATCGA CC GG TCGCAAGGTA GTTGTTTCTG TGAGACGGCG GC CA TACA CT TGTCCACTCC C GA AGGAA CA GTCCTCACAC AGCCCAGAGG CCCCTGCCTC CACTTGCTTC AAAGAAAGTG CA GCTT CC,CA ATCTCAATTA GGCAGAAGTA GCC CCTAA CT CTCCGCCCCA GC CT CGGC CT GGTCCTCCTC CAGGCCAGCA TA GGGGGTC C CAT CA AGGA A CCTGAAGGGG TTCCGCATCG AAACTCTTCG C CGAA C GTA AT C GAA AA C GGCTGAGCAC GCGGAGGTGC GA TGGT CGAG TGAGTGACAA CA GGT CCAA C AAGCACCCTC A GATA CGC CT CCCTGCCATT C CT CGGCA AG TGGTGCTGCA GTGCTOGGCA GAAGAAGAGC 4 1351 ATACAATTCC ACTGGAAAAA CTCCAACCAG ATAAAGATTC TGGGAAATCA or.**Iv'O S SHEET A WO89/01940 PCT/US88/02940 I WOz89/01940 PCT/US88/02940 51/93 1401 GGGCTCCTTC TTAACTAAAG GTCCA(CA GCTGAd) A CGCGCTGACT 1451 1501 1551 1601 1651 1701 1751 1801 1851 1901 195 1 2001 2051 2101 2151 2201 2251 2301 2351 2401 2451 2501 2551 2601 2651 2701 2751 2801 2851 2901 2951 3001 CAAGAAGAAG CTTAAGATAG GGAGGAGGTG ACCTGCTTCA A GTA GC C CCT GGGGAA GAC C GGACATGCAC ATCGTGGTGC GGGAACAGGT ACGGGCAGTG GTCTTGGATC TTACCCAGGA AC C C TCCCCC GGCCCTTGAA TGA TGAGAGC CC CA CCTCCC AAAGGTCTCG GGATGTGGCA AACATCAAGG GA TTTGAGAT GACAAACTAC AAGTGTATAA TTCCAACCTA GA GGA AAA CC TACTGCTGAC AAGACCCCAA GTGTTTAGTA AAAAGCTGCA TTATAAGTAG C CA CA CAGGC TACCTTTAGC A TA GTGC CTT CTTGTGGGAC AAGACTCAGA CAATTGCTAG GGGGCAGAGC CAGTGCAATG CT CTCCGTGT TGTCTTGCAG TAGCTTTCCA GGAGTTCTCC GCGAGCTGTG AC CTTTGAC C CC CTAA GCT C AGGCCTTGCC GC GAA A ACAG CA CTCA GCT C CTAAGCTGAT AAGCGGGAGA GTGTCTGCTG TT CTGC CCA C CTTTGTGAAG CTACAGAGAT TGTGTTAAAC TGGAACTGAT TGTTTTGCTC T CTCA ACA TT GGACTTTCCT A TA GAA CTCT CTGCTA TACA GCATAACAGT ATAGAGTGTC TTTTTAATTT CAAGGAAACT TTCCCCTGAT CATCAAGAAT TACTTACATC TGTGAAGTGG AGGACCAGAA TGTTCGGATT GACTGCCAAC TCTGACACCC CTGACCCTGA CCTTGGAGAG CCCCCCTGGT TAGGAGTCCA AGGGGTAAAA ACATACAGGG CTCAGCTGGA GCTCCAGGAT AGTGGCACCT AACCAGAAGA AGGTGGAGTT CAAAATAGAC STOP GAACCTCCAG CATAGTCTAf GAAAGAGG TTCCCAC.TCG CCTTTACAGT TGAAAAGCTG GTGGCAG(3CG GAGAGGGCTT CCTCCTCCAA TGAAGAACAA GGAAGTGTCT GTAAAACGGG CAGATGGGCA AGAAGCTCCC GCTCCACCTC TCAGTATGCT GGCTCTGGAA ACCTCACCCT GAAAGTTGCA TCAGGAAGTG AACCTGGTGG CAGAAAAATT TGACCTGTGA GGTGTGGGGA GCTGAGTTTG AAACTGGAGA ACAAGGAGGC AGGCGGTGTG GGTGCTGAAC CCTGAGGCGG AGTGACTCGG GACAGGTCCT GCTGGAATCC ATGGTCGACC CCGGTGCAGC CAATGGCCCT GAACCTTACT TCTGTGGTGT GACATAATTG TTAAAGCTCT AAGGTAAATA TAAAATTTTT TACTGATTCT AATTGTTTGT GTATTTTAGA GAATGGGAGC AGTGGTGGAA TGCCTTTAAI AGAAGAAATG CCATCTAGTG ATGATGAGGC CTACTCCTCC AAAAAAGAAG AGAAAGGTAG TCAGAATTGC TAAGTTTTTT GAGTCATGCT TGCTTGCTTTGCTATTTACA CCACAAAGGA AGAAAATTAT GGAAAAATAT TCTGTAACCT TATAATCATA ACATACTGTT TTTTCTTACT TGCTATTAAT AACTATGCTC AAAAATTGTG GTAAAGGGGT TAATAAGGAA TATTTGATGT GACTAGAGAT CATAATCAGC CATACCACAT TTGTAGAGGT W0!891/01940 WO'~89O194OPCT/US88/02940 52/93 3051 TTTACTTGCT 3101 AAATGAATGC 3151 TACAAATAAA 3201 ACTGCATTCT 3251 TCTGGATCCT 3301 GGTGCGGTTG 3351 GGCTCGCCAC 3401 CAGGCCCGTG 3451 TCCTTGCGGC 3501 CTAATGCAGG 3551 CTTCAACCCA 3601 CCOCACTTAT 3651 GCAGCGCTCT 3701 OATGATCGGC 3751 AAGCCTrTCGT 3801 ATTATCGCCG 3851 CGCGACGCGA 3901 GCGGCATCGG 3951 GACGACCATC 4001 AACTTCGATC 4051 CGAGCACATG 4101 GTCTGCCTCC 4151 CTGAATGGAA 4201 TGGAGCCAAT 4251 GGCAGAACAI 4301 ATCTCOGGCC 4351 AGCATCACAA 4401 CTATAAAGAI 4451 TGTTCCGACC 4501 GAAGCGTGGC 4551. TAGGTCOTTC 4601 CGACCOCTGC FIG'. /6(con/ d) TTAAAAAACC TCCCACACCT CCCCCTGAAC AATTGTTGTT GCAATAGCAT AGTTGTGOTT CTACGCCGGA C TGCGC CT A TTCGGGOC'TCA GCCGGGGGAC GOCGGTGCTO AGTCGCATAA GTCAGCTCCT GA CTOT CTT C GGGTCATTTT CTGTCOCTTG CA CTGGT C CC OCATGGCGOC GGCTGGATGG GA TOCC CGCG A GGGACA OCT ACTOGACCGC GAACGOGTTG CCGCGTTGCG GCCGGCGGCA *CAATTCTTGC *ATCCATCOCG GCGTTGCTOG AAATCGACGC -ACCAGGCGTT CTOC C GCTT A CTTTCTCAA :GCTCCAAGC7 :GCCTTATCCG GTTAA CTTGT CACAAATTTC TGTCCAAACT CGCATCGTGG T AT CGC CGA C TGAGCGCTTG TGTTGGGCGC AACGGCCTCA GGGAGAGCGT TCCGGTCGGC TTTATCATGC CGGCGAGGAC CGGTATT CG dC CA CCAAA C CGACGCGCTG C CTT CCC CA T TTGCAGOCA T CA AGGA TCO TGA 'kZGT C AC GCATGGATTG TCGCGGTGCA C CT CGCTAA C GGAGAACTGT TCCGCCATCT CGTTTTTCCA T CA AGTCA GA T CC C CCTGGA C COGATAC CT TGCTCACGCT GCTGTGTG iGTAACTATCC TTATTGCAGC A CAA ATA AAG CAT CA A TTA C C GOCAT CA C ATCACCGATG TTTCGGCGTO CATCT C CTTG AC CTA CTA CT CGACCGATGC GCGGGGCATG AACTCGTAGG COCTTTCGCT AAT CTTGCA C GTTT CGGCGA GGCTACGTCT TATGATTCTT TGCTOTCCAG CT CO COOCTC GGCOATTTAT TAGGCO CCGC TGGAGCCGGG GGA TTC A CCA GAATGCGCAA C CA GCAG0C CO TAGGCTCCGC GOTGC GA AA AGCTCC CT CO GTCCGCCTTT OTAGGTATCT i CACGAACCCC iTCTTGAOTCC CTGAAACATA TT AT A ATGGT CATTTTTTTC T CTTAT CA TO CG00CG0C CACA GGOAAGATCO GOTATOOTOG CATOCAC CAT GOCTGCTTC C CTT GA GA C A CTATCOT C G A CAGGTGC CO GOAOCGC GA C 0CC CTC OCT C GAAOCAOOCC TO CTG00C OTT CTCO CTT CCO GCAGOTA OAT TTACCAGCCT GC COC CT COO C CT ATA CC TT CC A CCTOGA C CTCCAAGAAT AC CA AC C CTT CA COCOG CG CCCCCTOACG CC CGA CA GA TGCGCTCTCC CT CC CTT C 0 CAOTTCGGTG CCGTTCAGCC AACCCGGTAA A 4651 GACACGACTT ATCGCCACTG OCAGCAGCCA CTOOTAACAG GATTAOCAGA SHE W089/01940 PCT/US88/02940 I WO' 89/01940 PCT/US88/02940 I I 1~.I I I I I .1 A 11 r I ,53/93 F/G. 16 (cont d) 4701 4751 4801 4851 4901 4951 5001 5051 5101 5151 5201 5251 5301 5351 5401 5451 5501 5551 5601 5651 5701 5751 5801 5851 5901 5951 6001 6051 6101 GCGAGGTATG C GG C TACA CT TTACCTTCGG GCTGGTAGCG AAAAGGATCT AGTGGAACGA AGGATCTTCA CTAAAGTATA GT GAGGC AC C T GA CTCCC CG CCC CA GTGCT TATCA GC AAT GCAACTTTAT A GT A AGTA GT CAGGCATCGT GGT TC C CAA C AGCGGTTAGC CAGTGTTATC AT GCCAT CCG A TT CTGA GA A CA CGGGATA A GGAAAACGTT ATCCAGTTCG TTA CTTT CA C GCAAAAAAGG CCTTTTTCAA GA TA CATA TT A CA TTT CC CC GA CATTAA CC TAGGCGGTGC A GA A GA CAG AAAA(AGTT GTGGTTTTTT CAA GA AGAT C AAACTCACGT C CTA GATC CT TATGAGTAAA TATCTCAGCG TCGTGTAGAT GCAATGATAC A AA CCA GCCA CCGCCTCCAT TCGCCAGT'TA GGTGTCA CGC GATCAAGGCG TCCTTCGGTC ACTCATGGTT TAAGATGCTT TAGTGTATGC TA CCGC GCCA CTTCGGGGCG A TGT AA C CCA CAGCGTTTCT GAATAAGGGC TATTATTGAA TGAATGTATT GAAAAGTGCC TATA AA A ATA TACAGAGTTC TATTTGGTAT GGTAGCTCTT TGTTTGCAAG CTTTGATCTT TAAGGGATTT TTTAAATTAA CTTGGT CTGA ATCTGTCTAT AACTACGATA CGCGAGA CCC GCC(QGAAGGG C CAGT CTA TT ATAGTTTGCG TCGTCGTTTG AGTTACATGA CTCCGATCGT ATGGCAGCA C TTCTGTGA CT GGC GACC GAG CATAGCAGAA A AA ACT C.CA CT CGTGCAC C GGGTGAGCAA GA C ACGGAA A GCA TTTA TC A TA GAA A AATA AC CTGA CGTC GGC GT ATC AC TTGAAGTGGT CTGCGCTCTG GATCCGGCAA CAGGAGATTA TTCT A C GOG TGGTCATGAG AAATGAAGTT CAGTTACCAA TTCGTTCATC CGGGAGGGCT ACGCTCACCG C CGA GCGCAG AATTGTTGCC CAACGTTGTT GTATGGCTTC T CCCC CATGT TGT CA GAA GT TGCATAATTC GGTGAGTACT TTGCTCTTGC CTTTAAAAGT A GGA TC TTA C CAACTGATCT AAACAGGAAG TGTTGAATAC GGGTTATTGT AACAAATAGG TAA GAA A CCA GAGGCCCTTT GGCCTAACTA CTGAAGCCAG ACAAACCACC CGCGC AGAA A TCTGACGCTC A TTAT CA AA A TTAAATCAAT TGCTTAATCA CATAGTTGCC TA CCAT CTGG GCTCCAGATT AAGTGGTCCT GGGAAGCTAG GCCATTGCTG A TTCA GCTC C TGTGCAAAAA AAGTTGGCCG TCTTACTGTC CAACCAAGTC CCGGCGTCAA GCTCATCATT CGCTGTTGAG TCAGCATCTT GCAAAATGCC TCATACTCTT CT CATGAGCG GGTTCCGCGC TTATTATCAT CGT CTT CA A SJBS TS"TUTE.