"Non-human Primate CD4 Polypeptides, Fusions Thereof, DNA encoding, and uses thereof"

FIELD OF THE INVENTION

The invention is in the field of recombinant genetics and pharmaceutical compositions.

BACKGROUND OF THE INVENTION

The human and simian immunodeficiency viruses HIV and SIV are the causative agents of Acquired Immune Deficiency Syndrome (AIDS) and Simian Immunodeficiency Syndrome (S1DS), respectively. See Curren, J. et al.. Science 329:1359-1357 (1985); Weiss, R. et al.. Nature 324:572-575 (1986). The HIV virus contains an envelope glycoprotein, gpl20 which binds to the CD4 protein present on the surface of helper T lymphocytes, macrophages and other cells. Dalgleish et al . Nature. 312:763 (1984). After the gpl20 binds to CD4, virus entry is facilitated by an envelope-mediated fusion of the viral target cell membranes.

During the course of infection, the host organism develops antibodies against viral proteins, including the major envelope glycoproteins gpl20 and gp41. Despite this humoral immunity, the disease progresses, resulting in a lethal immunosuppression characterized by multiple opportunistic infections, parasitemia, dementia and death. The failure of host anti-viral antibodies to arrest the progression of the disease represents one of the most vexing and alarming aspects of the infection, and augurs poorly for vaccination efforts based upon conventional approaches.

Two factors may play a role in the inefficacy of the humoral response to immunodeficiency viruses. First, like other RNA viruses (and like retroviruses in particular), the immunodeficiency viruses show a high mutation rate which allows antigenic variation to progress at a high rate in response to host immune surveillance. Second, the envelope glycoproteins themselves are heavily glycosylated molecules presenting few epitopes suitable for high affinity antibody binding. The poorly antigenic, "moving" target which the viral envelope presents, allows the host little opportunity for restricting viral infection by specific antibody production.

Cells infected by the HIV virus express the gpl20 glycoprotein on their surface. Gpl20 mediates fusion events among CD4 ⁺ cells via a reaction similar to that by which the virus enters the uninfected cell, leading to the formation of short-lived multinucleated giant cells. Syncytium formation is dependent on a direct interaction of the gpl20 envelope glycoprotein with the CD4 protein. Dalgleish et al.. supra, Klatzmann, D. et al.. Nature 312:763 (1984); McDougal, J.S. et al. Science. 131:382 (1986); Sodroski, J. et al .. Nature. 3 2:470 (1986); Lifson, J.D. et al.. Nature. 323:725 (1986); Sodroski, J. et al.. Nature. 321:412 (1986).

The human CD4 protein consists of a 372 amino acid extracellular region containing four im unoglobulin-like domains, a membrane spanning domain, and a charged intracellular region of 40 amino acid residues. Maddon, P. et aJL, Cell 42:93 (1985); Clark, S. et al .. Proc. Natl. Acad. Sci. (USA) 84:1649 (1987).

Evidence that CD4-gpl20 binding is responsible for viral infection of cells bearing the CD4 antigen includes the finding that a specific complex is formed between gpl20 and CD4. McDougal et al .. supra. Other workers have shown that cell lines, which were non-infective for HIV, were converted to infectable cell lines following transfection and expression of the human CD4 cDNA gene. Maddon et al .. Cell 47:333-348 (1986). PCT Application Publication Nos. WO 88/01304 (1988) and W089/01940 (1989) disclose that soluble forms of human CD4 comprising the immunoglobulin-like binding domains are useful for the treatment or prophylaxis of HIV infections.

In contrast to the majority of antibody-envelope interactions, the receptor-envelope interaction is characterized by a high affinity (K _a = 10^1/mole) immutable association. Moreover, the affinity of the virus for human CD4 is at least 3 orders of magnitude higher than the affinity of human CD4 for its putative endogenous ligand, the MHC class II antigens. A number of workers have disclosed methods for preparing hybrid proteins. For example, Murphy, United States Patent 4,675,382 (1987), discloses the use of recombinant DNA techniques to make hybrid protein molecules by forming the desired fused gene coding for a hybrid protein of diphtheria toxin and a polypeptide ligand such as a hormone, followed by expression of the fused gene.

Many workers have prepared monoclonal antibodies (Mabs) by recombinant DNA techniques. Monoclonal antibodies are highly specific well-characterized molecules in both primary

and tertiary structure. They have been widely used for in vitro immunochemical characterization and quantitation of antigens. Genes for heavy and light chains have been introduced into appropriate hosts and expressed, followed by reaggregation of the individual chains into functional antibody molecules (see, for example, Munro, Nature 312:597 (1984); Morrison, S.L., Science 229:1202 (1985); Oi et al .. Biotechnioues 4:214 (1986); Wood et al.. Nature 314:446-449 (1985)). Light- and heavy-chain variable regions have been cloned and expressed in foreign hosts wherein they maintained their binding ability (Moore et al .. European Patent Applica¬ tion 0088994 (published September 21, 1983)).

Chimeric or hybrid antibodies have also been prepared by recombinant DNA techniques. Oi and Morrison, Biotechnioues 4:214 (1986) describe a strategy for producing such chimeric antibodies which include a chimeric human IgG anti-leu3 antibody.

Gascoigne, N.R.J., et al .♦ Proc. Natl. Acad. Sci. (USA) 84:2936-2940 (1987) disclose the preparation of a chimeric gene construct containing a T-cell receptor α-chain variable (V) domain and the constant (C) region coding sequence of an immunoglobulin γ2a molecule. Cells transfected with the chimeric gene synthesize a protein product that expresses immunoglobulin and T-cell receptor antigenic determinants as well as protein A binding sites. This protein associates with a normal λ chain to form an apparently normal tetrameric (H L2» where H-heavy and L«light) immunoglobulin molecule that is secreted.

Sharon, J., et al., Nature 309:54 (1984), disclose construction of a chimeric gene encoding the variable (V) region of a mouse heavy chain specific for the hapten azophenylarsonate and the constant (C) region of a mouse kappa light chain (V^C.,). This gene was introduced into a mouse myeloma cell line. The chimeric gene was expressed to give a

protein which associated with light chains secreted from the myeloma cell line to give an antibody molecule specific for azophenylarsonate.

Morrison, Science £29:1202 (1985), discloses that variable light- or variable heavy-chain regions can be attached to a non-Ig sequence to create fusion proteins. This article states that the potential uses for the fusion proteins are three: (1) to attach antibody specifically to enzymes for use in assays; (2) to isolate non-Ig proteins by antigen columns; and (3) to specifically deliver toxic agents.

Recent techniques for the stable introduction of immunoglobulin genes into myeloma cells (Banerji, J., et al.. Cell 33:729-740 (1983); Potter, H., et al.. Proc. Natl. Acad. Sci. (USA) 81:7161-7165 (1984)), coupled with detailed structural information, have permitted the use of in vitro DNA methods such as utagenesis, to generate recombinant antibodies possessing novel properties.

PCT Application W087/02671 discloses methods for producing genetically engineered antibodies of desired variable region specificity and constant region properties through gene cloning and expression of light and heavy chains. The mRNA from cloned hybridoma B cell lines which produce monoclonal antibodies of desired specificity is isolated for cDNA cloning. The generation of light and heavy chain coding sequences is accomplished by excising the cloned variable regions and ligating them to light or heavy chain module vectors. This gives cDNA sequences which code for immunoglobulin chains. The lack of introns allows these cDNA sequences to be expressed in prokaryotic hosts, such as bacteria, or in lower eukaryotic hosts, such as yeast.

The generation of chimeric antibodies in which the antigen-binding portion of the immunoglobulin is fused to other moieties has been demonstrated. Examples of non- immunoglobul in genes fused to antibodies include

Staphylococcus aureus nuclease, the mouse oncogene c-mvc. and the Klenow fragment of E. coli DNA polymerase I (Neuberger, M.S., et al.. Nature 312:604-612 (1984); Neuberger, M.S., Trends in Biochemical Science. 347-349 (1985)). European Patent Application 120,694 discloses the genetic engineering of the variable and constant regions of an immunoglobulin molecule that is expressed in E. coli host cells. It is further disclosed that the immunoglobulin molecule may be synthesized by a host cell with another peptide moiety attached to one of the constant domains. Such peptide moieties are described as either cytotoxic or enzymatic. The application and the examples describe the use of a lambda-like chain derived from a monoclonal antibody which binds to 4- hydroxy-3-nitrophenyl (NP) haptens. European Patent Application 125,023 relates to the use of recombinant DNA techniques to produce immunoglobulin molecules that are chimeric or otherwise modified. One of the uses described for these immunoglobulin molecules is for whole-body diagnosis and treatment by injection of the antibodies directed to specific target tissues. The presence of the disease can be determined by attaching a suitable label to the antibodies, or the diseased tissue can be attacked by carrying a suitable drug with the antibodies. The application describes antibodies engineered to aid the specific delivery of an agent as "altered antibodies."

PCT Application W083/101533 describes chimeric antibodies wherein the variable region of an immunoglobulin molecule is linked to a portion of a second protein which may comprise the active portion of an enzyme. Boulianne et al.. Nature 312:643 (1984) constructed an immunoglobulin gene in which the DNA segments that encode mouse variable regions specific for the hapten trinitrophenol (TNP) are joined to segments that encode human u and kappa

regions. These chimeric genes were expressed to give functional TNP-binding chimeric IgM.

Morrison et al.. P.N.A.S. (USA) 81:6851 (1984), disclose a chimeric molecule utilizing the heavy-chain variable region exons of an anti-phosphoryl choline myeloma protein G, which were joined to the exons of either human kappa light-chain gene. The genes were transfected into mouse myeloma cell lines, generating transformed cells that produced chimeric mouse-human IgG with antigen-binding function. PCT Application Publication No. W089/02922 (1989), discloses chimeric antibody molecules comprising human CD4. Such chimeric antibody molecules may be administered to a subject infected with HIV to treat the HIV infection.

Despite the progress that has been achieved on determining the mechanism of HIV infection, a need continues to exist for methods of treating HIV viral infections.

SUMMARY OF THE INVENTION

The invention relates to a nucleic acid molecule specifying non-human primate CD4, or an HIV or SIV gpl20 binding fragment thereof.

In particular, the invention relates to a nucleic acid molecule specifying rhesus monkey CD4 comprising the following DNA sequence:

ATGAACCGGGGAATCCCTTTTAGGCACTTGCTTCTGGTGCTGCAACTGGCGCTACTC CCA MetAsnArgGlylleProPheArgHisLeuLeuLeuValLeuGlnLeuAlaLeuLeuPro

GCAGTCACCCAGGGAAAGAAAGTGGTGCTGGGCAAGAAAGGGGATACAGTGGAACTG ACC 120 AlaValThrGlnGlyLysLysValValLeuGlyLysLysGlyAspThrValGluLeuThr 15

TGTACAGCTTCGCAGAAGAAGAACACACAATTCCACTGGAAAAACTCCAACCAGATA AAG CysThrAlaSerGlnLysLysAsnThrGlnPheHisTrpLysAsnSerAsnGlnileLys

ATTCTGGGAATTCAGGGTCTCTTCTTAACTAAAGGTCCATCCAAGCTGAGCGATCGTGCT 240 IleLeuGlylleGlnGlyLeuPheLeuThrLysGlyProSerLysLeuSerAspArgAla 55

241 GACTCAAGAAAAAGCCTrTGGGACCAAGGATGCTTTTCCATGATCATCAAGAATCTTAAG 56 AspSerArgLysSerLeuTrpAspGlnGlyCysPheSerMetllelleLysAsnLeuLys

ATAGAAGACTCAGATACTTACATCTGTGAAGTGGAGAACAAGAAGGAGGAGGTGGAA HG 360

IIeGluAspSerAspThrTyrlleCysGluValGluAsnLysLysGluGluValGlu Leu 95

361 CTGGTGπCGGATTGACTGCCAACTCTGACACCCACCTGCTTGAGGGGCAAAGCCTGACC 96 LeuValPheGl LeuThrAlaAsnSerAspThrHisLeuLeuGluGlyGlnSerLeuThr

CTGACCTTGGAGAGCCCCCCTGGTAGTAGCCCCTCAGTGAAATGTAGGAGTCCAGGG GGT 480 LeuThrLeuGluSerProProGlySerSerProSerValLysCysArgSerProGlyGly 135

481 AAAAACATACAGGGGGGGAGGACCATCTCTGTGCCTCAGCTGGAGCGCCAGGATAGTGGC 136 LysAsnlleGlnGlyGlyArgThrlleSerValProGlnLeuGluArgGlnAspSerGly

ACCTGGACATGCACCGTCTCGCAGGACCAGAAGACGGTGGAGπCAAAATAGACATC GTG 600 ThrTrpThrCysThrValSerGlnAspGlnLysThrValGluPheLysIleAspIleVal 175

601 GTGCTAGCTTTCCAGAAGGCCTCCAGCACAGTCTATAAGAAAGAGGGGGAACAGGTGGAG 176 ValLeuAlaPheGlnLysAlaSerSerThrValTyrLysLysGluGlyGluGlnValGlu

TTCTCCπCCCACTCGCCTTTACACπGAAAAGCTGACGGGCAGTGGCGAGCTGTGG TGG 720 PheSerPheProLeuAlaPheThrLeuGluLysLeuThrGlySerGlyGluLeuTrpTrp 215

721 CAGGCGGAGAGGGCCTCCTCCTCCAAGTCTTGGATTACCTTCGACCTGAAGAACAAGGAA 216 GlnAlaGluArgAlaSerSerSerLysSerTrpIleThrPheAspLeuLysAsnLysGlu

GTGTCTGTAAAACGGGTTACCCAGGACCCCAAGCTCCAGATGGGCAAGAAGCTCCCG CTC 840 ValSerValLysArgValThrGlnAspProLysLeuGlnMetGlyLysLysLeuProLeu 255

841 CACCTCACCCTGCCCCAGGCCTTGCCTCAGTATGCTGGCTCTGGAAACCTCACGCTGGCC 256 HisLeuThrLeuProGlnAlaLeuProGlnTyrAlaGlySerGlyAsnLeuThrLeuAla

CTTGAAGCGAAAACAGGAAAGTTGCATCAGGAAGTGAACCTCGTGGTGATGAGAGCC ACT 960

LeuGluAlaLysThrGlyLysLeuHisGlnGluValAsnLeuValValMetArgAla Thr 295

961 CAGTTCCAGGAAAATTTGACCTGTGAAGTGTGGGGACCCACCTCCCCTAAGCTGACGCTG 296 GlnPheGlnGluAsnLeuThrCysGluValTrpGlyProThrSerProLysLeuThrLeu

AGCTTGAAACTGGAGAACAAGGGGGCAACGGTCTCGAAGCAGGCGAAGGCGGTGTGG GTG 1080 SerLeuLysLeuGluAsnLysGlyAlaThrValSerLysGlnAlaLysAlaValTrpVal 335

1081 CTGAACCCTGAGGCGGGGATGTGGCAGTGTCTGCTGAGTGACTCGGGACAGGTCCTGCTA 336 LeuAsnProGluAlaGlyMetTrpGlnCysLeuLeuSerAspSerGlyGlnValLeuLeu

GAATCCAACATCAAGGTTGTGCCCACATGGCCCACCCCGGTGCAGCCAATGGCCCTG ATT 1200

GluSerAsnIIeLysValValProThrTrpProThrProValGlnProMetAlaLeu Ile 375

1201 GTGCTGGGGGGCGTTGCGGGCCTCCTGCTTTTCACTGGGCTAGGCATCTTCTTCTGTGTC 376 ValLeuGlyGlyValAlaGlyLeuLeuLeuPheThrGlyLeuGlyllePhePheCysVal

AGGTGCCGGCATCGAAGGCGTCAAGCAGAGCGGATGTCTCAGATCAAGAGACTCCTC AGT 1320 ArgCysArgHisArgArgArgGlnAlaGluArgMetSerGlnileLysArgLeuLeuSer 415

1321 GAAAAGAAGACCTGCCAGTGCCCTCACCGGTTTCAGAAGACATGTAGCCCCATTTGA 1377 416 GlULysLysThrCysGlnCysProHisArgPheGlnLysThrCysSerProIleEnd 433

or a degenerate variant thereof.

