WO2000040964A1 - Inhibition of cd4-ccr5 interaction - Google Patents

Abstract

A method is provided for identifying an agent which inhibits the interaction of CD4 and CCR5 in a cell, including contacting a cell which expresses CD4 and CCR5 with an agent under conditions sufficient to allow interaction between said cell and said agent and evaluating the interaction of CD4 and CCR5. The interaction of CD4 and CCR5 in the cell contacted with the agent is compared to the interaction of CD4 and CCR5 in a control cell. A decreased interaction of CD4 and CCR5 in the cell contacted with the agent indicates the ability of the agent to inhibit infection with an immunodeficiency lentivirus. A method is also provided for preventing or inhibiting infection of a cell expressing CCR5 and CD4 with an immunodeficiency lentivirus, including contacting the cell with an agent that inhibits an interaction of CCR5 and CD4. A pharmaceutical composition is provided including at least one dose of an agent which inhibits an interaction of CD4 and CCR5 in a pharmaceutically acceptable carrier.

Description

INHIBITION OF CD4-CCR5 INTERACTION

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119 to U.S. Serial No. 60/115,182, filed January 8, 1999.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH This invention was made in part with funds from the National Institutes of Health, Intramural Program. The government may have certain rights in this invention.

FIELD OF THE INVENTION The present invention relates to chemokine receptors. The invention more specifically relates to cell surface proteins that participate in HIV infection which are useful for the development of anti-HIV therapeutics.

BACKGROUND OF THE INVENTION An HIV infection cycle begins with the entry of the virus into a target cell. Entry commences when an HIV envelope glycoprotein (Env) binds to a human CD4 molecule in a target cell membrane. This binding leads to fusion of virus and cell membranes, thus releasing the virus genome into the cell and initiating the infection cycle.

Recent studies have shown that the HIV fusion process occurs with a wide range of human cell types that either express human CD4 endogenously or that have been engineered to express human CD4. The fusion process, however, does not occur with nonhuman cell types engineered to express human CD4 even though these nonhuman cells still can bind Env. The disparity between human and nonhuman cell types exists because membrane fusion requires the coexpression of human CD4 and one or more cofactors specific to human cell types (reviewed in Dimitrov, D.S. and Broder, C.C. 1997, HIV and Membrane Receptors, Austin, TX: Landes Biosciences). Nonhuman cell types that have been engineered to express human CD4 but not the additionally required factor(s) are incapable of membrane fusion, and therefore are nonpermissive for HIV infection.

It has been hypothesized that the entry cofactors may directly associate with the CD4-HIV-1 envelope glycoprotein (Env) (gpl20-gp41) complex and therefore serve as coreceptors (Golding, H., et al., J. Virol. 67:6469-6475, 1993; Dimitrov, D.S., Nature Medicine 2:640-641, 1996). The identification of the entry cofactors as chemokine receptors (Feng, Y., et al, Science 272:872-877, 1996; Alkhatib et al, Science 272:1955-1958, 1996; Deng, H., et al., Nature 381 :661-666, 1996; Dragic, T., et al., Nature 381:667-673, 1996; Choe et al.,

Cell 85:1135-1148, 1996; Doranz et al., Cell 85:1149-1158, 1996) not only solved a long-standing puzzle concerning HIV tropism and pathogenesis, but also provided new tools for understanding the role of these molecules for the mechanism of HIV entry. It was demonstrated that gpl20, CD4 and the HIV-1 coreceptor CXCR4 can be co-immunoprecipitated suggesting that the complex between these three molecules plays a critical role in the initial stages of the entry process (Lapham et al., Science 274:602-605, 1996). In addition, gpl20 has been shown to associate with another HIV-1 coreceptor, CCR5 (Wu et al., Nature 384:179-183, 1996; Trkola et al., Nature 384:184-187, 1996). The gp 120-induced formation of a CD4-gp 120-coreceptor complex was further confirmed by membrane colocalization analyses (Ugolini et al., J. Immunol. 159:3000-3008, 1997; Iyengar et al., J.Virol. 72:5251-5255, 1998) and antibody inhibition assays (Ugolini et al., J. Immunol. 159:3000-3008, 1997). Convincing evidence for the existence of an interaction of CD4 and CXCR4 in the absence of gpl20 is lacking, although some lines of evidence suggest that CD4 can associate, although weakly, with CXCR4 even in the absence of gpl20 (Lapham et al, Science 274:602-605, 1996; Ugolini et al., J. Immunol 159:3000-3008, 1997; Mebatsion et al., Cell 90:841-847, 1997). It was also noted that a soluble form of CD4, containing its first and second domain, can displace the chemokines MIP-1 and MIP-1 (Wuet al., Nature 384:179-183, 1996) although the soluble form of the entire extracellular portion of CD4 was much less efficient in displacing MIP-1 (Wuet al., Nature 384:179-183, 1996).