-I j C!) 00 00 I WO 89/01940 WO'8901940PCT/US88/ 02940 615 j51/93 CTA OCT TTT CCA OTO A 3' FIG./18 OAT CTC ACT OCA XAG 3' 63 s 000 OTO ATA OTA A 3' S' GAT CTT ACT ATC A 3' 000 OCA GAG CCT OAC CCT 0AC CT? GGA GAG CCC C 3' CCO 000 0CC TCT CCA AOO TCA 000 TCA 0CC TCT 0 3' CCO OT AGT ACC CCC TCA OTO CAA TGA 3' OAT CTC AT? CCA CTO AGO 0CC TAC TAC 3' 7. OT ACT ACC CCC TCA OTO CAA TOT AGO AGT C 3' TAG CAC TCC TAC AT? OCA CTO ACC 0CC TAC TAC 3' CTA 000 OTA AAA ACA TAC AGO =0 OGA AGA CCT GA 3' OAT CTC AGO TCT TTC CCC CCC TOT ATO TT TTA CCC 3' OICA OCA TAO OAA CTO A 3' TOO CAC CTO GAC ATO CAC TOT CTT OCA OAT CTC AGT TOT OCA AGA CAG TOC ATO TCC AGO TO6C CAC TAT CCT GOA OCT 3' -j SUB-STITUTE SHEET I WO 89/01940 PCT/US88/02940 56193 pBG39 4 :66368 backDofl :snoluble T4#9 :AA #3 LYS :first 113 AA of T4 :basically up to ViJ1 bg394.seq Length: 5365 1 GAATTAATTC CAGCTTGCTG FIG. 19 51 101 151 201 251 301 351 1 451 501 551 1 1 751 801 851 901 951 1001 1051 1101 1151 1201. 1251 1301 TCCCCAGGCT GTCAGCAACC TGCAAAGCAT CCGCCCATCC T GG CTGA CT A CTGAGCTATT GT AT AGAA AC C GA AGGA GGC ACTCGCTCCA GGTGATTGGT GGCTATAAAA CTGTCTGCGA CGGTCTTTCC C TC CGCCA CC CTCTCGAGAA C GT GG CGGG C TGCTGATGAT GTGAGGTGTG T GALCAT CCA C TGGATCCAAG C CCA C TGGGC GTTTGAGAAG TCTGTGGGCT GC CA CAAT GA AC TGGCG CT C AAAAAGGGGA CCC CA GCA 66 A GGT GTG GA A GCATCTCAAT CGCCCCTAAC ATTTTTTTTA CC AGA AGTA G T CGGA CC ACT TAAGTGGGAG GGGTGTGAAG TTATAGGTGT GGGGGTGGGG GGGCCAGCTG AGTACTCTTG GAGGGACCTG AGGCGTCTAA GGCAGCGGGT GTAA TT AAAG GCAGGCTTGA TTTGCCTTTC CTTCGACTCG TCCTGGTTGC CAGCGGGCAA CA GGT C CCTA ACCGGGGAGT CTCCCAGCAG TACAGTGGAA TGGAATGTGT CAGAAGTATG A GT CCCC AGG TA GTCA GCA A TCCGCCCAGT TTTATGCAGA TGAGGAGGCT CT GAGA CGA A GGGTAGCGGT A CACA TGT CG A GG CCA CGTG GCGCGTTCGT TTGGGCTCGC GATCGGAAAC AGCGAGTCCG C CA GTCAC AG GGCGGTCGGG TAGGCGGTCT GATCGATCTG TCTCCACAGG A GGAA TT CC C A GA GCT CCAA GAA AGA CGC A CTGG C TCA GG CCCTTTTAGG C CA CTCA GGG CTGACCTGTA GTCAGTTAGG CAAAGCATGC CTCCCCAGCA C CATA GT C CC TCCGCCCATT GGC CGA GGC C TTTTTGGAGG GGCTCGCGTC C GTT GT CCA C CCCTCTTCGG AC CGGGTGTT C CT CA CTCT C GGTTGAGGAC C CGT CGG CCT C ATC GA C CGG TCGCAAGGTA GTTGTTTCTG TGAGACGGCG GC CATA CA CT T GT CCACT CC C GAA GGA ACA GT CCT CA CA C A GC CC AGA GG CCC CTGC CT C CACTTGCTTC AAAGAAAGTG C AG CTT CCCA A TAA AGATT C GTGTGGAAAG ATCTCAATTA 6 CA GA AGTA GC CC CTA ACT CTCCGCCCCA GCCTCGGCCT GGTCCTCCTC CAGGCCAGCA TAG66GGGTC C CATCAAGGAA C CTGA AGGGG TTCCGCATCG AAACTCTTCG C CGA AC GGTA ATCGGAAAAC GGCTGAGCAC GCGGAGGTGC GA TGGT CGA G TGAGTGACAA CA GGT CCAA C AAGCACCCTC A GA TA CGC CT CC CTGC CATT C CT CGGCA AG TGGTGCTGCA GTGCTGGGCA GA AGA AGAC T GGGA AATC A 1351 PAAAATTCC ACTGGAAAAA CTCCAACCAG I ;UBSTITUTE SHEET WO,89/01940 WO. 9/0 940PCT/US88/02940 57193 FIG. 19 (cont 'd) 1401 GGGCTCCTTC 1451 CAAGAAGAAG L501 CTTAAGATAG 1551 GGAGGAGGTG 1601 ACCTGCTTCA 1651 TGGTGTGACA 1701 TAAATATAAA 1751 GTTTGTGTAT 1801 GTGGAATGCC 1851 CTAGTGATGA 1901 AAGAAGAGAA 1951 TTTTTTGAGT 2001 TTTACACCAC 2051 AAATATTCTG 21U1 ACTGTTTTTT 2151 ATGCTCAAAA 2201 AAGGAATATT 2251 CCACATTTGT 23011 CTGAACCTGA 2351 TGCAGCTTAT 2401 ATAAAGCATT 2451 AA7GTATCTT 2501 CATCACCGGC 2551 CCGATGGGGA 2601 GGCGTGGGTA 2651 TCCTTGCATC 2701 ACTACTGGG( 2751 CGATGCCCT- 2801 GGCATGACT 2851 CGTAGGACA( 2901 TTCGCTGGAC 2951 TTGCACGCC( TTAACTAAAG CTTGTGGGAC AA GA CT CAGA CAATTGCTAG GGGGTGATAG TA ATT GGA CA ATTTTTAAGT T TT AGA TTC C TTTAATGAGG TGAGGCTACT A GGT AGAA GA CATGCTGTGT AAAGGAAAAA TAACCTTTAT C TT ACTC CA C A TT GTGT AC C TGATGTATAG A GA GG1TTTA AACATAAAAT A A TGGTTA CA TTTTTCACTG *ATCATGTCTG *GCCACAGGTG AGATCGGGC7 TGGTGGCAGG CAC CA TT C C -TGCTTCCTAA r GAGAGCCTTC k TCGTCGCCGC 1GTGCCGGCAC 1C GCGA CGA TC TCGCTCAAG( GTCCATCCAA GCTGAATGAT CGCGCTGACT CAAGGAAACT TACTTACATC TGTTCGGATT TA AGAT CTTT AA CTA CCTA C GTATAATGTG AAC CTA T5GA AAAACCTGTT GCTGACTCTC CCCCAAGGAC TTAGTAATAG GCTGCACTGC AAGTAGGCAT ACAGGCATAG TTTAGCTTTT TGCCTTGACT C TT GC TTTA A GAATGCAATT AA TA AA GCA A CA TT CTAGTT GATCCTCTAC CGGTTGCTGG C GCCA CTT CG ICCCGTGG CCG TGCGGCGGCG TGCAGGAGTC AAC CC AGTC A A CTTA TGA CI 1 CGCTCTGGGI 1 ATCGGCCTG1 'C TT CGTCA CI TTCCCCTGAT TGTGAAGTGG GA CTGC CAA C GT GA AGGA AC A GAGA TTTA A TTAAA CTA CT A CTGA TGA AT TTGCT CAGA A AACATTCTAC TTTCCTTCAG AACTCTTGCT TA TACA AGAA AACAGTTATA AGTGTCTGCT TAATTTGTAA A GA GAT CA TA AAAA CCTCC C GTTGTTGTTA TA GCA TC ACA GTGGTTTGTC GCCGGACGCA CGCCTATATC GGCTCATGAG GGGGACTGTT *GTGCTCAACG GCATAAGGGA GCTCCTTCCG *GTCTTCTTTA *CATTTTCGGC *CGCTTGCGG1 *GGTCCCGCCA CAT CAA GAAT A GGA CCA GAA T CTGA CACC C C TT A CTTCTG AGCTCTAAGG GATTCTAATT GGGAGCAGTG GAAATGC CAT TCCTCCAAAA AATTGCTAAG TGCTTTGCTA AATTATGGAA AT CATAA CAT A TT AA TAA CT A GGGGTT AAT AT CAGC CATA ACACCTCCCC ACTTGTTTAT A ATTT CA CA A CAAACTCATC TCGTGGCCGG GCCGACATCA CGCTTGTTTC GGGC GC CAT C GCCTCAACCT GAGC GT CGA C IGTGGGCGCGG T CA TGCA ACT GAGGACCGCT *ATTCGGAATC CCAAACGTTT Luc: :-GGCGAGAAG' CAGGCCATTA TCGCCGGCAT GGCGGCCGAC GCGCTGGGCT WO 89/01940 WO 8901940PCT/US88/02940 58/93 FIG. /9 (contld) 3051 ACGTCTTGCT GGCGTTCGCG ACGCGAGGCT GGATGGCCTT I H I' I I I I I I H 14 I H H [j [1 ['I 1~ 3101 ATTCTTCTCG C 3151 GTCCAGGCAG G 3201 CGGCTCTTAC C 3251 ATTTATGCCG C 3301 CGCCGCCCTA 1 3351 GCCGGGCCAC 3401 TCACCACTCC 3 451 GCGCAAACCA 3501 CAGCCGCACG 3551 CTCCGCCCCC 3601 GCGAAACCCG 3651 CCCTCGTGCG 3701 GCCTTTCTCC 3751 GTATCTCAGT 3801 AACCCCCCGT 3851 GAGTCCAACC 3901 TAACAGGATT 3951 AGTGU'TGGCC 40C01 GCTCTGCTGA 4051 