The invention also relates to a nucleic acid molecule specifying a soluble non-human primate CD4 fragment. In particular, the invention to a soluble rhesus CD4 fragment

(domain I) which binds HIV or SIV gpl20 comprising the following DNA sequence:

1 ATGAACCGGGGAATCCCTTTTAGGCACTTGCTTCTGGTGCTGCAACTGGCGCTACTCCCA -25 MetAsnArgGl lleProPheArgHisLeuLeuLeuValLeuGlnLeuAlaLeuLeuPro

GCAGTCACCCAGGGAAAGAAAGTGGTGCTGGGCAAGAAAGGGGATACAGTGGAACTG ACC 120 AlaValThrGlnGlyLysLysValValLeuGlyLysLysGlyAspThrValGluLeuThr 15

121 TGTACAGCπCGCAGAAGAAGAACACAC AATTCCACTGGAAAAACTCCAACCAGATAAAG 16 CysThrAl aSerGl nLysLysAsnThrGl nPheHi sTrpLysAsnSerAsnGl nil eLys

AπCTGGGAATTCAGGGTCTCTTCTTAACTAAAGGTCCATCCAAGCTGAGCGATCGT GCT 240 IleLeuGlylleGlnGlyLeuPheLeuThrLysGlyProSerLysLeuSerAspArgAla 55

241 GACTCAAGAAAAAGCCTTTGGGACCAAGGATGCTTTTCCATGATCATCAAGAATCTTAAG 56 AspSerArgLysSerLeuTrpAspGlnGlyCysPheSerMetllelleLysAsnLeuLys

ATAGAAGACTCAGATACTTACATCTGTGAAGTGGAGAACAAGAAGGAGGAGGTGGAA TTG 360 IIeGluAspSerAspThrTyrlleCysGluValGluAsnLysLysGluGluValGluLeu 95

361 CTGGTGTTCGGATTGACTGCCAACTCTGACACCCACCTGCTT 96 LeuValPheGlyLeuThrAlaAsnSerAspThrHisLeuLeu

or a degenerate variant thereof.

The invention also relates to a nucleic acid molecule specifying chimpanzee CD4, comprising the following DNA sequence:

1 ATGAACCGGGGAGTCCCTTTTAGGCACHGCTTCTGGTGCTGCAACTGGCACTCCTCCCA -25 MetAsnArgGlyValProPheArgHisLeuLeuLeuValLeuGlnLeuAlaLeuLeuPro

GCAGCCACTCAGGGAAAGAAAGTGGTGCTGGGCAAGAAAGGGGACACAGTGGAACTG ACC 120 AlaAlaThrGlnGlyLysLysValValLeuGlyLysLysGlyAspThrValGluLeuThr 15

121 TGTACAGCTTCCCAGAAGAAGAGCATACAATTCCACTGGAAAAACTCCAACCAGACAAAG 16 CysThrAlaSerGlnLysLysSerIIeGlnPheHisTrpLysAsnSerAsnGlnThrLys

ATTCTGGGAAATCAGGGCTCCHCTTAACTAAAGGTCCATCCAAGCTGAATGATCGCG TT 240 IleLeuGlyAsnGlnGlySerPheLeuThrLysGlyProSerLysLeuAsnAspArgVal 55

41 GACTCAAGAAGAAGCCTTTGGGACCAAGGAAACTTTACCCTGATCATCAAGAATCTTAAG 56 AspSerArgArgSerLeuTrpAspGlnGlyAsnPheThrLeuIlelleLysAsnLeuLys

ATAGAAGACTCAGATACTTACATCTGTGAAGTGGGGGACCAGAAGGAGGAGGTGCAATTG 360 IIeGluAspSerAspThrTyrlleCysGluValGlyAspGlnLysGluGluValGlnLeu 95

361 CTAGTGTTCGGATTGACTGCCAACTCTGACACCCACCTGCTTCAGGGGCAGAGCCTGACC 96 LeuValPheGlyLeuThrAlaAsnSerAspThrHisLeuLeuGlnGlyGlnSerLeuThr

CTGACCTTGGAGAGCCCCCCTGGTAGTAGCCCCTCAGTGCAATGTAGGAGTCCAAGG GGT 360 LeuThrLeuGluSerProProGlySerSerProSerValGlnCysArgSerProArgGly 135

481 AAAAACATACAGGGGGGGAAGACCCTCTCCGTGTCTCAGCTGGAGCTCCAGGATAGTGGC 136 LysAsnlleGlnGlyGlyLysThrLeuSerValSerGlnLeuGluLeuGlnAspSerGly

ACCTGGACATGCACTGTCTTGCAGAACCAGAAGAAAGTGGAGTTCAAAATAGACATC GTG 600 ThrTrpThrCysThrValLeuGlnAsnGlnLysLysValGluPheLysIleAspIleVal 175

601 GTGCTAGCTTTCCAGAAGGCCTCCAGCATAGTCTATAAGAAAGAGGGGGAACAGGTGGAG 176 ValLeuAlaPheGlnLysAlaSerSerlleValTyrLysLysGluGlyGluGlnValGlu

TTCTCCTTCCCACTCGCCTTTACAGTTGAAAAGCTGACGGGCAGTGGCGAGCTGTGG TGG 720 PheSerPheProLeuAlaPheThrValGluLysLeuThrGlySerGlyGluLeuTrpTrp 215

721 CAGGCGGAGAGGGCTTCCTCCTCCAAGTCTTGGATCACCTTTGACCTGAAGAACAAGGAA 216 GlnAlaGluArgAlaSerSerSerLysSerTrpIleThrPheAspLeuLysAsnLysGlu

GTGTCTGTAAAACGGGTTACCCAGGACCCTAAGCTCCAGATGGGCAAGAAGCTCCCG CTC 840 ValSerValLysArgValThrGlnAspProLysLeuGlnMetGlyLysLysLeuProLeu 255

841 CACCTCACCCTGCCCCAGGCCTTGCCTCAGTATGCTGGCTCTGGAAACCTCACCCTGGCC 256 HisLeuThrLeuProGlnAlaLeuProGlnTyrAlaGlySerGlyAsnLeuThrLeuAla

CTTGAAGCGAAAACAGGAAAGTTGCATCAGGAAGTGAACCTCGTGGTGATGAGAGCC ACT 840 LeuGluAlaLysThrGlyLysLeuHisGlnGluValAsnLeuValValMetArgAlaThr 295

961 CAGCTCCAGAAAAATTTGACCTGTGAGGTGTGGGGACCCACCTCCCCTAAGCTGATGCTG 296 GlnLeuGlnLysAsnLeuThrCysGluValTrpGlyProThrSerProLysLeuMetLeu

AGCTTGAAACTGGAGAACAAGGAGGCAAAGGTCTCGAAGCGGGAGAAGGCGGTGTGG GTG 1080 SerLeuLysLeuGluAsnLysGluAlaLysValSerLysArgGluLysAlaValTrpVal 335

-12-

1081 CTGAACCCTGAGGCGGGGATGTGGCAGTGTCTGCTGAGTGACTCGGGACAGGTCCTGCTG 336 LeuAsnProGluAlaGlyMetTrpGlnCysLeuLeuSerAspSerGlyGlnValLeuLeu

GAATCCAACATCAAGGTTCTGCCCACATGGTCCACCCCGGTGCAGCCAATGGCCCTG ATT 1200 GluSerAsnlleLysValLeuProThrTrpSerThrProValGlnProMetAlaLeuIle 375

1201 GTGCTGGGGGGCGTCGCCGGCCTCCTGCTTTTCATTGGGCTAGGCATCTTCTTCTGTGTC 376 ValLeuGlyGlyValAlaGlyLeuLeuLeuPhelleGlyLeuGlyllePhePheCysVal

AGGTGCCGGCACCGAAGGCGCCAAGCACAGCGGATGTCTCAGATCAAGAGACTCCTC AGT 1320 ArgCysArgHisArgArgArgGlnAlaGlnArgMetSerGlnileLysArgLeuLeuSer 415

1321 GAGAAGAAGACCTGCCAGTGCCCTCACCGGTπCAGAAGACATGTAGCCCCATTTGA 1377 416 GluLysLysThrCysGlnCysProHisArgPheGlnLysThrCysSerProIleEnd 433 or a degenerate variant thereof.

The invention also relates to a nucleic acid molecule specifying a soluble chimpanzee CD4 fragment (domain I) which binds HIV or SIV gpl20, comprising the following DNA sequence:

1 ATGAACCGGGGAGTCCCTTTTAGGCACHGCTTCTGGTGCTGCAACTGGCACTCCTCCCA -25 MetAsnArgGlyValProPheArgHisLeuLeuLeuValLeuGlnLeuAlaLeuLeuPro

GCAGCCACTCAGGGAAAGAAAGTGGTGCTGGGCAAGAAAGGGGACACAGTGGAACTG ACC 120 AlaAlaThrGlnGlyLysLysValValLeuGlyLysLysGlyAspThrValGluLeuThr 15

121 TGTACAGCTTCCCAGAAGAAGAGCATACAAπCCACTGGAAAAACTCCAACCAGACAAAG 16 CysThrAlaSerGlnLysLysSerlleGlnPheHisTrpLysAsnSerAsnGlnThrLys

AHCTGGGAAATCAGGGCTCCTTCTTAACTAAAGGTCCATCCAAGCTGAATGATCGCG TT 240 IIeLeuGlyAsnGlnGlySerPheLeuThrLysGlyProSerLysLeuAsnAspArgVal 55

241 GACTCAAGAAGAAGCCTTTGGGACCAAGGAAACTTTACCCTGATCATCAAGAATCTTAAG 56 AspSerArgArgSerLeuTrpAspGlnGlyAsnPheThrLeuIlelleLysAsnLeuLys

ATAGAAGACTCAGATACTTACATCTGTGAAGTGGGGGACCAGAAGGAGGAGGTGCAATTG 360 IIeGluAspSerAspThrTyrlleCysGluValGlyAspGlnLysGluGluValGlnLeu 95

361 CTAGTGTTCGGATTGACTGCCAACTCTGACACCCACCTGCπ 96 LeuValPheGlyLeuThrAlaAsnSerAspThrHisLeuLeu or a degenerate variant thereof.

The invention also relates to a nucleic acid molecule specifying chimpanzee CD4 with the cytoplasmic domain, comprising the following DNA sequence:

1 ATGAACCGGGGAGTCCCTTTTAGGCACTTGCTTCTGGTGCTGCAACTGGCACTCCTCCCA ^■ 25 MetAsnArgGl ValProPheArgHisLeuLeuLeuValLeuGlnLeuAlaLeuLeuPro

GCAGCCACTCAGGGAAAGAAAGTGGTGCTGGGCAAGAAAGGGGACACAGTGGAACTG ACC 120

AlaAlaThrGlnGlyLysLysValValLeuGlyLysLysGlyAspThrValGluLeu Thr 15

121 TGTACAGCTTCCCAGAAGAAGAGCATACAATTCCACTGGAAAAACTCCAACCAGAYAAAG 16 CysThrAlaSerGlnLysLysSerlleGlnPheHisTrpLysAsnSerAsnGlnThrLys

He

ATTCTGGGAAATCAGGGCTCCTTCTTAACTAAAGGTCCATCCAAGCTGAATGATCGC GYT 240 IleLeuGlyAsnGlnGlySerPheLeuThrLysGlyProSerLysLeuAsnAspArgVal 55 Ala

241 GACTCAAGAAGAAGCCTTTGGGACCAAGGAAACTTTMCCCTGATCATCAAGAATCTTAAG 56 AspSerArgArgSerLeuTrpAspGlnGlyAsnPheThrLeuIlelleLysAsnLeuLys

Pro

ATAGAAGACTCAGATACTTACATCTGTGAAGTGGGGGACCAGAAGGAGGAGGTGCAA TTG 360 IIeGluAspSerAspThrTyrI1eCysGluValGlyAspGlnLysGluGluValGlnLeu 95

361 CTAGTGTTCGGATTGACTGCCAACTCTGACACCCACCTGCTTCAGGGGCAGAGCCTGACC 96 LeuValPheGlyLeuThrAlaAsnSerAspThrHisLeuLeuGlnGlyGlnSerLeuThr

CTGACCTTGGAGAGCCCCCCTGGTAGTAGCCCCTCAGTGCAATGTAGGAGTCCAAGG GGT 360 LeuThrLeuGlUSerProProGlySerSerProSerValGlnCysArgSerProArgGly 135

481 AAAAACATACAGGGGGGGAAGACCCTCTCCGTGTCTCAGCTGGAGCTCCAGGATAGTGGC 136 LysAsnlleGlnGlyGlyLysThrLeuSerValSerGlnLeuGluLeuGlnAspSerGly

ACCTGGACATGCACTGTCTTGCAGAACCAGAAGAAAGTGGAGTTCAAAATAGACATC GTG 600 ThrTrpThrCysThrValLeuGlnAsnGlnLysLysValGluPheLysIleAspIleVal 175

601 GTGCTAGCTTTCCAGAAGGCCTCCAGCATAGTCTATAAGAAAGAGGGGGAACAGGTGGAG 176 ValLeuAlaPheGlnLysAlaSerSerlleValTyrLysLysGluGlyGluGlnValGlu

TTCTCCπCCCACTCGCCTTTACAGTTGAAAAGCTGACGGGCAGTGGCGAGCTGTGG TGG 720 PheSerPheProLeuAlaPheThrValGluLysLeuThrGlySerGlyGluLeuTrpTrp 215

721 CAGGCGGAGAGGGCπCCTCCTCCAAGTCTTGGATCACCTTTGACCTGAAGAACAAGGAA 216 GlnAlaGluArgAlaSerSerSerLysSerTrpIleThrPheAspLeuLysAsnLysGlu

GTGTCTGTAAAACGGGTTACCCAGGACCCTAAGCTCCAGATGGGCAAGAAGCTCCCG CTC 840 ValSerValLysArgValThrGlnAspProLysLeuGlnMetGlyLysLysLeuProLeu 255

841 CACCTCACCCTGCCCCAGGCCHGCCTCAGTATGCTGGCTCTGGAAACCTCACCCTGGCC 256 HisLeuThrLeuProGlnAlaLeuProGlnTyrAlaGlySerGlyAsnLeuThrLeuAl

CπGAAGCGAAAACAGGAAAGTTGCATCAGGAAGTGAACCTCGTGGTGATGAGAGCC ACT 840

LeuGluAlaLysThrGlyLysLeuHisGlnGluValAsnLeuValValMetArgAla Thr 295

961 CAGCTCCAGAAAAAHTGACCTGTGAGGTGTGGGGACCCACCTCCCCTAAGCTGATGCTG 296 GlnLeuGlnLysAsnLeuThrCysGluValTrpGlyProThrSerProLysLeuMetLeu

AGCHGAAACTGGAGAACAAGGAGGCAAAGGTCTCGAAGCGGGAGAAGGCGGTGTGGG TG 1080 SerLeuLysLeuGluAsnLysGluAlaLysValSerLysArgGluLysAl ValTrpVal 335

1081 CTGAACCCTGAGGCGGGGATGTGGCAGTGTCTGCTGAGTGACTCGGGACAGGTCCTGCTG 336 LeuAsnProGluAlaGlyMetTrpGlnCysLeuLeuSerAspSerGlyGlnValLeuLeu

GAATCCAACATCAAGGTTCTGCCCACATGGTCCACCCCGGTGCAGCCAATGGCCCTG ATT 1200 GluSerAsnlleLysValLeuProThrTrpSerThrProValGlnProMetAlaLeuIle 375

1201 GTGCTGGGGGGCGTCGCCGGCCTCCTGCTTTTCATTGGGCTAGGCATCTTCTTCTGTGTC 376 ValLeuGlyGlyValAlaGlyLeuLeuLeuPhelleGlyLeuGlyllePhePheCysVal

AGGTGCCGGCACCGAAGGCGCCAAGCASAGCGGATGTCTCAGATCAAGAGACTCCTC AGT 1320 ArgCysArgHisArgArgArgGlnAlaGlnArgMetSerGlnileLysArgLeuLeuSer 415 Glu

1321 GAGAAGAAGACCTGCCAGTGCCCTCACCGGTTTCAGAAGACATGTAGCCCCATTTGA 1377 416 GluLysLysThrCysGlnCysProHisArgPheGlnLysThrCysSerProIleEnd 433

wherein Y is C or T,

M is A or C, and

S is C or G; or a degenerate variant thereof.