Some individual HIV isolates, designated "macrophage-tropic," efficiently infect primary macrophages but not immortalized T-cell lines. Other isolates, designated "T-cell line-tropic," have the opposite property and infect immortalized T-cell lines more efficiently than they infect primary macrophages. Both types of isolates readily infect primary T-cells from the body, however. The selective tropism of these two types of isolates is thought to be due to their requirements for distinct cofactors that are differentially expressed on different CD4 positive cell types. It should be understood that other HIV strains are "dual-tropic" and have the ability to infect both macrophages and immortalized T-cell lines and are believed to be able to use more than one cofactor.

SUMMARY OF THE INVENTION The present invention is based on the discovery of an interaction of CD4 and

CCR5 in the absence of an immunodeficiency virus envelope protein (Env), and that this interaction is important for Env-mediated membrane fusion. A method is provided for identifying an agent which inhibits the interaction of CD4 and CCR5 in a cell, including contacting a cell which expresses CD4 and CCR5 with an agent under conditions sufficient to allow interaction between said cell and said agent and evaluating the interaction of CD4 and CCR5. The interaction of CD4 and CCR5 in the cell contacted with the agent is compared to the interaction of CD4 and CCR5 in a control cell. A decreased interaction of CD4 and CCR5 in the cell contacted with the agent indicates the ability of the agent to inhibit infection with an immunodeficiency lentivirus, such as HIV.

A method is provided for preventing or inhibiting infection of a cell expressing CCR5 and CD4 with an immunodeficiency lentivirus (e.g., HIV), including contacting the cell with an agent that inhibits an interaction of CCR5 and CD4.

A pharmaceutical composition is provided including at least one dose of an agent which inhibits an interaction of CD4 and CCR5 in a pharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a gel illustrating specific coimmunoprecipitation (CO-IP) of cell surface associated CD4 and CCR5 from 3T3 cells coexpressing thee two molecules. (A) Equal numbers of 3T3 cells expressing CD4 and CCR5 (+) or CD4 only (-) were biotinylated, process and either used as a whole-cell lysate (0.25% of total, gel I) or immunoprecipitated with an anti-CD4 mAb (OKT4) (gel II), anti-CXCR4 mAb (4G10) gel III), or anti-CCR5 mAb 5C7 (gels IV-

VI). The biotinylated proteins were detected by using streptavidin-horseradish peroxidase (gels I-IV). CD4 and CCR5 were detected in an aliquot of the same samples as in gel IV by using Western blotting with an anti-CD4 polyclonal antibody (T4-4) (gel V) or an anti-CCR5 polyclonal antibody (CKR5(C20)) (gel VI). M denotes molecular markers, and the numbers are in kDa). (B) Co immunoprecipitation of CD4 by the anti-CCR5 mAbs ml 80 (lanel) and ml 81 (Lane2). The coimmunoprecipitated CD4 and the immunoprecipitated CCR5 were detected by using Western blotting as in A. (C) Coimmunoprecipitation of CCR5 from 3T3.CD4.CCR5 cells with anti-CD4 antibodies.

3T3.CD4.CCR5 cells (lanes 1, 2, and 4) or 3T3.CD4 cells (lane 3) were used for immunoprecipitation by OKT4 (lanes 2 and 3) or by a control antibody (CG10) lane 4. Lane 1 shows form comparison immunoprecipitation with the antiCCR5 mAb 5C7. CD4 and CCR5 were detected by using Western blotting as in A. (D) Cell lysates were immunoprecipitated by the anti-CCR5 mAb 5C7, the immunoprecipitation product was analyzed by using a silver stain kit (lanes 1 and 2) and compared with proteins detected by streptavidin-horseradish peroxidase in biotinylated lysates (lanes 3 and 4). M denotes molecular weight marker, and + and - denote 3T3.CD4.CCR5 or 3T3.CD4 cells, respectively, *, bands caused by CD4 and CCR5. The two bands above and below CD4 are used by the 5C7 mAb heavy and light chain, respectively. Lane 2 represents lane 1 at higher sensitivity, where CCR5 is clearly seen. (E) CD4-CCR5 coimmunoprecipitation is not significantly affected by cholesterol depletion. 3T3.CD4.CCR5 cells were treated with 10 mM methyl-β-cyclodextrin for 1 hr at 37°C (which caused significant cytotoxicity) and used for immunoprecipitation by the anti-CCR5 antibody 5C7 (lane 2), and compared with untreated cells (lane 1) and 3T3.CD4 cells (lane 3). CD4 and CCR5 were detected by using Western blotting as in A. Bottom shows Western blotting of CD4 from whole-cell lysates.