CGGCAAACAA 4101 AGATTACGCG 4151 ACGGGGTCTG 4201 CATGAGATTA 4251 GAAGTTTTAA 4301 TACCAATGCT 4351 TTCATCCATA 4401 AGGGCTTACC 4451 TCACCGGCTC 4501 GCGCAGAAGT 4551 GTTGCCGGGA 4601 GTTGTTGCCA ;TTCCGGCGG C ~TAGATGACG A :AGCCTAACT T CTCGGCGAG C ACCTTGTCT C :TCGACCTGA A kAGAATTGGA C ACCCTTGGCAC CGGCGCATCT CTGACGAGCA ACAGGACTAT CT CT CCT GTT CTTCGGGAAG TCGGTGTAGG TCA GCC CGA C C GGT AAGA CA AGCAGAGCGA TAACTACGGC A GC C AGTTA C ACCACCGCTG CA GAAAAAA A ACGCTCAGTG TCAAAAAGGA AT CAATC TA A TAATCAGTCOA GTTGC C TGA C ATC TGGC C CC CA GA TTTAT C GGTCCTGCAA AG CTA GAGT A TTG.CTGCAGG ATCGGGATG CCATCAGGG CGATCACTG :ACATGGAAC ~CCTCCCCGC TGGAAGCCG CCAATCAAT IAACATATCC CGGGCCGCGT TCACA AAA AT A AAGA TA CCA ZCCGAC C CTGC CGTGGCGCTT TCGTTCGCTC CGCTGCGCCT CGACTTATCG GGTATGTAGG TACACTAGAA C TT CGGA AAA GTAGCC 3 GTGG GGA TCT CA A GAACGAAAAC T CTT CA CCTA A GTATA TAT C GGCACCTAT( T CC C CGT CG AGTGCTGCA AGCAATAAA( C TTT A TCCG( AGTAGTTCGI C AT CGTGGTI CCCGCGTTGC ACAGCTTCAA GACCGCTGAT GGGTTGGCAT GTTGCGTCGC GC GG CA CCT C TCTTGCGGAG ATCGCGTCCG TGCTGGCGTT CGACGCTCAA GGCGTTTCCC CGC TTA C CGG TCTCAATGCT CAAGCTGGGC TATCCGGTAA CCACTGGCAG C GGT GC TA CA GGACAGTATT A GA GTT GGT A ITTTTTTTGTT IAAGATCCTT7 TCACGTTAAG- GATCCTTTTA iAGTAAACTTC TCAGCGATC] r GTAGATAAC1 TGATACCGC( CAGCCAGCC( CTCCATCCA( CA GT TA ATA( 3 TCACGCTCG' CCCCATTATG A GG C CATGCT GGATCGCTCG C GTCA CGGCG GGATTGTAGG GGTGCATGGA GCTAACGGAT AACTGTGAAT CCATCTCCAG TTTCCATAGG GTCAGAGGTG CCTGGAAGCT A TA CCTGTCC CACGCTGTAG TGTGTGCACG CTATCGTCTT CAGCCACTGG GAGTTCTTGA T GGTATCTGC GCT OTT GA TC TGCAAGCAGC *GATCTTTTCT iGGATTTTGGT AATTAAAAAT iGTCTGACAGT -GTCTATTTCG ACGATACGGG 1 AGACCCACGC 1 GAAGGGCCGA 1 TCTATTAATT 1 TTTGCGCAAC T CGTTTGGTAT 4651 GGCTTCATTC AGCTCCGGTT CCCAACGATC AAGGCGAGTT ACATGATCCC 'SUBSTITUTE SHSET t WO 89/01940 W089/1940PCT/US88/02940 521935 FIG. 19 (cont'd) 4701 4751 4801 4851 4901 4951 5001 5051 5101 5151 5201 5251 5301 5351 CCATGTTGTG CAAAAA AGAAGTAAGT TGGCCC TAATTCTCTT ACTGTC AGTACTCAAC CAAGTC TCTTGCCCGG CGTCAA AAAAGTGCTC ATCATI TCTTACCGCT GTTGAC TGATCTTCAG CATCT AGGAAGGCAA AATGC( GAATAC*TCAT ACTCT TATTGTCTCA TGAGC( AATAGGGGTT CCGCGI AAACCATTAT TATCA CCCTTTCGTC TTCAA ~AGCG ~CAGT A T GC ;ATTC ~CACG GGAA IATCC rTTAC :GCAA TCCTT GGATA CACAT GTT AG CT CCT GTTATCACTC CATCCGTAAG TGAGAATAGT GGA TA ATA CC AACGTTCTTC AGTTCGATGT TTT CA CCA GC AAAAGGGAAT TTTCAATATT CATATTTGAA TTCCCCGAAA TCGGTCCTCC ATGGTTATGG ATGCTTTTCT GTATGCGGCG GC GCCACA TA GGGGCGAAAA AACCCACTCG GTTTCTGGGT AAGGGCGACA A TTGA AGCA T TGTATTTAGA A GTGC CA CCT GATCGTTGTC C AGC A CTGCA GTGACTGGTG A C CGA GTTGC GC AGA A CTTT CTCTCAAGGA T GCA C CCAA C GAGCAAAAAC C GGAA A TGTT TTATCAGGGT AAA ATAA AC A GA CGT CT AA G TGACA TTAACCTATA AAAATAGGCG TATCACGAGG SUBSTITUTEM SHa'Z7T i,~i 11 I 4 I 1. 14 I. Ps WO 89/01940 W089/1940PCT/US88/02940 GO/93 FIG. PB6~396 :8G368 backbone :soluble T4#12 :AA #3 LYS bg396.seq Length: 5518 1 GAATTAATTC CAGCTTGCTG TGGAATrGTGT GTCAGTTAGG GTGTGGAAAG 51 101 151 201 251 301. 351 40 1 451 501 551 601 1 701 751 801 851 901 951 1001 1051 1101 1151 1201 125-1 1301 1351 TCCCCAGGCT GTCA GCA AC C TGCAAAGCAT CCGCCCATCC TGGCTGACTA CT GAG CTAT T GTA TAGA AA C C GA A GGAGGC A CTCGCTCC A GGTGATTGGT GG CTATA AA A C TGT CTG CGA CGGTCTTTCC CTCCGCCACC CTCT CGA GA A CGTGGCGGGC TGCTGATGAT GTGAGGTGTG TGACATCCAC TGGATCCAAG CCCACTGGGC GTTT GAGA AG TCTGTGGGCT GCCACAATGA ACTGGCGCTC AAAAAGGGGA ATACAATTCC CCCCAGCAGG AGGTGTGGAA GCATCTCAAT CGCCCCTAAC ATTTTTTTTA CC AGA AGT AG T CGGA CCA CT TAAGTGGGAG GGGTGTGAAG TTATAGGTGT GGGGGT GGGG GGGCCAGCTG AGTACTCTTG GAGGGACCTG A GG CGT CTA A GGCAGCGGGT GTAATTAAAG GCAGGCTTGA TTTGCCTTTC CTTCGACTCG TCCTGGTTGC CAGCGGGCAA CAGGTCCCTA ACCGGGGAGT CTCCCAGCAG TA C AGTGGAA ACTGGAAAAA CAGAAGTATG A GT C CCCAGG TA GTCA GCA A TCCGCCCAGT TTT AT GCA GA TGAGGAGGCT CTGAGACGAA GGGTAGCGGT A CA CATGT CG A GGC CA CGTG GCGCGTTCGT TTGGGCTCGC GATCGGAAAC AGCGAGTCCG C C AGT CACAG GGCGGTCGGG TAGGCGGTCT GATCGATCTG T CT C CACA GG A GGA ATT CC C A GA GCTC CAA GAAAGACGCA CT GGCT CA GG CCCTTTTAGG C CA CTCA GGG CTGACCTGTA CTCCAACCAG CAAAGCATGC CTCCCCAGCA C CATA GTC CC TC CGCC C ATT GGC CGA GGC C TTTTTGGAGG GGCTCGCGTC C GTT GTC CA C C CCT CTTC GG A C CGG GTGTT C CT CA CTCTC GGTTGAGGA C C CGTC GGC CT CAT CGA C CGG TCGCAAGGTA GTTGTTTCTG T GAGA CGGCG GCCATACACT T GT CCA CTC C C GA AGGA ACA GTCCTCACAC A GC CCAGA GG CCC CTGC CTC CACTTGCTTC AAAGA A AGTG CAOC TT C CCA A TA A AGATT C AT CT CA ATT A GGCAGAAGTA GCCCCTAACT CTC CGC C CCA GCCTCGGCCT GGTCCTCCTC CAGGCCAGCA TAGGGGGTCC C AT CA AGGAA CCTGAAGGGG TTCCGCATCG AAACTCTTCG C CGA A CGGTA AT CGGAAAA C GGCTGAGCAC GCGGAGGTGC GATGGTCGAG TGA GTGA CA A CAGTC CAA C AA GCA C CCTC A GA TA CCCT CCCTGCCATT C CT CGGCA AG TGGTGCTGCA GTGCTGGGCA GAAGAAGAGC TGGGAAATCA 1401 GGG'CTCCTTC TTAACTAAAG GTCCATCCAA GCTGAATGAT CGCGCTGACT SUBS"IITUTE SHEET W6189/01940 WO 8901940PCT/US88/02940 61/93 1451 CAAGAAGAAG CTTGTGGGAC CAi FIG. 20 (cont'd) %GGAAACT TTCCCCTGAT CATCAAGiAAT 1501 1551 1601 1651 1701 175 1 1801 1851 1901, 195,1 200 1 205 1 2101 2151 2201 2251 2301 2351 2401 2451 2501 2551 2601 2651 2701 2751 2801 2851 2901 295 1 3001 C TTA AGA TAG GGAGGAGGTG ACCTGCTTCA A GT AOCCC CT GGG0A AGAC C GGACATGCAC C TOT GT GTG AGGTA A ATAT ATTGTTTGTG GTGOTGGAAT CATCTAGTGA AAAAAGAAGA AAGTTTTTTG C TAT TT ACAC GAAAAATATT CATACTGTTT A CT ATOCT CA AATAAGGAAT ATACCACATT C CC C TGAAC C TAT TGCA OCT CAAATAAAGC A TC A ATOTA T C GGC A TCA CC TCACCGATGG TTCGGCGTGG ATCTCCTTGC CCTACTACTGi GA C CGATGC C C GOGC A TGA ACTCGTAGGA kAGACTCAGA 1 CAATTGCTAG 1 GGGGCAGAGC CAGTGCAATG 7 CTCTCCGTGT TGTCTTGCAG ACATAATTGG AAAATTTTTA TATTTTAGAT GCCTTTAATG TGATGAGGCT GAAA GGTA GA AGTCATGCTG CA C A A AGA A C TOT AA CCTT TTTCTTACTC AAAATTGTGT ATTTGATGTA TGT AGAGGTT T GA AACA TAA TATAATOOTT ATTTTTTTCA C TTAT CATGT GGCGCCACAG GGAAGATCGG GTATGGTGGC A TGC ACC ATT GGCTGCTTCC CTTGAGAGCC CTATCGTCGC CAGGTGCCGG ACTTACATC GTTCOGATT TGA C CC TA FAG0GAOT CC A -TCAGCTGGA kACTGAGATC ACAAACTACC AGTGTATAAT TCCAAC CTAT A OGA AAA CCT AC TGC TGAC T A GA CCC CA AG TGTTTAGTAA AAAGCTGCAC TA TA AGT AGG CA CACA GGCA AC CTTTAGCT TAGTGCCTTG TTACTTGCTT AATOAATGCA ACAAATAAAG CTGCATTCTA CTGOATCCTC GTGCGGTTGC OCT CGCCA CT A GGC