The invention also relates to a nucleic acid molecule specifying a chimpanzee CD4 fragment, comprising the following DNA sequence:

1 ATGAACCGGGGAGTCCCTTTTAGGCACTTGCTTCTGGTGCTGCAACTGGCACTCCTCCCA -25 MetAsnArgGlyValProPheArgHisLeuLeuLeuValLeuGlnLeuAlaLeuLeuPro

GCAGCCACTCAGGGAAAGAAAGTGGTGCTGGGCAAGAAAGGGGACACAGTGGAACTG ACC 120

AlaAlaThrGlnGlyLysLysValValLeuGlyLysLysGlyAspThrValGluLeu Thr 15

121 TGTACAGCTTCCCAGAAGAAGAGCATACAATTCCACTGGAAAAACTCCAACCAGAYAAAG 16 CysThrAlaSerGlnLysLysSerlleGlnPheHisTrpLysAsnSerAsnGlnThrLys

He

ATTCTGGGAAATCAGGGCTCCTTCTTAACTAAAGGTCCATCCAAGCTGAATGATCGC GYT 240 IIeLeuGlyAsnGlnGlySerPheLeuThrLysGlyProSerLysLeuAsnAspArgVal 55 Ala

241 GACTCAAGAAGAAGCCTTTGGGACCAAGGAAACHTMCCCTGATCATCAAGAATCTTAAG 56 AspSerArgArgSerLeuTrpAspGl nGlyAsnPheThrLeuIl ell eLysAsnLeuLys

Pro

ATAGAAGACTCAGATACTTACATCTGTGAAGTGGGGGACCAGAAGGAGGAGGTGCAA TTG 360 IIeGluAspSerAspThrTyrlleCysGluValGlyAspGlnLysGluGluValGlnLeu 95

361 CTAGTGTTCGGATTGACTGCCAACTCTGACACCCACCTGCTT 96 LeuValPheGlyLeuThrAlaAsnSerAspThrHisLeuLeu

wherein Y is C or T, and M is A or C; or a degenerate variant thereof.

The invention also relates to a nucleic acid molecule specifying a gpl20 binding molecule capable of glycosylation which is related to human CD4 with the cytoplasmic domain, comprising the following DNA sequence:

1 ATGAACCGGGGAGTCCCTTTTAGGCACHGCHCTGGTGCTGCAACTGGCGCTCCTCCCA -25 MetAsnArgGl ValProPheArgHisLeuLeuLeuValLeuGlnLeuAlaLeuLeuPro

GCAGCCACTCAGGGAAAGAAAGTGGTGCTGGGCAAAAAAGGGGATACAGTGGAACTG ACC 120 AlaAlaThrGlnGlyLysLysValValLeuGlyLysLysGlyAspThrValGluLeuThr 15

121 TGTACAGCπCCCAGAAGAAGAGCATACAATTCCACTGGAAAAACTCCAACCAGAYAAAG 16 CysThrAlaSerGlnLysLysSerlleGlnPheHisTrpLysAsnSerAsnGlnThrLys

He

ATTCTGGGAAATCAGGGCTCCTTCTTAACTAAAGGTCCATCCAAGCTGAATGATCGC GCT 240 H eLeuGlyAsnGl nGlySerPheLeuThrLysGlyProSerLysLeuAsnAspArgAl a 55

241 GACTCAAGAAGAAGCCTπGGGACCAAGGAAACπCMCCCTGATCATCAAGAATCπAAG 56 AspSerArgArgSerLeuTrpAspGl nGlyAsnPheThrLeuIl ell eLysAsnLeuLys Pro

ATAGAAGACTCAGATACTTACATCTGTGAAGTGGAGGACCAGAAGGAGGAGGTGCAA TTG 360 II eGl uAspSerAspThrTyrll eCysGl uVal Gl uAspGl nLysGl uGl uVal Gl nLeu 95

361 CTAGTGTTCGGATTGACTGCCAACTCTGACACCCACCTGCTTCAGGGGCAGAGCCTGACC 96 LeuValPheGl LeuThrAlaAsnSerAspThrHisLeuLeuGlnGlyGlnSerLeuThr

CTGACCTTGGAGAGCCCCCCTGGTAGTAGCCCCTCAGTGCAATGTAGGAGTCCAAGG GGT 360 LeuThrLeuGlUSerProProGlySerSerProSerValGlnCysArgSerProArgGly 135

481 AAAAACATACAGGGGGGGAAGACCCTCTCCGTGTCTCAGCTGGAGCTCCAGGATAGTGGC 136 LysAsnlleGlnGlyGlyLysThrLeuSerValSerGlnLeuGluLeuGlnAspSerGly

ACCTGGACATGCACTGTCTTGCAGAACCAGAAGAAGGTGGAGTTCAAAATAGACATC GTG 600 ThrTrpThrCysThrValLeuGlnAsnGlnLysLysValGluPheLysIIeAspIleVal 175

601 GTGCTAGCTTTCCAGAAGGCCTCCAGCATAGTCTATAAGAAAGAGGGGGAACAGGTGGAG 176 ValLeuAlaPheGlnLysAlaSerSerlleValTyrLysLysGluGlyGluGlnValGlu

πCTCCTTCCCACTCGCCTTTACAGTTGAAAAGCTGACGGGCAGTGGCGAGCTGTGG TGG 720 PheSerPheProLeuAlaPheThrValGluLysLeuThrGlySerGlyGluLeuTrpTrp 215

721 CAGGCGGAGAGGGCTTCCTCCTCCAAGTCTTGGATCACCTTTGACCTGAAGAACAAGGAA 216 GlnAlaGluArgAlaSerSerSerLysSerTrpIleThrPheAspLeuLysAsnLysGlu

GTGTCTGTAAAACGGGTTACCCAGGACCCTAAGCTCCAGATGGGCAAGAAGCTCCCG CTC 840 ValSerValLysArgValThrGlnAspProLysLeuGlnMetGlyLysLysLeuProLeu 255

841 CACCTCACCCTGCCCCAGGCCTTGCCTCAGTATGCTGGCTCTGGAAACCTCACCCTGGCC 256 HisLeuThrLeuProGlnAlaLeuProGlnTyrAlaGl SerGlyAsnLeuThrLeuAl

CTTGAAGCGAAAACAGGAAAGTTGCATCAGGAAGTGAACCTGGTGGTGATGAGAGCC ACT 840 LeuGluAlaLysThrGlyLysLeuHisGlnGluValAsnLeuValValMetArgAlaThr 295

961 CAGCTCCAGAAAAATTTGACCTGTGAGGTGTGGGGACCCACCTCCCCTAAGCTGATGCTG 296 GlnLeuGlnLysAsnLeuThrCysGluValTrpGlyProThrSerProLysLeuMetLeu

AGCTTGAAACTGGAGAACAAGGAGGCAAAGGTCTCGAAGCGGGAGAAGGCGGTGTGG GTG 1080 SerLeuLysLeuGluAsnLysGluAlaLysValSerLysArgGluLysAlaValTrpVal 335

1081 CTGAACCCTGAGGCGGGGATGTGGCAGTGTCTGCTGAGTGACTCGGGACAGGTCCTGCTG 336 LeuAsnProGluAlaGlyMetTrpGlnCysLeuLeuSerAspSerGlyGlnValLeuLeu

GAATCCAACATCAAGGTTCTGCCCACATGGTCCACCCCGGTGCAGCCAATGGCCCTG ATT 1200 GluSerAsnlleLysValLeuProThrTrpSerThrProValGlnProMetAlaLeuIle 375

1201 GTGCTGGGGGGCGTCGCCGGCCTCCTGCTπTCAπGGGCTAGGCATCTTCTTCTGTGTC 376 ValLeuGlyGlyValAlaGlyLeuLeuLeuPhelleGlyLeuGlyllePhePheCysVal

AGGTGCCGGCACCGAAGGCGCCAAGCAGAGCGGATGTCTCAGATCAAGAGACTCCTC AGT 1320 ArgCysArgHisArgArgArgGlnAlaGluArgMetSerGlnileLysArgLeuLeuSer 415

1321 GAGAAGAAGACCTGCCAGTGCCCTCACCGGTTTCAGAAGACATGTAGCCCCATTTGA 1377 416 GluLysLysThrCysGlnCysProHisArgPheGlnLysThrCysSerProIleEnd 433

wherein Y is C or T, and

M is A or C; or a degenerate variant thereof; with the proviso that both Y is not T and M is not C at the same time.

The invention also relates to a nucleic acid molecule specifying a gpl20 binding molecule capable of glycosylation which is related to a human CD4 fragment, comprising the following DNA sequence:

1 ATGAACCGGGGAGTCCCππAGGCACπGCTTCTGGTGCTGCAACTGGCGCTCCTCCCA -25 MetAsnArgGlyValProPheArgHisLeuLeuLeuValLeuGlnLeuAlaLeuLeuPro

GCAGCCACTCAGGGAAAGAAAGTGGTGCTGGGCAAAAAAGGGGATACAGTGGAACTG ACC 120 AlaAlaThrGlnGlyLysLysValValLeuGlyLysLysGlyAspThrValGluLeuThr 15

121 TGTACAGCTTCCCAGAAGAAGAGCATACAATTCCACTGGAAAAACTCCAACCAGAYAAAG 16 CysThrAlaSerGlnLysLysSerIIeGlnPheHisTrpLysAsnSerAsnGlnThrLys

He

ATTCTGGGAAATCAGGGCTCCTTCTTAACTAAAGGTCCATCCAAGCTGAATGATCGC GCT 240 IIeLeuGlyAsnGlnGlySerPheLeuThrLysGlyProSerLysLeuAsnAspArgAla 55

241 GACTCAAGAAGAAGCCTTTGGGACCAAGGAAACTTCMCCCTGATCATCAAGAATCTTAAG 56 AspSerArgArgSerLeuTrpAspGlnGlyAsnPheThrLeuIlelleLysAsnLeuLys

Pro

ATAGAAGACTCAGATACTTACATCTGTGAAGTGGAGGACCAGAAGGAGGAGGTGCAA TTG 360 IIeGlUAspSerAspThrTyrlleCysGluValGluAspGlnLysGluGluValGlnLeu 95

361 CTAGTGTTCGGATTGACTGCCAACTCTGACACCCACCTGCTT 96 LeuValPheGlyLeuThrAlaAsnSerAspThrHisLeuLeu

wherein Y is C or T, and

M is A or C; or a degenerate variant thereof; with the proviso that both Y is not T and M is not C at the same time.

The invention also relates to a nucleic acid molecule specifying a fusion protein, comprising 1) a nucleic acid molecule specifying non-human primate CD4 or fragment thereof which binds HIV or SIV gpl20, and 2) a nucleic acid molecule specifying an immunoglobulin light or heavy chain, wherein the nucleic acid molecule which specifies the variable region of said immunoglobulin chain has been replaced with the nucleic acid molecule specifying said non-human primate CD4 or fragment thereof.

The invention also relates to a nucleic acid molecule specifying a fusion protein, comprising

1) a nucleic acid molecule specifying non-human primate CD4, or fragment thereof which binds HIV or SIV gpl20, linked to

2) a nucleic acid molecule specifying a cytotoxic polypeptide.

The invention also relates to vectors comprising the nucleic acid molecules of the invention.

The invention also relates to hosts transformed with the vectors of the invention. In particular, the invention relates to hosts which express complementary immunoglobulin light or heavy chains together with the expression product of said fusion protein nucleic acid molecule to give an immunoglobulin-like molecule which binds to HIV or SIV gpl20. The invention also relates to methods of producing non- human primate CD4, or fragment thereof which binds to HIV or SIV gpl20, which comprises cultivating in a nutrient medium under protein- producing conditions, a host strain transformed with a vector containing a nucleic acid molecule specifying a non-human primate CD4 or soluble fragment thereof which binds HIV or SIV gpl20, said vector further comprising expression signals which are recognized by said host strain and direct expression of said non-human primate CD4 or fragment thereof, and recovering the non-human primate CD4 or soluble fragment thereof so produced.

The invention also relates to a method of producing a fusion protein comprising non-human primate CD4, or fragment thereof which binds to gpl20, and an immunoglobulin light or heavy chain, wherein the variable region of the immunoglobulin chain has been substituted with non-human primate CD4, or fragment thereof which binds to HIV or SIV gpl20, which comprises

cultivating in a nutrient medium under protein- producing conditions, a host strain transformed with a vector specifying said fusion protein, said vector further comprising expression signals which are recognized by said host strain and direct expression of said fusion protein, and recovering the fusion protein so produced. In particular, the invention relates to a method of preparing a immunoglobulin-like molecule, wherein said host strain is a myeloma cell line which produces immunoglobulin light chains and said fusion protein comprises an immunoglobulin heavy chain of the class IgM, IgGl or IgG3, wherein an immunoglobulin-like molecule comprising said fusion protein is produced. The invention also relates to a method of preparing an immunoglobulin-like molecule, wherein said host produces immunoglobulin heavy chains of the class IgM, IgGl and IgG3 together with said fusion protein comprising an immunoglobulin light chain to give an immunoglobulin-like molecule which binds to HIV or SIV gpl20. The invention also relates to substantially pure non- human primate CD4. In particular, the invention relates to substantially pure rhesus CD4 comprising the following amino acid sequence:

MetAsnArgGlylleProPheArgHisLeuLeuLeuValLeuGlnLeuAlaLeuLeu Pro AlaValThrGlnGlyLysLysValValLeuGlyLysLysGlyAspThrValGluLeuThr CysThrAlaSerGlnLysLysAsnThrGlnPheHisTrpLysAsnSerAsnGlnileLys HeLeuGlylleGlnGlyLeuPheLeuThrLysGlyProSerLysLeuSerAspArgAla AspSerArgLysSerLeuTrpAspGlnGlyCysPheSerMetllelleLysAsnLeuLys IIeGluAspSerAspThrTyrlleCysGluValGluAsnLysLysGluGluValGluLeu LeuValPheGlyLeuThrAlaAsnSerAspThrHisLeuLeuGluGlyGlnSerLeuThr LeuThrLeuGlUSerProProGlySerSerProSerValLysCysArgSerProGlyGly LysAsnlleGlnGlyGlyArgThrlleSerValProGlnLeuGluArgGlnAspSerGly ThrTrpThrC sThrValSerGlnAspGlnLysThrValGluPheLysIIeAspIIeVal

ValLeuAlaPheGlnLysAlaSerSerThrValTyrLysLysGluGlyGluGlnValGlu PheSerPheProLeuAlaPheThrLeuGluLysLeuThrGlySerGlyGluLeuTrpTrp Gl AlaGluArgAlaSerSerSerLysSerTrpHeThrPheAspLeuLysAsnLysGlu ValSerValLysArgValThrGlnAspProLysLeuGlnMetGlyLysLysLeuProLeu HisLeuThrLeuProGlnAlaLeuProGlnTyrAlaGlySerGlyAsnLeuThrLeuAla LeuGluAlaLysThrGlyLysLeuHisGlnGluValAsnLeuValValMetArgAlaThr GlnPheGlnGluAsnLeuThrCysGluValTrpGlyProThrSerProLysLeuThrLeu SerLeuLysLeuGluAsnLysGlyAlaThrValSerLysGlnAlaLysAlaValTrpVal LeuAsnProGluAlaGlyMetTrpGlnCysLeuLeuSerAspSerGlyGlnValLeuLeu GluSerAsnIIeLysValValProThrTrpProThrProValGlnProMetAlaLeuIle ValLeuGlyGlyValAlaGlyLeuLeuLeuPheThrGlyLeuGlyllePhePheCysVal ArgCysArgHisArgArgArgGlnAlaGluArgMetSerGlnileLysArgLeuLeuSer GluLysLysThrCysGlnCysProHisArgPheGlnLysThrCysSerProIle.

The invention also relates to substantially pure chimpanzee CD4 comprising the following amino acid sequence:

MetAsnArgGlyValProPheArgHisLeuLeuLeuValLeuGlnLeuAlaLeuLeu Pro AlaAlaThrGlnGlyLysLysValValLeuGl LysLysGl AspThrValGluLeuThr CysThrAlaSerGlnLysLysSerlleGlnPheHisTrpLysAsnSerAsnGlnThrLys HeLeuGlyAsnGlnGlySerPheLeuThrLysGlyProSerLysLeuAsnAspArgVal AspSerArgArgSerLeuTrpAspGlnGlyAsnPheThrLeuIlelleLysAsnLeuLys IIeGluAspSerAspThrTyrlleCysGluValGlyAspGlnLysGluGluValGlnLeu LeuValPheGlyLeuThrAlaAsnSerAspThrHisLeuLeuGlnGl GlnSerLeuThr LeuThrLeuGlUSerProProGlySerSerProSerValGlnCysArgSerProArgGl LysAsnHeGlnGlyGlyLysThrLeuSerValSerGlnLeuGluLeuGlnAspSerGl ThrTrpThrCysThrValLeuGlnAsnGlnLysLysValGluPheLysIIeAspHeVal ValLeuAlaPheGlnLysAlaSerSerlleValTyrLysLysGluGlyGluGlnValGlu PheSerPheProLeuAlaPheThrValGlULysLeuThrGlySerGlyGluLeuTrpTrp GlnAlaGluArgAlaSerSerSerLysSerTrpHeThrPheAspLeuLysAsnLysGlu ValSerValLysArgValThrGlnAspProLysLeuGlnMetGlyLysLysLeuProLeu HisLeuThrLeuProGlnAlaLeuProGlnTyrAlaGlySerGlyAsnLeuThrLeuAla LeuGluAlaLysThrGlyLysLeuHisGlnGluValAsnLeuValValMetArgAlaThr GlnLeuGlnLysAsnLeuThrCysGluValTrpGlyProThrSerProLysLeuMetLeu

SerLeuLysLeuGluAsnLysGluAlaLysValSerLysArgGluLysAlaValTrpVal LeuAsnProGluAlaGlyMetTrpGlnCysLeuLeuSerAspSerGlyGlnValLeuLeu GluSerAsnlleLysValLeuProThrTrpSerThrProValGlnProMetAlaLeuIle ValLeuGlyGlyValAlaGlyLeuLeuLeuPhelleGlyLeuGlyllePhePheCysVal ArgCysArgHisArgArgArgGlnAlaGlnArgMetSerGlnIIeLysArgLeuLeuSer GluLysLysThrCysGlnCysProHisArgPheGlnLysThrCysSerProIle; or the glycosylated derivative thereof.

The invention also relates to a substantially pure non- human CD4 molecule comprising the following amino acid sequence:

MetAsnArgGl ValProPheArgHisLeuLeuLeuValLeuGlnLeuAlaLeuLeuPro AlaAlaThrGlnGlyLysLysValValLeuGlyLysLysGlyAspThrValGluLeuThr CysThrAlaSerGlnLysLysSerlleGlnPheHiSTrpLysAsnSerAsnGln-«?-L ys IIeLeuGlyAsnGlnGlySerPheLeuThrLysGlyProSerLysLeuAsnAspArg-#- AspSerArgArgSerLeuTrpAspGlnGlyAsnPhe-S-LeuIlelleLysAsnLeuLys IIeGluAspSerAspThrTyrlleCysGluValGlyAspGlnLysGluGluValGlnLeu LeuValPheGlyLeuThrAlaAsnSerAspThrHisLeuLeuGlnGlyGlnSerLeuThr LeuThrLeuGlUSerProProGlySerSerProSerValGlnCysArgSerProArgGly LysAsnlleGlnGlyGlyLysThrLeuSerValSerGlnLeuGluLeuGlnAspSerGly ThrTrpThrCysThrValLeuGlnAsnGlnLysLysValGluPheLysIIeAspHeVal ValLeuAlaPheGlnLysAlaSerSerlleValTyrLysLysGluGlyGluGlnValGlu PheSerPheProLeuAlaPheThrValGlULysLeuThrGlySerGlyGluLeuTrpTrp GlnAlaGluArgAlaSerSerSerLysSerTrpHeThrPheAspLeuLysAsnLysGlu

ValSerValLysArgValThrGlnAspProLysLeuGlnMetGlyLysLysLeuPro Leu

HisLeuThrLeuProGlnAlaLeuProGl TyrAlaGlySerGlyAsnLeuThrLeuAla

LeuGluAlaLysThrGlyLysLeuHisGlnGluValAsnLeuValValMetArgAla Thr

GlnLeuGlnLysAsnLeuThrCysGluValTrpGlyProThrSerProLysLeuMet Leu SerLeuLysLeuGluAsnLysGluAlaLysValSerLysArgGluLysAlaValTrpVal LeuAsnProGluAlaGlyMetTrpGlnCysLeuLeuSerAspSerGlyGlnValLeuLeu GluSerAsnIIeLysValLeuProThrTrpSerThrProValGlnProMetAlaLeuI1e ValLeuGlyGlyValAlaGlyLeuLeuLeuPhelleGlyLeuGlyllePhePheCysVal ArgCysArgHisArgArgArgGlnAla-%-ArgMetSerGlnileLysArgLeuLeuSer

GlULysLysThrCysGlnCysProHisArgPheGlnLysThrCysSerProIle,

wherein

-?- is Thr or He, -#- is Val or Ala,

-$- is Thr or Pro, and

-%- is Gin or Glu; or the glycosylated derivative thereof.

The invention also relates to a gpl20 binding molecule related to human CD4, comprising the following amino acid sequence:

MetAsnArgGlyValProPheArgHisLeuLeuLeuValLeuGlnLeuAlaLeuLeu Pro AlaAlaThrGlnGlyLysLysValValLeuGlyLysLysGlyAspThrValGluLeuThr CysThrAlaSerGlnLysLysSerIIeGlnPheHiSTrpLysAsnSerAsnGln-@-Lys IIeLeuGlyAsnGlnGlySerPheLeuThrLysGlyProSerLysLeuAsnAspArgAla AspSerArgArgSerLeuTrpAspGlnGlyAsnPhe-S-LeuIlelleLysAsnLeuLys IIeGluAspSerAspThrTyrlleCysGluValGluAspGlnLysGluGluValGlnLeu LeuValPheGlyLeuThrAlaAsnSerAspThrHisLeuLeuGlnGlyGlnSerLeuThr LeuThrLeuGluSerProProGlySerSerProSerValGlnCysArgSerProArgGl LysAsnlleGlnGlyGl LysThrLeuSerValSerGlnLeuGluLeuGlnAspSerGly ThrTrpThrCysThrValLeuGlnAsnGlnLysLysValGluPheLysIIeAspIIeVal ValLeuAlaPheGlnLysAlaSerSerlleValTyrLysLysGluGlyGluGlnValGlu PheSerPheProLeuAlaPheThrValGlULysLeuThrGlySerGlyGluLeuTrpTrp GlnAlaGluArgAlaSerSerSerLysSerTrpHeThrPheAspLeuLysAsnLysGlu ValSerValLysArgValThrGlnAspProLysLeuGlnMetGlyLysLysLeuProLeu HisLeuThrLeuProGlnAlaLeuProGlnTyrAlaGlySerGlyAsnLeuThrLeuAla LeuGluAlaLysThrGlyLysLeuHisGlnGluValAsnLeuValVal etArgAlaThr GlnLeuGlnLysAsnLeuThrCysGluValTrpGlyProThrSerProLysLeuMetLeu SerLeuLysLeuGluAsnLysGluAlaLysValSerLysArgGluLysAlaValTrpVal LeuAsnProGluAlaGlyMetTrpGlnCysLeuLeuSerAspSerGlyGlnValLeuLeu GluSerAsnlleLysValLeuProThrTrpSerThrProValGlnProMetAlaLeuIle ValLeuGlyGlyValAlaGlyLeuLeuLeuPhelleGlyLeuGlyllePhePheCysVal

ArgCysArgHisArgArgArgGlnAlaGluArgMetSerGlnileLysArgLeuLeuSer GluLysLysThrCysGlnCysProHisArgPheGlnLysThrCysSerProIle,

wherein -θ- is Thr or He, and

-$- is Thr or Pro; or the glycosylated derivative thereof; with the proviso that at least one of -(?- and -$- is Thr.

The invention also relates to non-human primate CD4 fragments which binds to HIV or SIV gpl20. Preferably, such non-human primate CD4 fragments are soluble in aqueous solution.

In particular, the invention relates to a soluble CD4 fragment which is derived from the rhesus monkey and comprises the following amino acid sequence:

MetAsnArgGlylleProPheArgHisLeuLeuLeuValLeuGlnLeuAlaLeuLeu Pro AlaValThrGlnGlyLysLysValValLeuGlyLysLysGlyAspThrValGluLeuThr CysThrAlaSerGlnLysLysAsnThrGlnPheHisTrpLysAsnSerAsnGlnileLys HeLeuGlylleGlnGlyLeuPheLeuThrLysGlyProSerLysLeuSerAspArgAla AspSerArgLysSerLeuTrpAspGlnGlyCysPheSerMetllelleLysAsnLeuLys IIeGluAspSerAspThrTyrlleCysGluValGluAsnLysLysGluGluValGluLeu LeuValPheGlyLeuThrAlaAsnSerAspThrHisLeuLeu.

The invention also relates to a soluble chimpanzee CD4 fragment comprising the following amino acid sequence:

MetAsnArgGl ValProPheArgHisLeuLeuLeuValLeuGlnLeuAlaLeuLeuPro AlaAlaThrGlnGlyLysLysValValLeuGlyLysLysGlyAspThrValGluLeuThr CysThrAlaSerGlnLysLysSerlleGlnPheHisTrpLysAsnSerAsnGlnThrLys IIeLeuGlyAsnGlnGlySerPheLeuThrLysGlyProSerLysLeuAsnAspArgVal AspSerArgArgSerLeuTrpAspGlnGlyAsnPheThrLeuIlelleLysAsnLeuLys IIeGluAspSerAspThrTyrlleCysGluValGlyAspGlnLysGluGluValGlnLeu LeuValPheGlyLeuThrAlaAsnSerAspThrHisLeuLeu.

The invention also relates to a gpl20 binding molecule capable of glycosylation comprising the following amino acid sequence:

MetAsnArgGlyValProPheArgHisLeuLeuLeuValLeuGlnLeuAlaLeuLeu Pro AlaAlaThrGlnGlyLysLysValValLeuGlyLysLysGlyAspThrValGluLeuThr CysThrAlaSerGlnLysLysSerlleGlnPheHiSTrpLysAsnSerAsnGln-@-Lys IleLeuGlyAsnGlnGlySerPheLeuThrLysGlyProSerLysLeuAsnAspArg-#- AspSerArgArgSerLeuTrpAspGlnGlyAsnPhe-$-LeuIleIleLysAsnLeuLys IIeGluAspSerAspThrTyrlleCysGluValGlyAspGlnLysGluGluValGlnLeu LeuValPheGlyLeuThrAlaAsnSerAspThrHisLeuLeu

wherein -@- is Thr or He,

-#- is Val or Ala, and

-$- is Thr or Pro; or the glycosylated derivative thereof.

The invention also relates to gpl20 binding molecule capable of glycosylation related to human CD4 fragments. In particular, the invention relates to a glycosylated human CD4 fragment comprising the following amino acid sequence:

MetAsnArgGlyValProPheArgHisLeuLeuLeuValLeuGlnLeuAlaLeuLeu Pro

AlaAlaThrGlnGlyLysLysValValLeuGlyLysLysGlyAspThrValGluLeu Thr CysThrAlaSerGlnLysLysSerlleGlnPheHiSTrpLysAsnSerAsnGln-5-Lys HeLeuGlyAsnGlnGl SerPheLeuThrLysGlyProSerLysLeuAsnAspArgAla AspSerArgArgSerLeuTrpAspGlnGlyAsnPhe-J-LeuIlelleLysAsnLeuLys IIeGluAspSerAspThrTyrlleCysGluValGluAspGlnLysGluGluValGlnLeu LeuValPheGlyLeuThrAlaAsnSerAspThrHisLeuLeu

wherein

-@- is Thr or He, and -$- is Thr or Pro; or the glycosylated derivative thereof; with the proviso that at least one of - - and -$- is Thr.

The invention also relates to fusion proteins, comprising non-human primate CD4 or gpl20 binding molecules of the invention, or HIV or SIV binding fragments thereof, linked to a cytotoxic polypeptide. The invention also relates to a fusion protein comprising non-human primate CD4 or gpl20 binding molecules of the invention, or fragments thereof which are capable of binding to HIV or SIV gpl20, fused at the C-terminus to a second protein which comprises an immunoglobulin heavy chain of the class IgM, IgGl or IgG3, wherein the variable region of said heavy chain immunoglobulin has been replaced with CD4, or HIV gpl20-binding fragment thereof.

The invention also relates to an immunoglobulin-like molecule, comprising: (1) a fusion protein of non-human primate CD4 or fragment thereof which binds to HIV or SIV gpl20 and an immunoglobulin heavy chain, linked to (2) an immunoglobulin light chain.

The invention also relates to a fusion protein comprising non-human primate CD4 or gpl20 binding molecules of the invention, or fragment thereof which binds to HIV or SIV gpl20, fused at the C-terminus to a second protein comprising an immunoglobulin light chain where the variable region has been deleted. The invention also relates to an immunoglobulin-like molecule comprising:

1) a fusion protein of non-human primate CD4 or gpl20 binding molecule of the invention, or fragment thereof which binds to HIV or SIV gpl20, and an immunoglobulin light chain, linked to 2) an immunoglobulin heavy chain.