C C T G CCTTGCGGCG TAATGCAGGA TT CA ACCC AG COCA CTTATG CA OCGCTC TO TGTGAAGTGG GACTGCCAAC CCTTGOAGAG4 AGGGGTAAAA GCTCCAGGAT TTTGTGAAGG TACAGAGATT GT GTT AA ACT GGAACTGATG GTT TT G CT CA CTCAACATTC GA CTTT C CTT TA GA ACTC TT T G CTATA CA A CATAACAGTT T AGA GTOT CT TTTTAATTTG A CTA GAGA TC TAAAAAACCT AT TGTT OTTO CA ATAGC AT C GTTGTOGTTT TA COCC GA C TG00CG0C CTA T TCGGGCTCAT C CGOOGA CT GCGGTG CTCA GTCGCATAAG TCAOCTCCTT A CTGT CTT CT GOT CATTTTC AGGACCAGAA T C TOACA CCC CCCCCCTOOT A CA TACA 000 A GT GOC A CCT AACCTTACTT TAAAGCTCTA A C TOATTC TA AATGGGAOCA GAAGAAATGC TA CT CCT CCA CA GA ATTGCT GCTTGCTTTG GAAAATTATG ATAATCATAA GCTATTAATA TAAAOGGGTT A TA A TCAGC C COCA CA CCTC TTAACTTOTT ACAAATTTCA GTCCAAACTC OCAT C OT GC AT CGCC GACA OAOCGCTTGT GTTGOGCGCC ACGGCCTCAA OGA GA0C GT C CCOOTOOOCG TTATCATOCA GGCGAGGACC 3051 GCTTTCGCT G GAGCGCOACG ATGATCGGCC TGTCGCTTGC GGTATTCOGA I- j U W0 89/01940 PCT/US88/02940 3101 3151 3201 3251 3301 3351 3401 3451~ 3501 3551 3 b 01 3651 3701 3751 3801 3851 3901 395 1 4001 4051 4101 4151 4201 4251 4301 4351 4401 4451 4501 4551 460 1 4651 62/,93 ATCTTGCACG TTTCGGCGAG GCTACGTCTT A TGATT CTT C GCTGTCCAGG TCGCGGCTCT GCGATTTATG A GGC C C C C GGAGCCGGGC GA TTCA CCA C AATGCGCAAA CAGCAGCCGC A OGC TC CGCC GTGGCGAAAC GCTC CCT CGT TCCGCCTTTC T AGGT A TCT C ACGAACCCCC CTTGA GT CCA T GG TA ACA GG TGAAGTGGTG TGCGCTCTGC ATC CGGC AA A A GC AGA TTA C TCTACGGGGT GGTCATGAGA AATGAAGTTI A OTT A C C A T CGTT CA TC C GGGAGGGCTI C OC TCA C CGC C GAGC GC AGA (cont'd) AGCCTTCGTC ACTGGTCCCG CCACCAAACG CC CT CGCT CA AAGC AGGC CA GCTGGCGTTC TCGCTTCCOG C A GOTAGA TG TA CCA GC CTA CC GGCTC GGC CTATACCTTG C A CCT CGAC C TCCAAGAATT C CAAC C CTTG ACGCGGCGCA C CCCT GA CGA C CGA CA GGA C OC GC TCT CCT TCCCTTCGGG A OTT CGGTGT C GTT CAG CC C ACCCGGTAAG ATTAGCAGAG GCCTAACTAC T GA AGC CA T CAAACCACCG GCGCA GA AA A CT GA CGCT CA *TTATCAAAAA *TAAATCAATC *GCTTAATCAG ATAGTTGCCT *ACCATCTGGC iCTCCAGATTI AGTGGTCCTC TTATCGCCGG GO GA CGC GAG C GGCA TC GGG A CGAC CAT CA A CTT CGAT CA GA GC ACAT GG T CTGC CT CC C TGAATGGAAG GGAGCCAA""C GCAGAACATA TCTCGGGCCG GC AT CACA AA TATA A AGA TA GTTCCGACCC A AG CGT GGCG AGT CGTT CG GACCGCTGCG A CA CGA CTTA C GAGGTA TOT GGCTACACTA TA C CTTC GGA CTGGTAG CGG AAAGGATCTC GTGOAACGAA GGATCTTCAC TAA AGT ATAT ITGAGGCACCT *GACTCCCCGT C C CAGTGCTG *ATCAGCAATA CAACTTTATC CATGGCGGCC GC TGGATOG C A TGC C CGC G GGGACAGCTT CTOGA C CGCT AACGGOTTGG CGCGTTGCGT CC GG C GCA C A ATT CTTGCG TC CAT CO CGT C GTT GCT G GC A ATC GA CGCT CCAGGCOTTT TGCCGCTTAC C TTT CT CAAT CTCCAAGCTG C CTTA TC CGG T CGC CA CT GO AGOCOOTOCT GAAGGACAOT AAAAGAGTTG TGOTTTTTTT A AGA AGATCC A ACT CAC OTT CTAGATCCTT A TGA GT AAA C AT CT CAGC GA C GTOT AGAT A iCAATOATACC AACCAGCCAC CGCCTCCATC GA CGCG 0TG C TT CCCC ATT TOC AGGC CAT CAAGGATCGC GATCGTCACG CATGGATTGT C GCGGT GCAT CTCGCTAACG GA GA A C TOT CCGCCATCTC OTT TT TCCAT C AAGT C AGAG CCCCCTOGAA C GOATA CC TO OCTCA CGC TO GGCTOTOTOC TAACTATCGT C AGC AOC CA C A CA GAOTT CT A TTTGGT AT C GTAGCTCTTO OTTTGCAAGC TTTGATCTTT AAGGGATTTT TTAAATTAAA TT GOTCTGA C TCTOTCTATT A CT A CGATAC OC GAGA C CCA i CCGGAAGGGC :CAGTCTATTA 47 ATTGr7GCCG GGAAO CTAOA GTAAGTAOTT CGCCAOTTAA TAGTTTOCOC SURSINTUTE SHEET [1 ii d it 4 WO, 89/01940 PTU8/24 PCT/US88/02940 (.3/93 FIG. 20 (cont'd") 4751 4801 4851 4901 4951 5001 5051 5101' 5151 5201 5251 5301 535 1 5401 5451 5501 AACGTTGTTG TATGGCTTCA CC CC CATGTT GT CA GA AGTA GCATAATTCT GTGAGTACTC TGCTCTTGCC TTTAAAAGTG GGATCTTACC AACTGATCT7 AACAGGAAGG G TT GA ATA CT GGTTATTGTC A CAA AT AGGG A AGA A ACCA T AGGCCCTTTC CCATTGCTGC TTCAGCTCCG GTGCAAAAAA AGTTGGCCGC CTTACTGTCA AAC CA AGTCA CGGCGTCAAC CT CAT CA TTG GCTGTTGAGA CA GCAT CTTT CAAAATGCCG CAT ACT CT TC TCATGAGCGG GTTCCGCGCA A GG CA TC GTG GTTCCCAACG GCGGTTAGCT A GT GTTA TC A TGCCATCCGT TT CTGA GAAT ACGGGATAAT GAAAACGTTC TCC AGTT CGA TA CTTTCAC C CAAAAAAGGG C TT TTT CAA T A TACA TATTT CATTTCCCCG GTGTCACGCT ATCAAGGCGA C CTT CGGTC C CTCATGGTTA AAGATGCTTT AGTGTATGCG ACCGCGCCAC TTCGGGGCGA T GT AACC CA C AGCGTTTCTG AATAAGGGCG ATTATTGAAG GAATGTATTT AAAAGTGCCA CGTCGTTTGG GTTACATGAT TCCGATCGTT T GGC AGC ACT T CT GTGA CTG GC GA CCGA GT ATAGGA GAA C AAACTCTCAA T CGTG CA CCC GGT GA GCAA A A CA CGGAAAT C ATTTAT CA G A GA AAAA TAA C CTGA CGTCT TATTATCATG ACATTAACCT ATAAAAATAG GCGTATCACG GT CT TCA A ~SjTTUTE ~IZZ1 M"T WO,89/01940 WO. 9/0 940PCT/US88/02940 6V 9Y3 PBG.393 rBG368 backbone :soluble T4#8 :AA #3 LYS z<per-fect" Stu/first 182 AA of :basically up to V2J2 bg393.seq Length: 5566 FIG.21 1 GAATTAATTC CAGCTTGCTG 51 101 151 201 251 301 351 401 451 501 551 601 651 701 751 801 851 901 951 1001 1051 1101 1151 1201 1251 1301 1351 TCCCCAGGCT GTCAGCAACC TGCAAAGCAT CCGCCCATCC TGGC TGA CTA CTGAGCTATT GTA TA GA AAC C GA AGGA GGC ACTCGCTCCA GGTGATTGGT GGCTATAAAA CT GT CTG CGA CGGT CTTTC C CTCCGCCACC CT CT CGAGAA C GTGGC GGGC TGCTGATGAT GTGAGGTGTG T GA CAT CCA C T OGA TC CAA G CC CA C TGGGC GTTTGAGAAG TCTGTGGGCT GCCACAATGA ACTGGCGCTC AAAAAGGGGA A TA CAA TTC C CCCC AGC AGO AGGTGTGGAA GCATCTCAAT CGCCCCTAAC ATTTTTTTTA CCAGAAGTAG TC GGA CCA CT TAAGTGGGAG GGGTGTGAAG TTATAGGTGT GGGGGT 0000 GGGCCAGCTG A GTA CT CTTG GA GGGA CC TO A GOC GT CTA A OOCAGCGGGT OTAA TT AAA 0 GCAGGCTTGA TTTGCCTTTC CTTC GACT CO TCCTGGTTGC CAGCGGGCAA CAGGTCCCTA ACCGGGGAGT CTCCCAGCAG TA CA GTGGAA ACTGGAAAAA TOGA ATGTGT C AGAA OT ATG AOT C CCCA GO TA GTC AGCA A TCCGCCCAGT TTTATGCAGA TGAGGAGGCT CT GA GA CGAA GGGTAGCGGT A CA CATGTCG A GGC CA CGTG GCGCGTTCGT TTGGGCTCGC GATCGGAAAC AG CGA GT CCG CCAGTCACAG GGCGGTCGGG TAGCGGTC T GATCGATCTG TCTCCA CA GO A GGA ATTCC C A GA GC TCCAA GAAAGACGCA C TGGC TCA GO CCCTTTTAGG CCACTCAGGG CTGACCTGTA CTCCAACCAG GTCAGTTAGG C AA AGC ATGC CTCCCCAGCA C CATA GTCC C TC COC CCA TT GGCCGAOGCC TTTTTGGAGG GGCTCGCGTC C GTT GTC CA C CCCTCTTCGG A CC GGGT OTT CCTCACTCTC GGTTGAGGAC C CGT CGGC CT CATC GA CC 00 TCGCAAGGTA GT TGT TTCT G T GA GA CGGCG GC CA TACA CT T GT C CACTC C C GA A GGA ACA GTCCTCACAC AOC CCAGA GG CCC CTGC CTC CACTTOCTTC AA AGA A AGTG CAGCTT C CCA ATAAAGATTC GTGTGGAAAG ATCTCAATTA GOCA GA AGT A GC CC CTA ACT CTCCOC C CCA GC CT CGGC CT GGTCCTCCTC CAGGCCAGCA TA GGGGGTC C CATCAAGGAA CCTGAAGGGG TTCCGCATCG AAACTCTTCO C CGAA CGGTA ATCGGAAAAC GGCTGAGCAC GCGGAGGTGC GA TGGT CGA G T GAT GA CA A CA GGT CCAA C AAGCACCCTC A GA TACGC CT CC CTGC CA TT CCTCGGCAAG TGGT GC TOCA GTGCTOOr'GGCA GA AGA AGA OC TGGGAAATCA $SUB-TITUTE aw!4rr WO 89/01940 WO 89/ 1940PCT/US88/02940 FIG. 21 .