The invention also relates to pharmaceutical compositions, comprising

1) a therapeutically effective amount of a non-human primate CD4, and 2) a pharmaceutically acceptable carrier.

The invention also relates to pharmaceutical compositions, comprising

1) a therapeutically effective amount of a soluble non- human CD4 fragment, and 2) a pharmaceutically acceptable carrier.

The invention also relates to pharmaceutical compositions comprising the proteins, glycoproteins, fusion proteins and immunoglobulin-like molecules of the invention.

The invention also relates to complexes between the substantially pure non-human primate CD4 and HIV or SIV gpl20.

The invention also relates to complexes comprising the non-human primate CD4 fragments of the invention and HIV or SIV gpl20.

The invention also relates to complexes comprising the fusion proteins and immunoglobulin-like molecules of the invention and HIV or SIV gpl20.

The invention also relates to complexes between the gpl20 binding molecules capable of glycosylation and HIV or SIV gpl20. The invention also relates to a method of treating HIV or

SIV infections, comprising administering to an animal in need of such treatment a therapeutically effective amount of substantially pure non-human primate CD4, or a soluble fragment thereof.

The invention also relates to a method of treating HIV or SIV infections, comprising administering to an animal in need of such treatment a therapeutically effective amount one of the fusion proteins of the invention. The invention also relates to a method of treating HIV or

SIV infections, comprising administering to an animal in need of such treatment a therapeutically effective amount one of the immunoglobulin-like molecules of the invention.

The invention also relates to a method of treating HIV or SIV infections, comprising administering to an animal in need of such treatment a therapeutically effective amount of the gpl20 binding molecules of the invention.

The invention also relates to a method for the detection of HIV or SIV gpl20 in a sample, comprising: (a) contacting a sample suspected of containing HIV or SIV gpl20 with the fusion protein or immunoglobulin-like molecule of the invention; and

(b) detecting whether a complex is formed. The invention also relates to a method for the detection of HIV or SIV gpl20 in a sample, comprising

(a) contacting a sample suspected of containing HIV or SIV gpl20 with substantially pure non-human primate CD4, or fragment thereof which binds to HIV or SIV gpl20, and

(b) detecting whether a complex has formed. The invention is related to the discovery that non-human primates have CD4 of differing amino acid sequence than human CD4. The invention is also related to the discovery that when non-human primate CD4 is expressed on the surface of human cells, strikingly fewer ultinucleated giant cells, or syncytia, are formed than when human CD4 is expressed on the surface of the cell. The invention is also related to the discovery that the presence of a glycine residue at position 87 in the non-human primate CD4 derived from the chimpanzee, instead of the glutamic acid residue as found in human CD4, is

responsible for the lack of syncytia formation. As a result, the CD4 molecule derived from the chimpanzee can now be used in therapeutic application without the potential of causing syncytia formation. The invention is also related to the unexpected discovery that chimpanzee CD4 contains two glycosylation sites (positions 32 and 66 (ASN)). This discovery allows for the preparation of glycosylated gpl20 binding molecules and fragments thereof which bind to gpl20 and likely have enhanced stability in vivo. Advantageously, the glycosylated gpl20 binding molecules and fragments thereof may be administered less frequently to an animal than human or other primate CD4 molecules which are not glycosylated. Thus, the invention also relates to primate (including human) CD4 molecules having one or more glycosylation sites, for example, the chimp sequence at amino acid reidues 34 and 68, at 34 only, and at 68 only. The invention also relates to other CD4 molecules with glycosylation sites at different positions, so long as the molecule retains binding to gpl20.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

THe invention is directed to nucleic acid molecules specifying non-human primate CD4, HIV gpl20 binding fragments thereof, HIV gpl20 binding soluble fragments thereof, fusion proteins thereof, and immunoglobulin-like molecules. The invention also relates to gpl20 binding molecules capable of being glycosylated, HIV gpl20 binding fragments thereof, fusion proteins thereof, and immunoglobulin-like molecules thereof. The nucleic acid molecules of the invention may be a DNA or RNA molecule.

By the term "soluble" is intended that the CD4 fragment is soluble in aqueous solutions which include, but are not

limited to, detergent-free aqueous buffers and body fluids such as blood, plasma and serum.

The invention is also directed to the expression of these novel nucleic acid molecules in transformed hosts to give proteins and glycoproteins. The invention also relates to the use of these proteins and glycoproteins to treat and diagnose

HIV infections.

In particular, the invention relates to expressing said nucleic acid molecules, which specify a fusion protein comprising an immunoglobulin light or heavy chain, in mammalian hosts which express complementary light or heavy chain immunoglobulins to give an immunoglobulin-like molecule which binds to HIV or SIV gpl20.

The CD4 proteins, glycoproteins, CD4 fragments, gpl20 binding molecules, fusion proteins and immunoglobulin-like molecules of the invention may be administered to an animal for the purpose of treating HIV or SIV infections. By the terms "HIV infections" is intended the condition of having AIDS, AIDS related complex (ARC) or where an animal harbors the AIDS virus, but does not exhibit the clinical symptoms of AIDS or ARC. By the terms "SIV infections" is intended the condition of being infected with simian immunodeficiency virus.

By the term "animal" is intended all animals which may derive benefit from the administration of the CD4 proteins, glycoproteins, CD4 fragments, gpl20 binding molecules, fusion proteins and immunoglobulin-like molecules of the invention. Foremost among such animals are humans, however, the invention is not intended to be so limited. By the term "fusion protein" is intended a fused protein comprising a CD4 molecule of the invention, or fragment thereof which is capable of binding to gpl20, linked at its C- terminus to an immunoglobulin chain wherein a portion of the N-terminus of the immunoglobulin is replaced with non-human

primate CD4. Alternatively, the CD4 molecule or fragment thereof may be linked to a cytotoxic polypeptide such as ricin or diphtheria toxin.

By the term "non-human primate" is intended any member of the suborder Anthropoidea except for the family Hominidae. Such non-human primates include the superfamily Ceboidea, family Cebidae (the New World monkeys including the capuchins, howlers, spider monkeys and squirrel monkeys) and family Callithricidae (including the marmosets); the superfamily Cercopithecoidea, family Cercopithecidae (including the macaques, mandrills, baboons, proboscis monkeys, mona monkeys, and the sacred hanuman monkeys of India); and superfamily Ho inoidae, family Pongidae (including gibbons, orangutans, gorillas, and chimpanzees). The rhesus monkey is one member of the macaques.

The nucleic acid molecules and proteins of the invention may be prepared according to the methods disclosed herein and according to well known methods of solid phase synthesis using the amino acid and DNA sequences disclosed herein. As described more fully in the examples below, the gly residue at position 87 of the CD4 derived from the chimpanzee differs from the Glu residue present in human CD4 which is responsible for syncytiu formation. This discovery allows for the preparation of new CD4 molecules which do not mediate syncytium formation. An example of such a protein related to the chimpanzee CD4 molecule comprises the following amino acid sequence:

MetAsnArgGl ValProPheArgHisLeuLeuLeuValLeuGlnLeuAlaLeuLeuPro AlaAlaThrGlnGlyLysLysValValLeuGlyLysLysGlyAspThrValGluLeuThr CysThrAlaSerGlnLysLysSerlleGlnPheHisTrpLysAsnSerAsnGln-?-Lys IIeLeuGl AsnGlnGlySerPheLeuThrLysGlyProSerLysLeuAsnAspArg-#- AspSerArgArgSerLeuTrpAspGlnGlyAsnPhe-S-LeuIlelleLysAsnLeuLys IIeGluAspSerAspThrTyrlleCysGluValGlyAspGlnLysGluGluValGlnLeu

LeuValPheGlyLeuThrAlaAsnSerAspThrHisLeuLeuGlnGlyGlnSerLeuThr LeuThrLeuGlUSerProProGlySerSerProSerValGlnCysArgSerProArgGly LysAsnlleGlnGlyGlyLysThrLeuSerValSerGlnLeuGluLeuGlnAspSerGly ThrTrpThrCysThrValLeuGlnAsnGlnLysLysValGluPheLysIIeAspIleVal ValLeuAlaPheGlnLysAlaSerSerlleValTyrLysLysGluGlyGluGlnValGlu PheSerPheProLeuAlaPheThrValGluLysLeuThrGlySerGlyGluLeuTrpTrp GlnAlaGluArgAlaSerSerSerLysSerTrpHeThrPheAspLeuLysAsnLysGlu ValSerValLysArgValThrGlnAspProLysLeuGlnMetGlyLysLysLeuProLeu HisLeuThrLeuProGlnAlaLeuProGlnTyrAlaGlySerGlyAsnLeuThrLeuAla LeuGluAlaLysThrGlyLysLeuHisGlnGluValAsnLeuValValMetArgAlaThr GlnLeuGlnLysAsnLeuThrCysGluValTrpGlyProThrSerProLysLeuMetLeu SerLeuLysLeuGluAsnLysGluAlaLysValSerLysArgGluLysAlaValTrpVal LeuAsnProGluAlaGlyMetTrpGlnCysLeuLeuSerAspSerGlyGlnValLeuLeu GluSerAsnlleLysValLeuProThrTrpSerThrProValGlnProMetAlaLeuIle ValLeuGlyGlyValAlaGlyLeuLeuLeuPhelleGlyLeuGlyllePhePheCysVal ArgCysArgHisArgArgArgGlnAla-%-ArgMetSerGlnIIeLysArgLeuLeuSer GluLysLysThrCysGlnCysProHisArgPheGlnLysThrCysSerProIle,

wherein -_ - is Thr or He,

-#- is Val or Ala,

-$- is Thr or Pro, and

-%- is Gin or Glu,

or the glycosylated derivative thereof.

The recombinant DNA molecules which encode this family of proteins and glycoproteins have the following sequence:

1 ATGAACCGGGGAGTCCCTTTTAGGCACTTGCTTCTGGTGCTGCAACTGGCACTCCTCCCA

GCAGCCACTCAGGGAAAGAAAGTGGTGCTGGGCAAGAAAGGGGACACAGTGGAACTG ACC 1 TGTACAGCTTCCCAGAAGAAGAGCATACAATTCCACTGGAAAAACTCCAACCAGAYAAAG

ATTCTGGGAAATCAGGGCTCCTTCTTAACTAAAGGTCCATCCAAGCTGAATGATCGC GYT

241 GACTCAAGAAGAAGCCTTTGGGACCAAGGAAACTTTWCCCTGATCATCAAGAATCTTAAG

ATAGAAGACTCAGATACHACATCTGTGAAGTGGGGGACCAGAAGGAGGAGGTGCAAT TG 361 CTAGTGTTCGGATTGACTGCCAACTCTGACACCCACCTGCTTCAGGGGCAGAGCCTGACC

CTGACCπGGAGAGCCCCCCTGGTAGTAGCCCCTCAGTGCAATGTAGGAGTCCAAGG GGT

481 AAAAACATACAGGGGGGGAAGACCCTCTCCGTGTCTCAGCTGGAGCTCCAGGATAGTGGC

ACCTGGACATGCACTGTCTTGCAGAACCAGAAGAAAGTGGAGTTCAAAATAGACATC GTG

601 GTGCTAGCHTCCAGAAGGCCTCCAGCATAGTCTATAAGAAAGAGGGGGAACAGGTGGAG TTCTCCHCCCACTCGCCTTTACAGTTGAAAAGCTGACGGGCAGTGGCGAGCTGTGGTGG

721 CAGGCGGAGAGGGCTTCCTCCTCCAAGTCTTGGATCACCTTTGACCTGAAGAACAAGGAA

GTGTCTGTAAAACGGGTTACCCAGGACCCTAAGCTCCAGATGGGCAAGAAGCTCCCG CTC

841 CACCTCACCCTGCCCCAGGCCTTGCCTCAGTATGCTGGCTCTGGAAACCTCACCCTGGCC

CTTGAAGCGAAAACAGGAAAGTTGCATCAGGAAGTGAACCTCGTGGTGATGAGAGCC ACT 961 CAGCTCCAGAAAAATTTGACCTGTGAGGTGTGGGGACCCACCTCCCCTAAGCTGATGCTG

AGCTTGAAACTGGAGAACAAGGAGGCAAAGGTCTCGAAGCGGGAGAAGGCGGTGTGG GTG

1081 CTGAACCCTGAGGCGGGGATGTGGCAGTGTCTGCTGAGTGACTCGGGACAGGTCCTGCTG

GAATCCAACATCAAGGTTCTGCCCACATGGTCCACCCCGGTGCAGCCAATGGCCCTG ATT

1201 GTGCTGGGGGGCGTCGCCGGCCTCCTGCTTTTCATTGGGCTAGGCATCTTCTTCTGTGTC AGGTGCCGGCACCGAAGGCGCCAAGCASAGCGGATGTCTCAGATCAAGAGACTCCTCAGT

1321 GAGAAGAAGACCTGCCAGTGCCCTCACCGGTTTCAGAAGACATGTAGCCCCATTTGA

wherein Y is C or T, W is A or C, and

S is C or G; or a degenerate variant thereof.

In general, for the preparation of fusion proteins comprising an immunoglobulin, that portion of immunoglobulin which is deleted is the variable region. The fusion proteins

of the invention may also comprise immunoglobulins where more than just the variable region has been deleted and replaced with the CD4 molecule or HIV gpl20 binding fragment thereof. For example, the Vμ and CHI regions of an immunoglobulin chain may be deleted. In practice, any amount of the N-terminus of the immunoglobulin heavy chain can be deleted as long as the remaining fragment mediates cell death by antibody effector function or other mechanism. The minimum sequence required for binding complement encompasses domains CH2 and CH3. Joining of Fc portions by the hinge region is advantageous for increasing the efficiency of complement binding.

The CD4 molecules of the invention and fusion proteins thereof may comprise the complete CD4 sequence, the 372 amino acid extracellular region and the membrane spanning domain, or just the extracellular region. Moreover, the fusion proteins may comprise fragments of the extracellular region which retains binding to HIV gpl20. The extracellular domain of CD4 consists of four contiguous regions each having amino acid and structural similarity to the variable and joining (V-J) domains of immunoglobulin light chains as well as related regions in other members of the immunoglobulin gene superfami¬ ly. These structurally similar regions of CD4 are termed the Vj, V2, V3 and V4 domains. See PCT Application Publication Number WO 89/02922 (published October 3, 1988). Thus, the non-human primate CD4 and fusion proteins thereof may comprise any combination of such binding regions. In general, any fragment of the CD4 proteins and glycoproteins of the invention may be used as long as they retain binding to gpl20. Gpl20 binding CD4 fragments may be obtained by cutting the DNA sequence which encodes chimpanzee CD4 at the Nhe site at position 603 (to give a molecule which encodes two binding domains) or the BspMl site at position 405 (to give a molecule which encodes one domain). Alternatively, the DNA molecule

encoding rhesus CD4 may be cut at the Nhe site at position 603 (to give a molecule which encodes two domains) or the BspMl site at position 405 (to give a molecule which encodes one domain). Other fragments may be obtained using, for example, an exonuclease. The DNA fragment can then be incorporated into a cloning vector and introduced into a host, followed by screening the transformed host for the presence of a protein which binds gpl20. Methods for screening clones for specific binding activity are well known to those of ordinary skill in the art. Preferably, such CD4 fragments are soluble in aqueous solution.

Where the fusion protein comprises an immunoglobulin light chain, it is necessary that no more of the Ig chain be deleted than is necessary to form a stable complex with a heavy chain Ig. In particular, the cysteine residues necessary for disulfide bond formation must be preserved on both the heavy and light chain moieties.