(cont'd) 1401 GGGCTCCTTC TTAACTAAAG GTCCATCCAA 1451 1501 1551 1601 1651 1701 1751 1801 1851 1901 1951 2001 2051 2101 2151 2201 2251 2301 2351 2401 2451 2501 2551 2601 2651 2701 2751 2801 2851 2901 2951 CAA GA AGA AG C TT A AGATA G GGAGGAGGTG ACCTGCTTCA AGTAGCCCCT GGGGAAGACC GGACATGCAC ATCGTGGTGC GTGGTGTGAC GTAAATATAA TGTTTGTGTA GGTGGA ATGC TCTAGTGATG AA AGAA GA GA GTTTTTTGAG A TTT ACA CCA AAAATATTCT TA CTGTTTTT TAT GCT C AAA TAAGGAATAT AC CA CATTTG CC TGAA CC TG TT GC A GCTT A AATAAAGCAl CAA TGT AT C GCATCACCG( AC CGA TGGGC CGGCGTGGG' CTCCTTGCA' TACTACTGG( C CGA TGC CC' CTTGTGGGAC AA GA CTC AGA CAATTGCTAG GGGGCAGAGC C AGTGC A ATG CTCT C CGTGT TGT CTTG CA G T AG CTTTCC A A TA ATTGGA C AATTTTTAAG TTTTAGATTC CTTTAATGAG AT GAGG CTA C AA GGT AGAA G T CAT GCTGT G CAAAGGAAAA GTAACCTTTA T CT TACT CC A AATTGTGTAC TTGATGTATA TA GAGGTTTI AAACATAAAA TAATGGTTAC TTTTTTCACI TATCATGTC- 1CGCCACAGG& 1 AAGATCGGG-( r ATGGTGGCAI r GCACCATTC, 1 CTGCTTCCT, r TGAGAGCCT C AA GGA AACT TA CTTA CA TC TGTTCGGATT C TGA C CCTGA TA GGA GT CCA CTCAGCTGGA AACCAGAAGA GTGAGATCTT AAA CTA C CTA TGTATAATGT CAA CCT AT GG GAAAACCTGT TGCTGACTCT ACCCCAAGGA TTTA GTA A TA AGCTGCACTG TAAGTAGGCA CA CAGGCA TA CTTTAGCTTT GTGCCTTGAC ACTTGCTTTA TGAATGCAAM AAA TA AAGC A rGCATTCtAGI r GGATCCTCTA r GCGGTTGCTC -TCGCCACTTC 1 GCCCGTGGtC( CTTGCGGCGG( A ATGCAGGAG' T CAACCCAGTI GCTGAATGAT TTCCCCTGAT TGTGAAGTGG GACTGCCAAC CCTTGGAGAG A GGGGTA A AA G CT CCA GGAT AGGTGGAGTT TGTGAAGGAA CA GAGA TTTA GTTAAA CTA C AACTGATGAA TTTGCTCAGA CAACATTCTA C TTT C CTTCA GA ACT CTTCC CTATA CA AGA TAACAGTTAT GAGTGTCTGC TTAATTTGTA TA GAGA TCA T AAAAACCTCC *TGTTGTTGTT A TAC A TC A C -TGTGGTTTGI CGCCGGACGC IGCGCCTATAI GGGC TCA TG GGGGGACTG" ZGGTGCTCAA( r CGCATAAGG( C AGCTCCTTCi C TGTCTTCTT' CGCGCTGACT C AT CA AGAAT A GGA C CAGA A T CTGA CACC C C CC C CCTGGT A CA TA CAGGG A GT GGC A CCT CAAAA TAGA C C CTTA CTTCT A AGCT CTAA G TGATTCTAAT TGGG A GC AGT A GA A ATGCCA CTCCTCCAAA GAATTGCTAA TTGCTTTGCT AAATTATGGA AATCATAACA TA TT AATAA C A A GGTTA A A'A TC AGC CAT CA CA CCT CCC AACTTGTTTA AA ATTT CACA *CCAAACTCAT ATCGTGGCCG *CGCCGACATC GCGCTTGTTT rTGGGCGC CAT :GGCC TCA AC C 1 AGAGCGTCGA :GGTGGGCGCG T ATCATGCAAC 3001 GOCATOACT ATCGTCGCCG CACTTATGA WO 89/01940 PCT/US88/02940 WO 89/01940 PCT/US88/02940 FIG. 2/ (cont'd) GGCA GCGCTCTGGG TCATTTTCGG 3051 TCGTAGGACA GGTGCC 3 "0 1 3151 3201 3251 3301 3351 3401 3451 3501 3551 3601 3651 3701 3751 3801 3851 3901 3951 4001 405 1 4 101 4151 4201 4251 4301 4351 4401 4451 4501 4551 4601 465 1 TTTCGCTGGA CTTGCACGCC TCGGCGAGAA TACGTCTTGC GATTCTTCTC TGrCCAGGCA GCGGCTCTTA GATTTATGCC GCGCCGCCCT AGCCGGGCCA TT CACCA CT C TGCGCAAACC GCAGCCGCAC GCTC CGC CC C GGC GAA ACrC TCCCTCGTGC C GCC TTT C r C GGTATCTCAG GAACCCCCCG TGA GT CC AA C GT AA C AGGA T AAGTGGTGGC CGCTCTGCTG C CGGCA A ACA CA GATT A CGC TA CGGGGTC T TCATGAGATT T GA AGT TTT A TTACCAATGC GTT CATC CAT GA GGGC TTA C CTCACCGGC7 GCGCGACGAT CTCGCTCAAG GCAGGCCATT TGGCGTTCGC GCTTCCGGCG GGTAGATGAC CCAGCCTAAC GCCTCGGCGA ATACCTTGTC C CT CGA CCTG CAAGAATTGG AACCCTTGGC GC GGC GC A TC C CTGA CGAGC GACAGGACTA GCT CT CCT GT C CTT CGGGA A TTCGGTGTAG TTC AGC C CGA C CGGTA AGA C TAGCAGAGCG CTAA CTA CGG A AG CC AOTT A AACCACCGCT GCA GA AA AA A GACGCTCAGI ATCAAAAAGC AATCAATCTA TTAATCAGTC AGTTGCCTGA CAT CTGGCC C GATCGGCCTG C CTTC GTCA C AT CGC CGGC A GACGCGAGGC GCATCGGCAT GACCATCAGG TT CGA TC ACT GCACATGGAA TGCCTCCCCG A AT GGA AGC C A GC CAAT CAA A GA A CATATC TCGGGCCGCG ATCACAAAAA TAAAGATACC TC CGA C CCTG GCGTGGCGCT GTCGTTCGCT C CGCTG CGC C A CGA CTT ATC AGGTATGTAG C TACA ZT AGA CCTTCGGAAA GGTAGCbGTG AGGATCTCAA GGAACGAAAA iATC TTCAC CT kAAGTATATAT i AGGCACCTAI kCTCCCCGTCC :CAGTGCTGCA TCGCTTGCGG TGGTCCCGCC TGGCGGCCGA TGGATGGCCT GCCCGCGTTG GA CAG CTTC A GGACCGCTGA C GGGTT GG CA CGTTGCGTCG GGC GG CA CCT TTCTTGCGGA CATCGCGTCC TTGCTGGCGT TCGACGCTCA A GG CGTTTC C CC GCTT A CCG TTCTCAATGC CCAAGCTGGG TTATCCGGTA GCCACTGGCA GC GGTG C TA C AGGACAGTAT A AGA GTT GGT GTTTTTTTGT GAAGATCCTT CT CA CGTTA A A GA TC CTT TT GA GTAA A CTT CTCAGCGATC iTGTA GA TAA C A TGA TA C CGC C GA GGA CCGC TATT(.GGAAT ACCAAACGTT CGCGCTGGGC TCCCCATTAT CA GG CCATGC AGGATCGCTC T CGTC A CGGC TGGATTGTAG CGGTGCATGG CGCTAACGGA GAACTGTGAA GCCATCTCCA TTTTC CA TAG A GTCA GA GGT CC CT GGA AGC GATACCTGTC TCACGCTGTA C TGT GTGC A C A CTAT CGT CT GCAGCCACTG A GA GTT CTTG TTGGTATCTG AGCTCTTGAT TTGCAAGCAG TGATCTTTTC GGGATTTTGG AAA TT A AAAA GGTCTGACAG TGTCTrATTTC TACGATACGG GA GACCCA CG CCAGATTTAT CAGCAATAAA CCAGCCAGCC GGAAGGGCCG t WO' 89/0 1940 PCT/US88/0294o WU89/01940 PCT/US88/02940 G7/93 FIG. 21 (con t'd) 470.1 4751 4801 4851 4.90 1 4951 5001 5051 5101 5151 5201 5251 5301 5351 5401 5451 5501 5551 AGCGCAGAAG TGTTGCCGGG C GT TGTT GC C TGGCTTCATT CCCATGTTGT CAGAAGTAAG ATAATTCTCT GA GT ACT CAA CT CTTGC CCG TAAAAGTGCT AT CTTA C CGC CT GAT CTT CA CAGGAAGGCA TGAATACTCA TTATTGTCTC AAAT AGGGGT GAAACCATTA GCCCTTTCGT TGGTCCTGCA AAGCTAGAGT ATTGCTGCAG CAGCTCCGGT GCAAAAAAGC TTGGCCGCAG TACTGTCATG C C AAGT CATT GCGTCAACAC C AT CA TTGGA T GTT GA GA TC GCATCTTTTA AAATGCCGCA TA CT CTTC CT AT GA GCGGA T TCCGCGCACA TTAT CA TGA C CTTC A A A CTTTAT CCG AAGTAGTTCG GCATCGTGGT TCC CAA CGAT GGTTAG CTC C T GTTATCA CT C CATC CGTAA CTGAGAATAG GGGATAATAC AAACGTTCTT CA GTT CGA TG C TT TC AC CA G A AA A AGCA A TTTTCAATAT A CA TATTT GA TTTCCC CGA A CCTCCATCCA CCAGTTAATA GTCACGCTCG CA AG C GAGT TTC OGT CCT C CATGOTTATG GA TOC TTTT C TGTATGCGGC COCOCCACAT C OGG C GAA A TAACCCACTC CGTTTCTOGGG TAAGGGCOAC TA TTGAA GC A ATGTATTTAG AA GT GC CAC C GTCTATTAAT GTTTGCOCAA TCGTTTOGTA TACATOATCC CGATCGTTGT GCAGCACTGC TOTGACTOOT GAC CGA GTTG AOCAOAACTT ACTCTCAAGG OT GCA C CCAA TGAGCAAAAA ACGGAAATGT TTTATCAGOO AA AA ATAAA C TGACGTCTAA ATTAACCTAT AAAAATAGGC GTATCACGAG AH~ 'S WO, 89/01940 WO89/1940PCT/US88/02940 68 /93 fBC395 .G368 bacJLWore :scluoie T4#10 :AA #3 =LYrS -first 131 AA of T4 bg395.seQ Length. 5413 FIG 22 151 251 Js 1 401 451 1 1 701 7 S1 801 851 1 951 100) 1051 1201 1251 130 1 1351 14 C I GAATTAATTC CAGCTTGCTG TCCCCAGGCT GTCA GCA ACC rGCAAAGC'AT C CGCCC AT CC TGGCTGA CTA C TGAGCTATT GTATAUCAAAC C GA AGCGAGGC ACTClGCTCCA GGTGATTGGT GGCTATAAAA C TGT CTGC GA C GGT CTTTC C CTCCGCCACC CT CT CGAGAA CGTGGCGGGC TGCTGATGAT GTGAGGTGTG TGA CA TCC AC TGOATCC AA G CC CA CTGGGC GTTTGAGAAG TC7GTGGGCT GCCACAATGA ACTGGCGCTC AAAAAGGGGA ATACAATTCC GGGCTCCTTC CCC CAGCAGG AGGTGTGGAA GCATCTCAA T CGCCCCTAAC ATTTTTTTTA CCAGAAGTAG TCGGACCACT TAAGTGGGAG GGG7GTGAAG TTATAGGTGT GGGGGTGGGG GGGCCAGCTG A GT ACT CTTG GA GGG ACC TG AGGCGTCTAA GGCAGCGGGT GTAATTAAAG GCAGGCTTGA TTTGCCTTTC C TT CGA CTCG TCCTGGTTGC CA GC GGGC AA CAGGTCCCTA AC CGGGGAGT CTCCCAGCAG TA C AGTGGAA ACTGGAAAAA TTAACTAAAG TGGAATGTGT CA GA AGTAT G A GT CCCCA GG TA GT C AGCA A T C C GCC CAGT TTTATGCAGA TGAGGAGGCT C TGA GA CGAA GGGTAGCGGT A CA C ATGT CG A GGC CA CGTG GCGCGTTCGT TTGGGCTCGC GATCGGAAAC A GCGA GT CCG CCAGTCACAG GGCGGTCGGG TAGGCGGTCT GATCGATCTG T CTC CA C AGG A GGA ATTC C, A GA GCT CCA A GA AAGA CGCA C TGGCT CA GG CCCTTTTAGG C CA CTCA GGG CTGACCTGTA CTC CAA C CAG GTCCATCCAA GTCAGTTAGG CAAAGCATGC CTCCCCAGCA CC ATA GTCC C TCCGCCCATT GGC CGA GGC C TTTTTGGAGG GGCTCGCGTC C GT TGTC CA C C CCT CTT CGG ACCGGGTGTT C CT CACTCTC GGTTGAGGAC C CGT CGGC CT CA TCGA C CGG TCGCAAGGTA GTTGTTTCTG T GAGA CGGCG GCCATACACT TGTCCACTCC A GGA ACA ,TC CT CA CA C AGCCCAGAGG CCC CTGCCTC CA CTTGCTT C AAAGA A AGTG CA GCTT C CCA ATAAAGATTC GCTGAATGAT GTGTGGAAAG ATCTCAATTA GGCAGAAGTA GCCCCTAACT CTC CGC C CCA GC CT CGG CC T GGTCCTCCTC CA GGC CAGC A TA GGGGGTC C C ATC A AGGAA C CTGA AGG GG TTCCGCATCG AAACTCTTCG CCGAACGGTA ATCGGAAAAC GGC TGA GCA C GCGGAGGTGC GA TGGT CGA G T GA GTGA CA A CAGGTCCA Z AAGCACCCTC A GA TA CGC CT CCCTGCCATT C CT CGGCAAG TGGTGCTGCA GTGCTGGGCA GAAGAAGAGC TGGGAA ATCA CGCGCTGACT W089/01940 PCT/1JS88/02940 WO 89/01940 PCT/US88/02940 09 93 1451 CAAGAAGAAG CTTGT F I G 22(con/'d) GGGAC CAAGGAAACT TTCCCCTGAT CAT CA AGAAT 1501 1551 1601 1651 1701 1751 1801 1851 1901 1951 2001 2051 2101 2151 2 20 1 2251 2301 2351 2401 2451 2501 2551 260 1 2651 2701 27 5 2801 2851 2901 2951 3001 3051 CTTAA GA TAG GGAGGAGGTG A CCTGC TTCA A GTA GC C CCT GT GTGACA TA AATATAAAAT TTGTGTATTT G GA A TGC CTT AGTGATGATG GAA GA GA AAG TTTT GACT CA TA CA CCACAA ATATTCTGTA TGTTTTTTCT G CT C A A A A GGAATATTTG ACATTTGTAG GAACCTGAAA CAGCTTATAA AAAGCATTTT TGTATCTTAT TCACCGGCGC GA T CGGAA C CGTGGGTATC CTTGCATGC/ TACTGGGCT( A TGC C CTT G) CAT GA CTAT( TAGGACAGGC CGCTGGAGC( GCA CCCCCTi GCGAGA AG 2 AAGACTCAGA T CAATTGCTAG T GGGGCAGAGC C CAGTGCAATG A ATTGGACAAA C TTTTAAGTGT TAGATTCCAAC TAATGAGGAAA AGGCTACTGC CT AGA AGA CC TCCTGTGTTT AGGAAAAAGC ACCTTTATAA TA CT CCACA C TGTCT A C CTT ATCT A TA GTG AGGTTTTACT CA TA A AATGA TGGTTACAAA TTTCACTGCA CATCT CT GGA CA CAGCGTGCG iA TCGCCCCT CC ;GTGGCAGGCC C CCATT CCTTG 1CTTCCTAATG GAGCCTTCAA :CT CCCGCA C r GCCGGCAGCG 1 CGACGATGAT 2GCTCAAGCCT A GGCCATTATC ACTTACATC T *GTTCGGATT G TGACCCTGA C ~GATCTTTGT C TACCTACAG A TAATGTGTT A CTATGGAAC I k.ACCTGTTTTC rGACTCTCAA CCAAGGACTT AGTAA TAGA A TGCA CTGC TA GTAGGCATAA A GG CATA GAG TAG CT TTT'TA C CTT GA CTAG TGCTTTAAAA ATGCAATTGT TAAAGCAATA TT CT AGTT CT TCCTCTACGC GTTCCTCGCC C CA CTTC GGG CCTGGCCGGG CGGCGGCGGT CAGGAGTCGC CCCAGTCAGC TTATGACTCT CT CTGGGT CA CGCCCTGTCG T CGTCA C TGG CC CGCA T C GTGAAGTGG A IACTGCCAAC T CTTGGAGAG C hAAGGAACCT T ~GATTTAAAG C IAACTACTGA I ~GATGAATGG C ICTCAGAAGA CATTCTACTC TCCTTCAGAA- CTCTTGCTTG TA CA AGAAAA CA OTT A TAAT TCTCTGCTAT ATTTGTAAAG A GA TCA TAAT AAC CTC CCA C T OTTGCT TAA C CCATCACAAA GCT TTCT CCA COCA CGCATC C CT ATAT CC CTCA TGAGC C GGACT OTT CC OCT CAA CGC A TAACOCA GA TC CTT CC OCT C TT CTTTAT C TTTTC GOC GA CTTGCGGTAT TCC CGCCCA CC CGCC CGA CGC GCACCACAA ,CTGACACCC 2CCCCC COOT ACTTCTOTC TCTAAGGTA TC TA ATT CT ~AGCAGTGGT A TO CCATCT :TCCAAAAAA CTC TA AGTT CTTTGCTATT TTATGGAAAA CA TA ACATA C TAA TA ACTAT 2GGTTAATAA CAGCCCA TAC C A CCTCCCC CT TTCTTTATTC TTTCACAAAT AACTCATCAA CTGCC OCA C GACA TCA CC C TT GTTTC CC CCCCAT CTC CTCAACCTAC CGTCCA C CC GOGCCCOC A TGCAA CT CC GGACCGCTTT TCGGAATCTT A AA C TTT CC CCTCOCCTAC WO 89/01940 W089/1940PCT/US88/02940 70/93 3101 GTCTTGCTG CGTTCGCGAC GCGAG(CTGG ATGGCCTTCC CCATTATGAT 3 15 1 3201 3251 3301 3351 3401 3451 3501 3551 3601 3651 3701 375r1 3801 3851 3901 395 1 1 4051 4 10 1 4.15 1 420 1 4251 4301 435 1 440. 4451 450] 4551 460 1 4651 TCTTCTCGCT C C AGGCAGGT GGOTOTTACCA TTA7GCCGCC C CGC CCTATA CGGGC CA CCT ACCACTCCAA GC AA AC CAA C GCCGCACGCG C CGCCC C CCT GA AACC CGA C CTCGTGCGCT CTTTCTCCCT ATCTCAGTTC CCCCCCGTTC GTCCAACCCG AC AGGA TTAG IGOT GGCCTA I CTGCTGAAO G C. A AACA AA C aTTACGCGCA GGGTCTGA C TGAGATTATC A OT TTT AA AT CC4ATGCTTA C A T C C A FA G7 GGCTTAC CAl A CCGGCTC CA GCAGAAGTGC TOCCGGGAAC T GTT S C C AT7 TCCGGCGGCA A GA TGA CGA C GCCTAACTTC TCGGCGAGCA CCTTGTCTGC C GA CCT OAAT GAATTGGAGC CCTTGGCAGA GCGCATCTCG GACGAGCATC AGGACTATAA CTCCTGTTCC TCGOGAAGCO GGTGT AGGT C AGCCCGACCG GTA AGA C ACG C AGAOC GA GG ACT ACOOCTA C CA GTTA CCT CAC C GCT GOT GAA AA A AAGO OCT CAOTGGA AAAA AGOAT C CA AT CTA AAG ATCAOTGAGO *TOCCTOACTC *CTGGCCCCAG GATTTATCAO iTCCTOC A ACT iCT AGA GTAAG GCTGCAGGCA rCGGGATGCC CATCAGGGAC GATCACTGGA CATGGAA COO CTCCCCGCGT 0 A AGC CGGC CAT C AATT C ACA TAT CCAT GGCCGC OTTO ACAAAAATCG A GA TACCA GO GA CCC TOCCO TOOCOCTTTC OTTCOCT C CA CTGCOCCTTA A CTT A TCOC C TATOTAGOCO CA CTA GAA GO TCGGAAAAAO AOC GOTGGTT ATCT C AAGA A ACOAAAACTC TT CA CCTA GA T AT ATAT GAO CA CCTAT CT C CC C T C TGT TOCTOCAATG CAATAAACCA TTATCCGCCT TAGTTCGCCA TCGTGGTGTC COCOTTOCAG AG CTT CA A G CCOCTGATCG OT TGC AT GO TO C TCO CG GGCACCTCOC TTO C GA GA A C OCOT C CGCC CTGGCGTTTT A COCTCA AGT COT TT CC C CC C TTA C CGOAT TCAATGCTCA A OCTG000CT 0 TCCOOTAACT ACTOOCAOCA OTOC TA C AGA ACAOTATTTG AOTTOGTAOC TTTTTGTTTG GA T CCTTT GA ACOTTAAOGGG TCCTTTTAAA TAAACTTGOT AGCOATCTOT A GA TAA CTA C ATAC CO CGAO OCCAGCCOGA CCATCCAOTC GTTAATAGTT AC OCTCOT CO OCCATGCTOT ATCGCTCGCO T CA CGGCOAT ATTOT AGGC 0 TO CA TOGA OC TAACGGATTC CTOT GAA TG0C AT CTC C AOCA TCCATAGOCT CA GAGGT GC TGiGAAGCTCC A CCTOT C C C COC T T AGGT TGTGCACOAA AT