When expressed in a host, e.g., a mammalian cell, the fusion protein may associate with other light or heavy Ig chains secreted by the cell to give a functioning immunoglobulin-like molecule which is capable of binding to gpl20. The gpl20 may be in solution, expressed on the surface of infected cells, or may be present on the surface of the HIV virus itself. Alternatively, the fusion protein may be expressed in a mammalian cell which does not secrete other light or heavy Ig chains. When expressed under these conditions, the fusion protein may form a homodimer.

Genomic or cDNA sequences may be used in the practice of the invention. Genomic sequences are expressed efficiently in myeloma cells, since they contain native promoter structures.

The constant regions of the antibody cloned and used in the chimeric immunoglobulin-like molecule may be derived from any mammalian source. They may be complement binding or ADCC

active. The constant regions may be derived from any appropriate isotype, including IgGl, IgG3, or IgM.

The joining of various DNA fragments, is performed in accordance with conventional techniques, employing blunt-ended or staggered-ended termini for ligation, restriction enzyme digestion to provide appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and ligation with appropriate ligases. The genetic construct may optionally encode a leader sequence to allow efficient expression of the fusion protein. For example, the leader sequence utilized by Maddon et al.. Cell 42:93-104 (1985) for the expression of human CD4 may be used.

For cDNA isolation, cDNA libraries may be screened, for example, by use of a complementary probe or by assay for the expressed CD4 molecule of the invention using a CD4-specific antibody. Methods for preparing antibodies by immunizing animals with an antigen are taught, for example, by Kohler and Milstein, Nature (London) 256:495 (1975); Kohler et al.. Eur. J. Immunol. 6:511 (1976); Kohler et al .. Eur. J. Immunol. 6:292 (1976); or Hammerling et al.. in: Monoclonal Antibodies and T-Cell Hvbridomas. Elsevier, N.Y., pp.563-681 (1981). The invention further relates to monoclonal and polyclonal antibodies which are specific for the non-human CD4 proteins, glycoproteins of the invention, and the soluble and non- soluble fragments thereof.

The non-human primate CD4 may be derived from any member of the suborder Anthropoidea except for the family Hominidae. Preferably, the non-human primate CD4 is derived from the rhesus monkey or chimpanzee, although the invention is not intended to be so limited. One of ordinary skill in the art can obtain the CD4 from any additional primate by isolation of the poly-A containing RNA of mitogen stimulated peripheral blood mononuclear cells obtained from the particular animal.

After preparation of cDNA with, for example, reverse tran- scriptase, the cDNA may be ligated into an appropriate cloning vector and used to transform an appropriate host. The clones may then be screened with a monoclonal antibody directed to the rhesus monkey or chimpanzee CD4 of the invention followed by selection of positive clones, or by hybridization with the chimp or rhesus CD4 cDNAs.

To express the CD4 molecules and fusion hybrid proteins of the invention, transcriptional and translational signals recognized by an appropriate host element are necessary. Eukaryotic hosts which may be used include mammalian cells capable of culture in vitro, particularly leukocytes, more particularly myeloma cells or other transformed or oncogenic lymphocytes, e.g., EBV-transformed cells. Advantageously, mammalian cells are used to express the glycosylated CD4 proteins. Alternatively, non-mammalian cells may be employed, such as bacteria, fungi, e.g., yeast, filamentous fungi, or the like.

Preferred hosts for fusion protein production are mammalian cells, grown in vitro in tissue culture or in vivo in animals. Mammalian cells provide post translational modification to immunoglobulin protein molecules which provide for correct folding and glycosylation of appropriate sites. Mammalian cells which may be useful as hosts include cells of fibroblast origins such as VERO or CH0-K1 or cells of lymphoid origin, such as the hybridoma SP2/0-AG14 or the myeloma P3x63Sgh, and their derivatives. For the purpose of preparing an immunoglobulin-like molecule, a plasmid containing a gene which encodes a heavy chain immunoglobulin, wherein the variable region has been replaced with one of the CD4 βolecules of the invention, may be introduced, for example, into J558L myeloma cells, a mouse plasmacytoma expressing the lambda-1 light chain but which does not express a heavy chain (see Oi et al .. P.N.A.S. (USA) 80:825-829

(1983)). Other preferred hosts include COS cells, BHK cells and hepatoma cells.

The constructs may be joined together to form a single DNA segment or may be maintained as separate segments, by themselves or in conjunction with vectors.

Where the protein is not glycosylated, any host may be used to express the protein which is compatible with replication and transcription of sequences in the expression plasmid. In general, vectors containing replication and transcription controlling sequences are derived from species compatible with a host cell are used in connection with the host. The vector ordinarily carries a replication origin, as well as specific genes which are capable of providing phenotypic selection in transformed cells. The expression of the non-human primate CD4 molecules and fusion proteins can also be placed under control with other regulatory sequences which may be homologous to the organism in its untransformed state. For example, lactose-dependent E. coli chromosomal DNA comprises a lactose or lac operon which mediates lactose utilization by elaborating the enzyme beta-galactosidase. The lac control elements may be obtained from bacterial phage lambda placδ, which is infective for E. coli. The lac promoter-operator system can be induced by IPTG.

Other promoters/operator systems or portions thereof can be employed as well. For example, colicin El, galactose, alkaline phosphatase, tryptophan, xylose, tax, and the like can be used.

For mammalian hosts, several possible vector systems are available for expression. One class of vectors utilize DNA elements which are derived from animal viruses such as bovine papilloma virus, polyo a virus, adenovirus, vaccinia virus, baculovirus, retroviruses (RSV, MMTV or MOMLV), or SV40 virus. Cells which have stably integrated the DNA into their chromo¬ somes may be selected by introducing one or more markers which

allow selection of transfected. host cells. The marker may provide for prototropy to an auxotrophic host, biocide resistance, e.g., antibiotics, or heavy metals such as copper or the like. The selectable marker gene can be either directly linked to the DNA sequences to be expressed, or introduced into the same cell by cotransformation. Additional elements may also be needed for optimal synthesis of mRNA. These elements may include splice signals, as well as transcriptional promoters, enhancers, and termination signals. The cDNA expression vectors incorporating such elements includes those described by Okayama, H., Mol. Cel . Biol.. 3:280 (1983) and others.

Once the vector or DNA sequence containing the constructs has been prepared for expression, the DNA constructs may be introduced to an appropriate host. Various techniques may be employed, such as protoplast fusion, calcium phosphate precipitation, electroporation or other conventional techniques. After the fusion, the cells are grown in media and screened for the appropriate activity. Expression of the gene(s) results in production of the desired protein. If the expressed product is a fusion protein, it may then be subject to further assembly with an immunoglobulin light or heavy chain to form an immunoglobulin-like molecule.

The host cells for CD4 protein and glycoprotein, CD4 fragment, and immunoglobulin production may be immortalized cells, primarily myeloma or lymphoma cells. These cells may be grown in appropriate nutrient medium in culture flasks or injected into a synergistic host, e.g., mouse or a rat, or immunodeficient host or host site, e.g., nude mouse or hamster pouch. In particular, the cells may be introduced into the abdominal cavity of an animal to allow production of ascites fluid which contains the immunoglobulin-like molecule. Alternatively, the cells may be injected subcutaneously and the chimeric antibody is harvested from the blood of the host.

The cells may be used in the same manner as hybrido a cells. See Diamond et al .. N. Eno. J. Med. 204:1344 (1981), and Kennatt, McKearn and Bechtol (Eds.), Monoclonal Antibodies: Hvbridomas: -- A New Dimension in Biologic Analysis. Plenum, 1980.

The CD4 proteins, glycoproteins, CD4 fragments, fusion proteins and immunoglobulin-like molecules of the invention may be isolated and purified in accordance with conventional conditions, such as extraction, precipitation, chromatography, affinity chromatography, electrophoresis or the like. For example, the CD4 proteins, glycoproteins and fragments may be purified by passing a solution thereof through a column having gpl20 immobilized thereon (see U.S. patent No. 4,725,669). The bound CD4 molecule may then be eluted by treatment with a chaotropic salt or by elution with aqueous acetic acid (1 M).

The Ig fusion proteins may be purified by passing a solution containing the fusion protein through a column which contains immobilized protein A or protein G which selectively binds the Fc portion of the fusion protein. See, for example, Reis, K.J., et al.. J. Immunol. 112:3098-3102 (1984); PCT Application, Publication No. W087/00329. The chimeric antibody may the be eluted by treatment with a chaotropic salt or by elution with aqueous acetic acid (1 M).

Alternatively the non-human primate CD4 proteins and glycoproteins, fragments, fusion proteins and immunoglobulin- like molecules may be purified on anti-CD4 antibody columns, or on anti-immunoglobulin antibody columns to give a substantially pure protein.

By the term "substantially pure" is intended that the protein is free of the impurities that are naturally associated therewith. Substantial purity may be evidenced by a single band by electrophoresis.

In one embodiment of the invention, cDNA sequences which encode the CD4 molecules of the invention, or a fragment

thereof which binds gpl20, may be ligated into an expression plasmid which codes for an antibody wherein the variable region of the gene has been deleted. Methods for the preparation of genes which encode the heavy or light chain constant regions of immunoglobulins are taught, for example, by Robinson, R. et al .. PCT Application, Publication No. W087- 02671. The cDNA sequence encoding the CD4 molecule or fragment may be directly joined to the cDNA encoding the light or heavy Ig contant regions or may be joined via a linker sequence. Preferably, the linker sequence does not encode a protein product which gives rise to an antigenic reaction in the individual .

Preferred immunoglobulin-like molecules which contain the CD4 molecules of the invention, or fragments thereof, contain the constant region of an IgM, IgGl or IgG3 antibody.

The CD4 proteins, glycoproteins, fragments, fusion proteins and immunoglobulin-like molecules, and pharmaceutical compositions thereof may be used for the treatment or prophylaxis of HIV viral infections. This method comprises administering to an animal an effective amount of the CD4 proteins, glycoproteins, fragments, fusion proteins and immunoglobulin-like molecules, and pharmaceutical compositions thereof, which are capable of specifically forming a complex with gpl20 so as to render the HIV or SIV, with which the individual is infected, incapable of infecting T4 ⁺ cells.

The fusion protein and immunoglobulin-like molecule may complex to gpl20 which is expressed on infected cells. Although the inventor is not bound by a particular theory, it appears that the Fc portion of the fusion protein or immunoglobulin-like molecule may bind with complement to

ediate destruction of the cell. In this manner, infected cells are destroyed so that additional viral particle production is stopped.

For the purpose of treating HIV infections, the non-human primate CD4 molecules or fragments thereof, fusion proteins or immunoglobulin-like molecules of the invention may additionally contain a radiolabel, therapeutic agent or cytotoxic polypeptide which enhances destruction of the HIV particle or HIV-infected cell. Examples of radioisotopes which can be bound to the proteins, glycoproteins, fusion proteins, and immunoglobulin- like molecules of the invention for use in HIV-therapy are 125 _If 131τ, 90 _Y> 67 _Cu , 217 _Bi> 211 _Atj 212 _Pbj 47 _{Sc> and} 109p _d>

Optionally, a label such as boron can be used which emits a and β particles upon bombardment with neutron radiation.

For in vivo diagnosis radionucleotides may be bound to the CD4 proteins, glycoproteins or fragments thereof, fusion proteins or immunoglobulin-like molecules either directly or by using an intermediary functional group. An intermediary group which is often used to bind radioisotopes, which exist as metal l i c cations , to antibodies is diethylenetria inepentaacetic acid (DTPA). Typical examples of metallic cations which are bound in this manner are 99 ^m Tc 123^ 111m, 131 _If 97 _R|J> 67 _Cu> 67 _Ga , _and 68 _Ga . Moreover, the CD4 proteins and glycoproteins or fragments thereof, fusion proteins and immunoglobulin-like molecules may be tagged with an NMR imaging agent which include paramagnetic atoms. The use of an NMR imaging agent allows the in vivo diagnosis of the presence of and the extent of HIV infection within a patient using NMR techniques. Elements which are particularly useful in this manner are 157 _r j _} 55^,, lδ∑øy, ⁵² Cr, and ⁵⁶ Fe.

Introduction of the nucleic acid molecules of the invention by gene therapy may also be contemplated, for

example, using retroviruses or other means to introduce the genetic material specifying the fusion proteins into suitable target tissues. In this embodiment, the target tissues having the nucleic acid molecules of the invention may then produce the CD4 molecules or fusion protein in vivo.

The nucleic acid molecules specifying the CD4 molecules or fragments thereof may be used to reconstitute the immune system of an individual suffering from HIV. For example, the bone marrow cells of an HIV-infected individual may be removed and the he atopoietic stem cells, either as part of a mixed population or a purified fraction, may be infected or transfected with a virus or DNA construct that specifies the non-human primate CD4 or fragment thereof. Production of human CD4 may be shut down by including within the same or different genetic construct, a gene which interferes with the expression of human CD4. Such a gene may take many forms, for example, it may encode RNA that binds to a regulatory protein (since the non-human primate CD4 may be under other control, its expression will not be affected); an antisense RNA that binds selectively to the human CD4 gene; or a DNA-binding protein that has had its regulatory region amputated. The modified stem cells would then be injected back into the patient where they will migrate to the bone marrow. Preferably, the marrow would have been previously cleared of normal hematopoietic cells by irradiation or with a toxic drug. See Baltimore, D. Nature 335:395-396 (1988).

Methods for the transfection of hematopoietic cells are well known and taught, for example, by Wetherall, D.J., Nature 331:13-14 (1988); Dick, J.E., Ann. N. Y. Acad. Sci . 507:242- 251 (1987); Eglitis, D.B. et al.. Science 230:1395-1398 (1985); Gillio, A. et al.. Ann. N.Y. Acad. Sci. 511:406-417 (1987). Methods for the transfection of cells with anti-sense RNA are taught, for example, by Ha bor, J.E. et al .. Proc. Natl. Acad. Sci. (USA) 85:4010-4014 (1988); Sanford, J.C., jh

Theor. Biol. 130:469-480 (1988); Izant, J.G. et al.. Science 229:345-352 (1985); and Hambor, J.E. et al.. J. EXP. Med. 168:1237-1245 (1988).

The non-human primate CD4, and soluble and non-soluble fragments thereof which bind HIV or SIV gpl20, may also be used in vivo to treat HIV infection by blocking infection of human CD4 bearing lymphocytes and syncytium formation. See Lui, M. et al .. J. Clin. Invest.82:2176-2180 (1988) or Fischer, R.A. et al.. Nature 331:76-78 (1988) for a discussion on the use of human CD4 and soluble fragments thereof to block HIV infection of CD4 bearing lymphocytes and syncytium formation.

Fusion proteins comprising the CD4 proteins, glycoproteins and fragments thereof, and a therapeutic agent can also be used to treat HIV infected individuals by killing HIV-infected cells in vivo. Therapeutic agents may include, for example, cytotoxic polypeptides such as the bacterial toxins diphtheria toxin or ricin. Methods for producing fusion proteins comprising fragment A of diphtheria toxin are taught in U.S. Patent 4,675,382 (1987) which is incorporated by reference herein. Diphtheria toxin contains two polypeptide chains. The B chain binds the toxin to a receptor on a cell surface. The A chain actually enters the cytoplasm and inhibits protein synthesis by inactivating elongation factor 2, the factor that translocates ribosomes along mRNA concomitant with hydrolysis of ATP. See Darnell, J., et al .. in Molecular Cell Biology. Scientific American Books, Inc., page 662 (1986). Alternatively, a fusion protein comprising ricin, a toxic lectin, may be prepared. Methods for the preparation of a fusion protein comprising human CD4 linked to active regions of Pseudomonas endotoxin A and the use thereof to selectively kill HIV infected cells are taught by Chaudhary, V.K. et al.. Nature 335:369-372 (1988), which is incorporated by reference herein.

The dose ranges for the administration of the CD4 proteins, glycoproteins and fragments thereof, fusion proteins and immunoglobulin-like molecules are those which are large enough to produce the desired effect whereby the symptoms of HIV or SIV infection are ameliorated. The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, sex and extent of disease in the patient, counter indications, if any, immune tolerance and other such variables, to be adjusted by the individual physician. Dosage can vary from .001 mg/kg to 50 mg/kg, preferably 0.1 mg/kg to 1.0 mg/kg, of the CD4 molecule of the invention, gpl20 binding molecule, or fragment thereof, fusion protein, or immunoglobulin-like molecule, in one or more administrations daily, for one or several days. The immunoglobulin-like molecule can be administered parenterally by injection or by gradual perfusion over time. They can be administered intravenously, intraperitoneally, intramuscularly, or subcutaneously. Preparations for parenteral administration include sterile or aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present, such as, for example, antimicrobials, anti-oxidants, chelating agents, inert gases

and the like. See, generally, Remington's Pharmaceutical Science. 16th Ed., Mack Eds., 1980.

The invention also relates to a method for preparing a medicament or pharmaceutical composition comprising the components of the invention, the medicament being used for therapy of HIV or SIV infection in animals.

The proteins and glycoproteins of the present invention may also be used in combination with other therapeutics used in the treatment of AIDS, ARC and HIV infection. For example, the proteins and glycoproteins may be co-administered with anti-retroviral agents that block reverse transcriptase such as AZT, DDI, HPA-23, phosphonofor ate, sura in, ribavirin and deoxycytidine. Alternatively, the proteins and glycoproteins of the invention may be co-administered with such anti-viral agents as interferons, including alpha interferon, gamma interferon, omega interferon, or glucosidase inhibitors such as castanospermine. Such combination therapies may advantageously utilize lower dosages of those agents so as to minimize toxicity and enhance the effectiveness of the treatment.

The detection and quantisation of antigenic substances and biological samples frequently utilizes immunoassay techniques. These techniques are based upon the formation of the complex between the antigenic substance, e.g., gpl20, being assayed and an antibody or antibodies in which one or the other member of the complex may be detectably labeled. In the present invention, the CD4 proteins, glycoproteins or fragments thereof, immunoglobulin-like molecules or fusion proteins may be labeled with any conventional label. Thus, the CD4 protein, glycoprotein or fragment thereof, fusion protein or immunoglobulin-like molecule can also be used in assay for HIV or SIV viral infection in a biological sample by contacting a sample, derived from an animal suspected of having an HIV or SIV infection, with the CD4

protein, glycoprotein or fragment thereof, fusion protein or immunoglobulin-like molecule, and detecting whether a complex with gpl20, either alone or on the surface of an HIV- infected cell, has formed. For example, a biological sample may be treated with nitrocellulose, or other solid support which is capable of immobilizing cells, cell particles or soluble protein. The support may then be washed with suitable buffers followed by treatment with the CD4 protein, glycoprotein or fragment thereof, fusion protein, or immunoglobulin-like molecule, any of which may be detectably labeled. The solid phase support may then be washed with a buffer a second time to remove unbound protein and the label detected.

In carrying out the assay of the present invention on a sample containing gpl20, the process comprises: a) contacting a sample suspected of containing gpl20 with a solid support to effect immobilization of gpl20, or cell which expresses gpl20 on its surface; b) contacting said solid support with the detectably labeled CD4 protein, glycoprotein or fragment thereof which binds to HIV gpl20, immunoglobulin-like molecule or fusion protein molecule of the invention; c) incubating said detectably labeled molecule with said support for a sufficient amount of time to allow the detectably labelled molecule to bind to the immobilized gpl20 or cell which expresses gpl20 on its surface; d) separating the solid phase support from the incubation mixture obtained in step c); and e) detecting the bound detectably labeled molecule and thereby detecting and quantifying gpl20.

Alternatively, the detectably labeled CD4 protein, glycoprotein or fragment thereof, immunoglobulin-like molecule or fusion protein - gpl20 complex in a sample may be separated from a reaction mixture by contacting the complex with an

immobilized antibody or protein which is specific for an immunoglobulin or, e.g., protein A, protein G, anti-IgM or anti-IgG antibodies. Such anti-immunoglobulin antibodies may be monoclonal or polyclonal. The solid support may then be washed with suitable buffers to give an immobilized complex. The label may then be detected to give a measure of gpl20 and, thereby, the presence of HIV.

This aspect of the invention relates to a method for detecting HIV or SIV viral infection in a sample comprising: (a) contacting a sample suspected of containing gpl20 with a fusion protein comprising non- human primate CD4 or fragment thereof that binds to HIV gpl20 and the Fc portion of an immunoglobulin chain, and (b) detecting whether a complex is formed.

The invention also relates to a method of detecting gpl20 in a sample, further comprising:

(c) contacting the mixture obtained in step (a) with an Fc binding molecule, such as an antibody, protein A, or protein G, which is immobilized on a solid phase support and is specific for the fusion protein, to give a gpl20 fusion protein-immobilized antibody complex (d) washing the solid phase support obtained in step (c) to remove unbound fusion protein, and

(e) and detecting the label on the fusion protein.

Of course, the specific concentrations of detectably labeled immunoglobulin-like molecule (or fusion protein) and gpl20, the temperature and time of incubation, as well as other assay conditions may be varied, depending on various factors including the concentration of gpl20 in the sample, the nature of the sample, and the like. Those skilled in the art will be able to determine operative and optimal assay

conditions for each determination by employing routine experimentation.

Other such steps as washing, stirring, shaking, filtering and the like may be added to the assays as is necessary for the particular situation.

One of the ways in which the CD4 protein, glycoprotein or fragment thereof, immunoglobulin-like molecule or fusion protein can be detectably labeled is by linking the same to an enzyme. This enzyme, in turn, when later exposed to its substrate, will catalize the formation of a product which can be detected as, for example, by spectrophotometric, fluorometric or by visual means. Enzymes which can be used to detectably label the CD4 protein, glycoprotein or fragment thereof, immunoglobulin-like molecule or fusion protein of the present invention include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxi¬ dase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose- Vl-phosphate dehydrogenase, glucoa ylase and acetylcholine esterase.

The CD4 protein, glycoprotein or fragment thereof, immunoglobulin-like molecule or fusion protein of the present invention may also be labeled with a radioactive isotope which can be determined by such means as the use of a gamma counter or a scintillation counter or by autoradiography. Isotopes which are particularly useful for the purpose of the present invention are: ³ H, ¹² 5ι, 131^ 32 _P} 35 _s> 14 _c> 51 _Crj 36 _cl> ⁵⁷ Co, ⁵⁸ Co, ⁵⁹ Fe and ⁷⁵ Se.

It is also possible to label the CD4 protein, glycoprotein or fragment thereof, immunoglobulin-like molecule or fusion protein with a fluorescent compound. When the fluorescently labeled immunoglobulin-like molecule is exposed

to light of the proper wave length, its presence can then be detected due to the fluorescence of the dye. Among the most commonly used fluorescent labelling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, β-phthaldehyde and fluorescamine.

The CD4 protein, glycoprotein or fragment thereof, immunoglobulin-like molecule or fusion protein of the invention can also be detectably labeled using fluorescence emitting metals such as IM-lu, or others of the lanthanide series. These metals can be attached to the CD4 protein, glycoprotein or fragment thereof, immunoglobulin-like molecule or fusion protein, using such metal chelating groups as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA). The CD4 protein, glycoprotein or fragment thereof, immunoglobulin-like molecule or fusion protein of the present invention also can be detectably labeled by coupling it to a chemi luminescent compound. The presence of the chemiluminescent-tagged CD4 protein, glycoprotein or fragment thereof, immunoglobulin-like molecule or fusion protein is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.

Likewise, a bioluminescent compound may be used to label the CD4 protein, glycoprotein or fragment thereof, immunoglobulin-like molecule or fusion protein of the present invention. Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Important biolum-

inescent compounds for purposes of labeling are luciferin, luciferase and aequorin.

Detection of the CD4 protein, glycoprotein or fragment thereof, immunoglobulin-like molecule or fusion protein may be accomplished by a scintillation counter, for example, if the detectable label is a radioactive gamma emitter, or by a fluorometer, for example, if the label is a fluorescent material. In the case of an enzyme label, the detection can be accomplished by colorimetric methods which employ a substrate for the enzyme. Detection may also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.

The assay of the present invention is ideally suited for the preparation of a kit. Such a kit may comprise a carrier means being compartmentalized to receive in close confinement therewith one or more container means such as vials, tubes and the like, each of said container means comprising the separate elements of the immunoassay. For example, there may be a container means containing a solid phase support, and further container means containing the detectably labeled CD4 protein, glycoprotein or fragment thereof, immunoglobulin-like molecule or fusion protein. Further container means may contain standard solutions comprising serial dilutions of analytes such as gpl20 or fragments thereof to be detected. The standard solutions of these analytes may be used to prepare a standard curve with the concentration of gpl20 plotted on the abscissa and the detection signal on the ordinate. The results obtained from a sample containing gpl20 may be interpolated from such a plot to give the concentration of gpl20.

The CD4 protein, glycoprotein or fragment thereof, immunoglobulin-like molecule or fusion protein of the present invention can also be used as a stain for tissue sections. For example, a labeled molecule comprising CD4 protein or

glycoprotein or HIV gpl20 binding fragment thereof, may be contacted with a tissue section, e.g., a brain biopsy specimen. This section may then be washed and the label detected. The following examples are illustrative, but not limiting the method and composition of the present invention. Other suitable modifications and adaptations which are obvious to those skilled in the art are within the spirit and scope of this invention.

EXAMPLES EXAMPLE 1 ISOLATION OF CHIMPANZEE AND RHESUS MONKEY CD4 cDNAs

cDNA clones encoding the CD4 antigens of the Chimpanzee (P_an troglodytes) and the Rhesus Monkey (Maccaca mulatta) were isolated, sequenced, and expressed. Non-human primate CD4 cDNAs were synthesized from the poly-A containing RNA of mitogen stimulated peripheral blood mononuclear cells obtained from these animals. cDNA expression libraries were made in the vector CDM8 and CD4 cDNAs we isolated by four rounds of immunoselection as previously described by Seed et al . , Proc.

Natl. Acad. Sci (USA) 84:3365-3369 (1987). Sequencing was carried out using the dideoxynucleotide chain termination technique on single and double stranded templates. The DNA and amino acid sequences of the Chimpanzee and Rhesus Monkey

CD4 are shown below. Also shown is a comparison of the respective sequences to human CD4.

RHESUS CD4 CODING SEQUENCE AND PREDICTED AMINO ACID SEQUENCE SHOWING DIFFERENCES FROM HUMAN SEQUENCES

ATGAACCGGGGAATCCCTTTTAGGCACTTGCTTCTGGTGCTGCAACTGGCGCTACTC CCA MetAsnArgGlylleProPheArgHisLeuLeuLeuValLeuGlnLeuAlaLeuLeuPro

Val

G C 1

GCAGTCACCCAGGGAAAGAAAGTGGTGCTGGGCAAGAAAGGGGATACAGTGGAACTG ACC 120

AlaValThrGlnGlyLysLysValValLeuGlyLysLysGlyAspThrValGluLeu Thr 15 Ala C T A

121 TGTACAGCTTCGCAGAAGAAGAACACACAATTCCACTGGAAAAACTCCAACCAGATAAAG 16 CysThrAlaSerGlnLysLysAsnThrGlnPheHisTrpLysAsnSerAsnGlnileLys

Serlle

G T

ATTCTGGGAAπCAGGGTCTCTTCπAACTAAAGGTCCATCCAAGCTGAGCGATCGT GCT 240 H eLeuGlyll eGl nGlyLeuPheLeuThrLysGlyProSerLysLeuSerAspArgAl a 55 Asn Ser Asn A CTC AT C

A

241 GACTCAAGAAAAAGCCTTTGGGACCAAGGATGCTTTTCCATGATCATCAAGAATCTTAAG 56 AspSerArgLysSerLeuTrpAspGlnGlyCysPheSerMetllelleLysAsnLeuLys Arg Asn ProLeu

G AA CC C . . . * .

ATAGAAGACTCAGATACTTACATCTGTGAAGTGGAGAACAAGAAGGAGGAGGTGGAA TTG 360 IIeGluAspSerAspThrTyrlleCysGluValGluAsnLysLysGluGluValGluLeu 95

AspGln Gin G C C

61 CTGGTGTTCGGATTGACTGCCAACTCTGACACCCACCTGCTTGAGGGGCAAAGCCTGACC 96 LeuValPheGlyLeuThrAlaAsnSerAspThrHisLeuLeuGluGlyGlnSerLeuThr

Gin

CTGACCTTGGAGAGCCCCCCTGGTAGTAGCCCCTCAGTGAAATGTAGGAGTCCAGGGGGT 480 LeuThrLeuGlUSerProProGlySerSerProSerValLysCysArgSerProGlyGly 135

Gin Arg C A

481 AAAAACATACAGGGGGGGAGGACCATCTCTGTGCCTCAGCTGGAGCGCCAGGATAGTGGC 136 LysAsnlleGlnGlyGlyArgThrlleSerValProGlnLeuGluArgGlnAspSerGly Lys Leu Ser Leu

ACCTGGACATGCACCGTCTCGCAGGACCAGAAGACGGTGGAGTTCAAAATAGACATC GTG 600

ThrTrpThrCysThrValSerGlnAspGlnLysThrValGluPheLysIleAspHeV al 175

Leu Asn Lys T T A A

601 GTGCTAGCTTTCCAGAAGGCCTCCAGCACAGTCTATAAGAAAGAGGGGGAACAGGTGGAG 176 ValLeuAlaPheGlnLysAlaSerSerThrValTyrLysLysGluGlyGluGlnValGlu

He

TTCTCCTTCCCACTCGCCTTTACACTTGAAAAGCTGACGGGCAGTGGCGAGCTGTGG TGG 720 PheSerPheProLeuAlaPheThrLeuGlULysLeuThrGlySerGlyGluLeuTrpTrp 215 Val

G

721 CAGGCGGAGAGGGCCTCCTCCTCCAAGTCTTGGATTACCTTCGACCTGAAGAACAAGGAA 216 GlnAlaGluArgAlaSerSerSerLysSerTrpHeThrPheAspLeuLysAsnLysGlu

GTGTCTGTAAAACGGGTTACCCAGGACCCCAAGCTCCAGATGGGCAAGAAGCTCCCG CTC 840 ValSerValLysArgValThrGlnAspProLysLeuGlnMetGlyLysLysLeuProLeu 255

841 CACCTCACCCTGCCCCAGGCCTTGCCTCAGTATGCTGGCTCTGGAAACCTCACGCTGGCC 256 HisLeuThrLeuProGlnAlaLeuProGlnTyrAlaGlySerGlyAsnLeuThrLeuAla

CTTGAAGCGAAAACAGGAAAGπGCATCAGGAAGTGAACCTCGTGGTGATGAGAGCCACT 960 LeuGl uAl aLysThrGl LysLeuHi sGl nGl uVal AsnLeuVal Val MetArgAl aThr 295