COT CTTGA OCCACTOOTA GTTCTTOAAO GTATCTOCGC TCTTOATCCO CAAGCAOCAO TCTTTTCTAC A TTTTGTC A TTAAAAATOA C TO A CAOTT A CT ATT TC OTT GA TA C0GOAG ACCCACGCTC AOGGCCGAGC TATTAATTGT TO CGC A AC TTTGGTATOG 4701 CITC.TTCAG CTCCGGTTCC CAACGATCAA GGCGAGTTAC ATGATCCCCC WG 89/01940 WO 8901940PCT/US88/02940 71/93 FIG. 22(cori/'d) 4751 4801 4851 4901 495] 5001 S 05 1 5101 5151 5201 5251 5301 5351 540 1 ATGTTGTGCA AAAA AAGTAAGTTG GCCC ATTCTCTTAC TGT( TACTCAACCA AGTC TTGCCCGGCG TCAA AAGTGCTCAT CAT TTACCGCTGT TGA( ATCTTCAGCA TCT GAAGGCAAAA TGCi ATACTCATAC TCT TTGTCTCATG AGC4 TAGGGGTTCC GCG ACCATTATTA TCA CTTTCGTCTT CAA ~AGCGGT ICAGTGT :ATGCCA :ATTCTG kCACGGG rGGAAAA IA T CCAG FTTTA CTT CGCA AA A TCCTTTT GGATACA CA CA TTT T AGCT CC TT C TATCACTCAT TCCGTAAGAT A GA ATA GTGT ATAATACCGC C GTT CTT CGG TTCGATGTAA TCACCAGCGT A AGGG AA TA A T CAATATTAT TAI TTGAATG CCC CGAAAAG GGTCCTCCGA GGTTATGGCA GCTTTTCTGT A TGC GGC GA C GCCACATAGC GGCGAAAACT CCCACTCGTG TTCTGGGTGA GGGCGACACG TGAAGCATTT TATTTAGAAA T GCCA C CTGA TCGTTGTCAG GCACTGCATA GA CTGGTGAG CGAGTTGCTC A GA A CTTT AA CTCAAGGATC CACCCAACTG G CA AA A ACAG GAAATGTTGA ATCAGGGTTA AATAAACAAA C GTC TA AGA A TGACATT AACCTATAAA AATAGGCGTA TCACGAGGCC SUTiTUTE SHEET WG,89/01940 W089/1940PCT/US88/02940 7Z/93~ FIG 23 43 t. I I p 4 I WO,89/01940 PCT/US88/02940 7,3/9)3 C/df C/al 16-/0 rs 74.2 4,377hkbp 5 T4. cla I F G.24., 1 U! 111111 WO 89/01940 PCTJUS88/ 02940 7W/93 (1 13) 6,qlE (srop) I3amal/ FIG. SUBSTITUTE SHEET A trp cla z ori P196-10 rST4. js(61) rsT4.2 4377iP TIC 9 o.Y 374 dLvm- S(5 TOP) .BrilI E MII fluid I B .4 m 14-11gla 1 HijndUlI(61) /1 md hr (62) 3 3 46 bp bp al(8 9 bp) p2II-)f I-) 36oobp FIG 26 74 WO 89/01940 7(o/93 PCT/US88/02940 ftp QFIG. 27 A. p (3) 4*1f3,1 MW)4 0 T4 Irm/ Xh o AY5 To S'fe cbrecled mtlc'edesa to change crn Asn cL+ aminno acid posififon to at L4-5IIn3 74-66 C/o I 1 tLc direcled mnufae-feGesi #0 cleld-e Gin a~nd CGlY cut amm~io a.ci pC oS(~lt~s*~1 2 US/fl3 T4AI& -87 rs 7 t ih SUBSTITUTZ Sjir I'- C%0 000 InC In- 100 00 le -7 W-0 89/01940 PCT/US88/02940 78/ 93 r' 49 (r5 74.113) v/84 3 77 0 14 4 68 24 7 FIG. 29A SIjN~TUTE SHEET -U I7 WO-89/01940 79/93 rsT4. 9(S f848 //o PCT/US88/02940 FIG 298 t WO89/01940 PCT/US88/ 02940 I WO'89/01940 PCT/US88/02940 80/93 FIG 30 (a) (b) rsT4:.1/3 r- o 20 4-'0 60 80 Frccon Numbe~r o 20 40 60 80 l00 (c) A A c D E Ka 6 3 -1 tI ti 1HEE 1.1 W-0 89101940 PCT/US88/02940 3 /1193 N)L jau/9~) .2U *ZRISSTITUTOE SHEET WO'89/01940 W089/1940PCT/US-88/02940 8Z/93 3 G MW (Id) -97 4 AA* 43 ij~ ~zj> L' FIG'.32 1Th~JT1~ ~m I WO 89/01940 W089/1940PCT/US88/02940 8,3/93 02534667 8 3/0/1I MW &9e-rs T4,7 F1/G.33 'SUB T T V i"-u'tAz t -7 WO 89/01940 PCT/US88/02940 84J/93 (zi lo 00 11 o 0 0 g K 0 @0T I I hf Ii 14 U U UO11.1~qul/ '34P2STITUTE SHEET F16.35 go- rs T4. 17 4 A/A ~-0 0- /T4.3/0K1474 A 7 509 A9 12/ZO/,AT4A /0 ,/56 3/25 .625 I '156 .3/25 .625 tg5 2,5 /ogaq o2 ov2 a pp-~ WO 89/0 1940 PCT/US88/02940 q Lk N IN SUEZ All UTZ Is I WO 89/01940 W089/1940PCT/US88/02940 87/9c3 K N T!TUTR sHrtf*-.. I U WO 89/01940 PCT/US88/02940 88/93 is ~i1 I I I:: >-118STfTUTE SH4EET I WO 89/01940 PCT/US88/02940 WO,89/01940 PCT/US88/02940 59 3 LJ%~ I 0 ii S-U- SITUSv 1. N0 C91/66 CELLZ 1,5101V 45,A V 020 H9Clst2TA 197Lf 3 t5-4 r 4 9 ZvH s74 r trl/1A64071-1 05ull5,-/lcnto 2! q PRE WO 89/01940 PCT/US88/02940 51/953 '4 NN t'Q/SflJ //9jo &og//uf SUB-1--fR iai I -j "L I WO 89/01940 W089/1940PCT/US88/02940 92193 FIG. 42 polyA 1 Mhez I a hea,,7 17U 4 C/aSe le 377 z 1,a V4,Q poll,4 SUBSTITUTEMi SHEET r- j WO 89/01940 9,3/9,3 PCT/US881 02940 -bva/e,j rsT4 KJd< /a 9 M 112- 377 or cedce/I FIG. 43 SUBSTITUTZ. Sh- ~B i INTERNATIONAL SEARCH REPORT International Application No. PCT/US88/02940 I. CLASSIFICATION OF SUBJECT MATTER (it several classification symbols apply, indicate all) 6 According to International Patent Classification (IPC) or to both National Classification and IPC TPCC4>: C0.7H 15/12. C12Q 1/70, C12Q 1/02, see attachment. U.S. CL.: 5.36/'2.7; 435/,29,39,68 9.1 .70 .172.3. po II. FIELDS SEARCHED Minimum Documentation Searched 7 Classification System Classification Symbols 4 3 2 9 3 9 6 8,91,170,172.1,172.3,240,253, 320; 30/35.0,412; 514/2; 424/85; 536/27; 935/6, U.S. 9, 11,12,15,22.,23,24,59,60,65,66 Documentation Searched other than Minimum Documentation to the Extent that such Documents are Included In the Fields Searched 8 Chemical Abstract Data Base _(CAS) 1967-1988; Biosis Data Base 1969-1988 Keywords: CD4, T4, TCell, AIDS, HTLV, HTLVI,HTLVIII, see attachment. IIl. DOCUMENTS CONSIDERED TO BE RELEVANT 9 Category Citation of Document, 11 with indication, where appropriate, of the relevant passages 1 2 Relevant to Claim No. 13 Y SCIENCE, Volume 234, issued 1986 13-20, November, (Washington, DC., 29-33 SATTENTAU ET AL), "Epitopes and of the CD4 Antigen and HIV Infection" 48-52 See pages 1120-1123. See particularly page 1120 Y SCIENCE, Volume 234, issued 1986, 13-20, November, (Washington, D.C. U.S.A) 29-33, HOXIE ET AL), "Alterations and in T4 (CD4) Protein and mRNA 48-52 synthesis in Cells Infected with HIV" see pages 1123-1127. See particularly page 1123. Y,p PROCEEDINGS NATIONAL ACADEMY OF 1-4,25-27, SCIENCES, U.S.A, Volume 84, 34-36 and issued 1987 December (Washington, 39-46 MADDON ET AL.), "Structure and Expression of the Human and Mouse T4 Genes", See pages 9155-9159, See particularly page 9155 and 9156. Special categories of cited documents: 10 later document published after the international filing date or priority date and not in conflict with the application but document defining the general state of the art which is not or priority date and not prconict with the application but considered to be of particular relevance cited to understand the principle or theory underlying the invention earlier document but published on or after the International document of particular relevance; the claimed invention filing date cannot be considered novel or cannot be considered to document which may throw doubts on priority claim(s) or involve an inventive step which is cited to establish the publication date of another document