961 CAGTTCCAGGAAMTTTGACCTGTGAAGTGTGGGGACCCACCTCCCCTAAGCTGACGCTG 296 Gl nPheGl nGl uAsnLeuThrCysGl uValTrpGlyProThrSerProLysLeuThrLeu

Leu Lys Met

AGCTTGAAACTGGAGAACAAGGGGGCAACGGTCTCGAAGCAGGCGAAGGCGGTGTGG GTG 1080 SerLeuLysLeuGluAsnLysGlyAlaThrValSerLysGlnAlaLysAlaValTrpVal 335

Glu Lys ArgGlu A A G A

1081 CTGAACCCTGAGGCGGGGATGTGGCAGTGTCTGCTGAGTGACTCGGGACAGGTCCTGCTA 336 LeuAsnProGluAlaGlyMetTrpGlnCysLeuLeuSerAspSerGlyGlnValLeuLeu

GAATCCAACATCAAGGTTGTGCCCACATGGCCCACCCCGGTGCAGCCAATGGCCCTG ATT 1200

GluSerAsnIIeLysValValProThrTrpProThrProValGlnProMetAlaLeu Ile 375

Leu Ser

C T

1201 GTGCTGGGGGGCGTTGCGGGCCTCCTGCTTTTCACTGGGCTAGGCATCTTCTTCTGTGTC

376 ValLeuGlyGlyValAlaGlyLeuLeuLeuPheThrGlyLeuGlyllePhePheCysVal He - C C T

AGGTGCCGGCATCGAAGGCGTCAAGCAGAGCGGATGTCTCAGATCAAGAGACTCCTC AGT 1320 ArgCysArgHisArgArgArgGlnAlaGluArgMetSerGlnileLysArgLeuLeuSer 415

1321 GAAAAGAAGACCTGCCAGTGCCCTCACCGGTTTCAGAAGACATGTAGCCCCATTTGA 1377 416 GluLysLysThrCysGlnCysProHisArgPheGlnLysThrCysSerProIleEnd 433

CHIMP CD4 CODING SEQUENCE AND PREDICTED AMINO ACID SEQUENCE SHOWING DIFFERENCES FROM HUMAN SEQUENCES

1 ATGAACCGGGGAGTCCCTTTTAGGCACTTGCTTCTGGTGCTGCAACTGGCACTCCTCCCA -25 MetAsnArgGlyValProPheArgHisLeuLeuLeuValLeuGlnLeuAlaLeuLeuPro

1

GCAGCCACTCAGGGAAAGAAAGTGGTGCTGGGCAAGAAAGGGGACACAGTGGAACTG ACC 120 AlaAlaThrGlnGlyLysLysValValLeuGlyLysLysGlyAspThrValGluLeuThr 15

* AAAAAAAAA

121 TGTACAGCTTCCCAGAAGAAGAGCATACAATTCCACTGGAAAAACTCCAACCAGACAAAG 16 CysThrAlaSerGlnLysLysSerlleGlnPheHisTrpLysAsnSerAsnGlnThrLys He

T

ATTCTGGGAAATCAGGGCTCCTTCTTAACTAAAGGTCCATCCAAGCTGAATGATCGC GTT 240 IIeLeuGlyAsnGlnGlySerPheLeuThrLysGlyProSerLysLeuAsnAspArgVal 55

Ala C

AAAAAAAAA

241 GACTCAAGAAGAAGCCTTTGGGACCAAGGAAACTTTACCCTGATCATCAAGAATCTTAAG 56 AspSerArgArgSerLeuTrpAspGlnGlyAsnPheThrLeuIlelleLysAsnLeuLys

Pro CC

ATAGAAGACTCAGATACTTACATCTGTGAAGTGGGGGACCAGAAGGAGGAGGTGCAATTG 360 IIeGluAspSerAspThrTyrlleCysGluValGl AspGlnLysGluGluValGlnLeu 95

Glu A

361 CTAGTGTTCGGATTGACTGCCAACTCTGACACCCACCTGCTTCAGGGGCAGAGCCTGACC 96 LeuValPheGlyLeuThrAlaAsnSerAspThrHisLeuLeuGlnGlyGlnSerLeuThr

CTGACCTTGGAGAGCCCCCCTGGTAGTAGCCCCTCAGTGCAATGTAGGAGTCCAAGG GGT 360 LeuThrLeuGlUSerProProGlySerSerProSerValGlnCysArgSerProArgGly 135

481 AAAAACATACAGGGGGGGAAGACCCTCTCCGTGTCTCAGCTGGAGCTCCAGGATAGTGGC 136 LysAsnlleGlnGlyGlyLysThrLeuSerValSerGlnLeuGluLeuGlnAspSerGly

ACCTGGACATGCACTGTCTTGCAGAACCAGAAGAAAGTGGAGTTCAAAATAGACATC GTG 600

ThrTrpThrCysThrValLeuGlnAsnGlnLysLysValGluPheLysIleAspIle Val 175

601 GTGCTAGCTTTCCAGAAGGCCTCCAGCATAGTCTATAAGAAAGAGGGGGAACAGGTGGAG 176 ValLeuAlaPheGlnLysAlaSerSerlleValTyrLysLysGluGlyGluGlnValGlu

TTCTCCπCCCACTCGCCTTTACAGTTGAAAAGCTGACGGGCAGTGGCGAGCTGTGG TGG 720

PheSerPheProLeuAlaPheThrValGlULysLeuThrGlySerGlyGluLeuTrp Trp 215

721 CAGGCGGAGAGGGCTTCCTCCTCCAAGTCTTGGATCACCTTTGACCTGAAGAACAAGGAA 216 GlnAlaGluArgAlaSerSerSerLysSerTrpHeThrPheAspLeuLysAsnLysGlu

GTGTCTGTAAAACGGGTTACCCAGGACCCTAAGCTCCAGATGGGCAAGAAGCTCCCG CTC 840 ValSerValLysArgValThrGlnAspProLysLeuGlnMetGlyLysLysLeuProLeu 255

41 CACCTCACCCTGCCCCAGGCCTTGCCTCAGTATGCTGGCTCTGGAAACCTCACCCTGGCC 256 HisLeuThrLeuProGlnAlaLeuProGlnTyrAlaGlySerGlyAsnLeuThrLeuAla

CTTGAAGCGAAAACAGGAAAGTTGCATCAGGAAGTGAACCTCGTGGTGATGAGAGCC ACT 840 LeuGluAlaLysThrGlyLysLeuHisGlnGluValAsnLeuValValMetArgAlaThr 295

*

961 CAGCTCCAGAAAAATTTGACCTGTGAGGTGTGGGGACCCACCTCCCCTAAGCTGATGCTG 296 GlnLeuGlnLysAsnLeuThrCysGluValTrpGlyProThrSerProLysLeuMetLeu

AGCTTGAAACTGGAGAACAAGGAGGCAAAGGTCTCGAAGCGGGAGAAGGCGGTGTGG GTG 1080 SerLeuLysLeuGluAsnLysGluAlaLysValSerLysArgGluLysAlaValTrpVal 335

1081 CTGAACCCTGAGGCGGGGATGTGGCAGTGTCTGCTGAGTGACTCGGGACAGGTCCTGCTG 336 LeuAsnProGluAlaGlyMetTrpGlnCysLeuLeuSerAspSerGlyGlnValLeuLeu

GAATCCAACATCAAGGTTCTGCCCACATGGTCCACCCCGGTGCAGCCAATGGCCCTG ATT 1200 GluSerAsnlleLysValLeuProThrTrpSerThrProValGlnProMetAlaLeuHe 375

1201 GTGCTGGGGGGCGTCGCCGGCCTCCTGCTTTTCATTGGGCTAGGCATCTTCTTCTGTGTC 376 ValLeuGlyGlyValAlaGlyLeuLeuLeuPhelleGlyLeuGlyllePhePheCysVal

AGGTGCCGGCACCGAAGGCGCCAAGCACAGCGGATGTCTCAGATCAAGAGACTCCTC AGT 1320 ArgCysArgHisArgArgArgGlnAlaGlnArgMetSerGlnileLysArgLeuLeuSer 415

Glu G

1321 GAGAAGAAGACCTGCCAGTGCCCTCACCGGTTTCAGAAGACATGTAGCCCCATTTGA 1377 416 GluLysLysThrCysGlnCysProHisArgPheGlnLysThrCysSerProIleEnd 433

The chimpanzee CD4 antigen is 99% homologous to its human counterpart, possessing 5 amino acid substitutions in the 433 amino acid predicted mature polypeptide, while the rhesus monkey CD4 is 92% homologous having 34 divergences from the human CD4 amino acid sequence. Antigen expression was effected transiently in CDM8 as well as stably using the retroviral vector pMNCS.

EXAMPLE 2 CHARACTERIZATION OF THE HUMAN CD4 DOMAIN WHICH IS REQUIRED FOR HIV MEDIATED SYNCYTIUM FORMATION

These non-human primate CD4 antigens were expressed on human cells which were thereby rendered susceptible to infection by HIV, but formed strikingly fewer multinucleated giant cells, or syncytia, than their counterparts expressing the human CD4 antigen. Using in vitro utagenesis this phenotype was localized to a single amino acid difference between the chimpanzee and human CD4 glycoproteins. This amino acid substitution quantitatively affects the ability of HeLa cells to form syncytia when these antigens are expressed in concert with the external and trans membrane proteins (EMP and TMP) of the human immunodeficiency virus type I (HIV). This was achieved by transiently expressing six trans-species hybrid CD4 antigens, which contain each of the three nonconservative extracellular amino acid sequence changes between the two species alone and in pairs, followed by infection with the Vaccinia:(HIV env) recombinant virus VSC25. The presence of a glycine residue at position 87, as found in chimpanzee CD4, instead of the glutamic acid residue found in human CD4, essentially eliminates the formation of multinucleated syncytia. Conversely the transfer of the human glutamic acid residue at position 87 to the chimpanzee CD4 confers the ability to form syncytia in the presence of HIV EMP and TMP. In contrast the absence or presence of either of the two amino acid substitutions which create glycosylation sites unique to the chimpanzee CD4 antigen, at amino acids 34 and 68 in the first immunoglobulin variable region homologous domain, has little or no effect on the extent of syncytium formed in this assay. We expect that all of these hybrid CD4 glycoproteins will show equal affinity for HIV EMP, since none of these amino acid sequence differences are in the HIV binding site defined earlier.

If syncytium formation is an important mechanism of HIV induced disease this blockade of HIV mediated syncytium formation may account for the resistance of the chimpanzee to the pathology of the acquired immune deficiency syndrome (AIDS) despite prolonged infection by HIV.

EXAMPLE 3 PREPARATION OF CD4-IG CDNA CONSTRUCTS

The Extracellular portion of the chimpanzee or rhesus monkey coding sequence (encoding the signal peptides and amino acids 1-372 of the mature glycoproteins) is fused at three locations to a human IgGl heavy chain constant region gene by means of a synthetic splice donor linker molecule. To exploit the splice donor linker, a BamHI linker having the sequence CGCGGATCCGCG is first inserted at amino acid residue 395 of the CD4 precursor sequence (nucleotide residue 1295). A synthetic splice donor sequence

GATCCCGAGGGTGAGTACTA GGCTCCCACTCATGATTCGA bounded by BamHI and Hindi11 complementary ends is created and fused to the Hindlll site in the intron preceding the CHI domain, to the Espl site in the intron preceding the hinge domain, and to the Banl site preceding the CH2 domain of the IgGl genomic sequence. Assembly of the chimeric genes by ligation at the BamHI site affords molecules in which either the variable (V) region, the V+CH1 regions, or the V, CHI and hinge regions are replaced by CD4. In the last case, the chimeric molecule is expected to form a monomer structure, while in the former, a dimeric molecule is expected. Immunoprecipitation of the fusion proteins with a panel of monoclonal antibodies directed against CD4 epitopes will show that all of the epitopes are preserved. A specific high affinity association is demonstrated between the chimeric molecules and HIV envelope proteins expressed on the surface

of cells transfected with an attenuated (reverse transcriptase deleted) proviral construct, or infected with a vaccinia:HIV env recombinant virus.

EXAMPLE 4 PREPARATION OF THE FUSION PROTEINS FROM SUPERNATANTS

OF COS CELLS

COS cells grown in DME medium supplemented with 10% Calf Serum and gentamicin sulfate at 15 μg/ml are split into DME medium containing 10% NuSerum (Collaborative Research) and gentamicin to give 50% confluence the day before transfection. The next day, CsCl purified plasmid DNA is added to a final concentration of 0.1 to 2.0 μg/ml followed by DEAE Dextran to 400 μg/ml and chloroquine to 100 μM. After 4 hours at 37 ^* C, the medium is aspirated and a 10% solution of dimethyl sulfoxide in phosphate buffered saline is added for 2 minutes, aspirated, and replaced with DME/10% Calf Serum. 8 to 24 hours later, the cells are trypsinized and split 1:2.

For radiolabeling, the medium is aspirated 40 to 48 hours after transfection, the cells are washed once with phosphate buffered saline, and DME medium lacking cysteine or methionine is added. 30 minutes later, 35s_ι _aD eled cysteine and methionine are added to final concentrations of 30-60 μci and 100-200 μci respectively, and the cells allowed to incorporate label for 8 to 24 more hours. The supernatants are recovered and examined by electrophoresis on 7.5% polyacrylamide gels following denaturation and reduction, or on 5% polyacrylamide following denaturation without reduction. The IgG-CD4 fusion proteins form dimer structures. The CD4-IgM fusion proteins form large multimers beyond the resolution of the gel system without reduction, and monomers of the expected molecular mass with reduction.

Unlabeled proteins are prepared by allowing the cells to grow for 5 to 10 days post transfection in DME medium

containing 5% NuSeru and gentamicin as above. The supernatants are harvested, centrifuged, and purified by batch adsorption to either protein A trisacryl, protein A agarose, goat anti-human IgG antibody agarose, rabbit anti-human IgM antibody agarose, or monoclonal anti-CD4 antibody agarose. Antibody agarose conjugates are prepared by coupling purified antibodies to cyanogen bromide activated agarose according to the manufacturer's recommendations, and using an antibody concentration of 1 mg/ml. Following batch adsorption by shaking overnight on a rotary table, the beads are harvested by pouring into a sintered glass funnel and washed a few times on the funnel with phosphate buffered saline containing 1% Nonidet P40 detergent. The beads are removed from the funnel and poured into a small disposable plastic column (Quik-Sep QS-Q column, Isolab), washed with at least 20 column volumes of phosphate buffered saline containing 1% Nonidet P40, with 5 volumes of 0.15 M NaCl, 1 mM EDTA (pH 8.0), and eluted by the addition of either 0.1 M acetic acid, 0.1 M acetic acid containing 0.1 M NaCl, or 0.25 M glycine-HCl buffer, pH 2.5.

EXAMPLE 5 BLOCKAGE OF SYNCYTIUM FORMATION BY THE FUSION PROTEINS

Purified or partially purified fusion proteins are added to HPB-ALL cells infected 12 hours previously with a vaccinia virus recombinant encoding HIV envelope protein. After incubation for 6-8 more hours, the cells are washed with phosphate buffered saline, fixed with formaldehyde, and photographed. All of the full-length CD4 immunoglobulin fusion proteins will show inhibition of syncytium formation.

Having now fully described this invention, it will be appreciated by those skilled in the art that the same can be performed with any wide range of equivalent parameters of

composition, conditions, and methods of preparing such recombinant molecules, vectors, transformed hosts and proteins without departing from the spirit or scope of the invention or any embodiment thereof.