of particular relevance; the claimed invention citation or other special reason (as specified) cannot be considered to involve an inventive step when the document referring to an oral disclosure, use, exhibition or document is combined with one or more other such docu- other means ments, such combination being obvious to a person skilled document published prior to the International filing date but in the art. later than the priority date claimed document member of the same patent family IV. CERTIFICATION Date of the Actual Completion of the International Search Date of Mailing of this International Search Report 2: NOVEMBER 1988 03 FEB 1989 International Searching Authority Sgnature of Auorar a ISA/US RICRARD C. PEET Form PCT/ISA/210 (second sheet) (Rev.11-87) ii ii '1J 1 PCT/tJS88/0 2940 A~ttachment to PCT/ISA/210 Classification of Subject Matter IPC: C12Q 1/06, C12P 21/00, C12P 19/34, C12P 1/04, C12N 15/00, C.L2N 7/00; C07K 13/00, C07K 3/00; A61K 37/687 A61K 39/00, A61K 45/02 US.CL.: 240, 320; 530/350, 412; 514/2; 424/85 r-I. Fields Searched Keywords: ARC, Surface, receptor, therap?, purif?, Immunoassay, Detection, Pharmaceutical Composition, Lymphocyte, Igg, Polyvalent, Solub?, gene, Clon?, Protein, Polypeptide, Fusion, Expression, Vector, Plasmid, Surface Protein, Surface Antigen, Acquired Immune Deficiency Syndrome, Retrovirus I International Application No. PCT/ILJS88294O SIII. DOCUMENTS CONSIDERED TO BE RELEVANT (CONTINUED FROM THE SECOND SHEET) I Category Citation of Document, with indication, where appropriate, of the relevant passages Relevant to Claim No Y x Y P, Y Y Y ,P PROCEEDINGS NATIONAL ACADEMY OF SCIENCES, U.S.A, Volume 84, issued 1987, June (Washington, CHANH ET "Monoclonal Anti-idiotypic Antibody Mimics the CD4 Receptor and Binds Human Immunodeficiency Virus" See pages 3891-3895. See particularly page 3891. CELL, Volume 47, issued 1986, November, (Cambridge, Mass., U.S.A) MADDON ET AL), "The T4 Gene Encodes the AIDS Virus Receptor and is Expressea in the Immune System and the Brain", See pages 333-348, See particularly pages 333-335. CHEMICAL ABSTRACTS, Volume 107, no. 15, issued 1987 October 12 (Columbus, Ohio, T.L. LENTZ et al, "Rabies virus binding to cellular membranes measured by enzyme immunoassay' see page 359, column 1, the abstract no. 131853f, Muscle Nerve, 1985, 336-345 (Eng). CHEMICAL ABSTRACTS, Volume 106, no. 21, issued 1987, May 25, (Columbus, Ohio, J.P. ZIMMER ET AL., 'Diphenylhydantoin (DPH)"blocks HIV-receptor on T-lymphocyte surface', see page 123, column 1, the abstract no. 168522c, Blut, 1986, 53(6), 447-450 (Eng). BIOLOGICAL ABSTRACTS, Volume 85, no. 4, issued 1988, April 15 (Philadelphia, PA, U.S.A), A.G. DALGLEISH ET AL., 'Neutralization of HIV isolates by anti-idiotypic antibodies which mimic the T4 (CD4) epitope: A potential AIDS vaccine' see page 222, abstract no. 37595, Lancet 2 (8567): 1047-1050 (Eng). 13-24, 29-33 and 48-b2 1,3-6 and 25-27 2,7-24 and 28-50 16-18 32-33 and 13-15, 19-20, 29-30, 48-49 and 51-52 13-15, 19-20, 29-30, 48-49, and 51-s2 Form PCT/tSA121o(extrasti) (Rev. 11-7) WO089/01940 PCT US88/02940 II International Application No: PCT/ US8 8 0 2940 q MIC OORGANISMS Optional Sheet in connection with the microorganism rierred to @n pag 95 lirps 29-35 o h ecito A. IDENTIFICATION OF DEPOSIT 96, lines 19-21 iiFurthez, depoosits ared dentited an an dditieanl sheet 3additional sheets attached Name of depositary Institution 4 In Vitro International1, Inc. Address of depositary Institution (including postal code and country)' 611 Hanmirids Ferry Road, Linthicum, Maryland 21090 United States of Amnerica oats, of depoait 5Accession Number 4 See attached additional sheets See attached additional sheets B. ADDITIONAL INEDICATIONS I (leave blank It not appicable). This Intormation is continued on a separote attached shoartE In resoect of those designations in which a European patent is sought samples of the dep:osited microorganisms will be made available until the publication of the mention of the grant of the European patent or until the date on which the application has been refused or withdrawn or is deemed to be withdrawn, only by the issue of such a sampole to an expert nominated by the person recp.estinj the sampole (Rule 28(4 EPC). C. DIESIGNATED STATES FOR WHICHI NDICATIONS ARA MADE 1 (it the indlicationa are not ]or all designated States) U 0. SELPARATE FURNISHING Of INDICATIONS I (leave blank It not applicable) The Indications listed below will be submitted to the International Bureau later S (Specity the general nature of the indications e.g.. -Accession Number *t Deposit") A. This shoot was received With the International application when rilied (to be checked by the receiving Office) (Authorized Offcer) The date of receipt (from the applicant) by the Iternational Bureau 16 wae 13~ JANUAFY 1989 (Authorized Officar)__ 13. 01,M5 Form PCTIRO/134 (January 15511) WO089/01940 PCT/US88/02940 Additional Sheet 1 of 3 To Form PCT/RO/1 34 Continuation Of Box A IIDENTIFICATION OF DEPOSITS IABG378: E.coli MC1061/pBG378 1199-7: E.coli MC1061/p1.99-7 170-2: E.coli JA221/p1.70-2 EC100: E.coli JM83/pEC100 BG377: E.coli MC1061/pBG377 BG380: E.coli MC1OG1/pBG38O BG381: E.coli MC1061/pBG381 DATE OF DEPOSITS 2 September 1987 ACCESSION NUMBERS IVI 10143 IVI 10144 IVI 10145 IVI 10146 IVI 10147 IVI 10148 IVI 10149 WO 89/01940 PCT/US88/ 02940 Additional Sh~eet 2 of 3 To Form PCT/RO/13 4 Cbnt±nuation Of Box A IDENTIFICATION OF DEPOSITS BG-3 91: BG-392: BG-393: BG-394: BG-3 96: 2 03-5 E.coli MC1061/pBG391 E.coli MC1061/pBG392 E.coli MC1061/pBG393 E.coli MC1061/pBG394 E.coli MC1061/pBG396 E.coli SG936/p203-5 DATE OF DEPOSITS 6 January 1988 ACCESSION NUMBERS IVI IVI IVI IVI IVI IVI 10151 10152 10153 10154 10155 10156 WO 89/01940 PCT/1JS88/02940 WO 89/01940 PCT/US88/02940 Additional Sheet 3 of 3 To Form PCT/RO/13 4 Cbontinuation Of Box A IDENTIFICATION OF DEPOSITS 211-11: 214-10: 215-7: E.coli A89/pBG211-11 E.coli A89/pBG214-10 E.coli A89/pBG215-7 DATE OF DEPOSITS 24 August 1988 ACCESSION NUMBERS IVI 10183 IVI 10184 IVI 10185