FIG. 2. shows coimmunoprecipitation of CD4 and CCR5 in primary cells. (A) Human CD4 T cells expressing high (+) and very high (++) amounts of CCR5 were used for immunoprecipitation with the anti-CCR5 mAb 5C7 (Center) or fusion with HeLa cells expressing the HIV-1 JRFL Env (Left and Right). The coimmunoprecipitated CD4 (Center Top) and immunoprecipitated CCR5 (Center Middle) were detected by using Western blotting as in Fig. 1 A. The CD4 Western blotting of whole-cell lysates is shown (Bottom) as a measure of the level of CD4. The average number of syncytia for ++ cells was 92 + 10.5, for + cells was 29 + 7, and for control HeLa cells was 6 + 3. The average diameter of syncytia from the ++ cells was about 4-fold larger than that for the + cells. (B) Human macrophages (Left, lane 1) and monocytes (Left, lane 2) wee used for CCR5 immunoprecipitation. CD4 coimmunopreceipitation was detected as in A. The CD4 Western blotting of whole-cell lysates is shown (Bottom) as a measure of the level of CD4. (Right) Fusion of these cells with

HeLa cells expressing the HIV-1 JRFL Env as quantitated by the β- galactosidase assay. The control represents HeLa cells that do not express HIV-1 Env.

FIG. 3. shows the effect of gpl20 on the CCR5-CD4 and CXCR4-CD4 association. (A) 3T3.CD4.CXCR4 cells were incubated with the anti-CXCR4 mAb 4G10 in the absence (-) or presence (+) (5 g/ml) of HIV-1 IIIB gpl20. The CD4 and CXCR4 were detected by using Western blotting with either the polyclonal anti-CD4 Ab T4-4 or 4G10. (B) 3T3.CD4.CCR5 cells were incubated with the anti-CCR5 mAb 5C7 in the absence or presence (5 g/ml) of HIV4 89.6 gpl20. The CD4 and CCR5 were detected by using Western blotting as described in Fig. 1 A. (C) The same as in B but gpl20 from the R5 HIV-1 JRFL was used instead of the dual tropic 89.6.

FIG. 4. demonstrates the involvement of the first two domains of CD4 and the second extracellular loop of CCR5 in the CD4-CCR5 association. (A) Coimmunoprecipitation of CD4-CD8 hybrid molecules containing the first two domains of CD4 by an anti-CCR5 mAb. The A2.01.T4.T8 cells expressing the hybrid CD4-CD8 molecule were infected with a recombinant vaccinia virus (vvCCR5-l 107) (lane 1), encoding the gene for CCR5, and a control wild-type (WR) vaccinia virus (lane 2). The anti-CCR5 mAb 5C7 was used to immunoprecipitate CCR5 and T4-4 for detection of CD4 by Western blotting.

(B) The amount of CD4-CD8 molecules in A2.01.T4.T8 cells infected with the CCR5 (lane 1) or WR (lane 2) vaccinia virus was not significantly different as demonstrated by Western blot with an anti-CD4 Ab (T4-4).

(C) Coimmunoprecipitation of CCR5 by an anti-CD4 mAb (OKT4) is inhibited in the presence of another anti-CD4 mAb (CG7). For comparison, lane 1 shows CCR5 immunoprecipitated by 5C7. Lanes 2, 3, and 4 represent the amount of CCR5 coimmunoprecipitated by a mixture of OKT4 (ascites fluid

3.5 1/ml) and increasing the concentrations of the anti-CD4 mAb CG7 (0, 5, and 10 g/ml, respectively). (D) CD4 coimmunoprecipitation by 5C7 is inhibited in the presence of another anti-CCR5 mAb (2D7) (Wu et al, J. Exp. Med., 186:1373-1381 (1997)) directed to the second extracellular loop. Equal amounts (3 g/ml) of 5C7, which does not affect HIV entry, were mixed with increasing amounts (0, 3, and 6 g/ml) (lanes 1, 2, and 3, respectively) of the HIV-1 blocking mAb 2D7 and used for immunoprecipitation. The sample obtained from 9 x 10⁶ 3T3.CD4.CCR5 cells was divided into two portions, and the smaller one (1/6 of total) was used for Western blot of CD4 by T4-4, and the rest were used for Western blot of CCR5 by the CKR5(C20). (E) The

CCR5-terminus-specific mAb 5C7 (lane 2) immunoprecipitates CCR5 much more efficiently than the CCR5 ecl-2-specific mAb 2D7 (lane 1). Equal amounts (4 g/ml) of these two mAbs were used for immunoprecipitation of CCR5 in 3T3.CD4.CCR5 cells. The molecular markers are shown on the right side (lane M). (F and G) Differential inhibition by two anti-CCR5 mAbs, ml 82 and ml 83 (which do not immunoprecipitate CCR5 as measured by the assay) of the CCR5 coimmunoprecipitation by the anti-CD4 mAb OKT4. Lanes 1, 2, and 3 represent 1, 2 and 4 g/ml of ml82 and ml83, respectively. CCR5 was detected by Western blotting with CKR5(C20). FIG. 5. shows the colocalization of CD4 and CXCR4 (Left) or CCR5 (Right). A CXCR4 (or CCR5)-myc tag-expressing HeLa cell, double stained for CD4 (green) and CXCR4 (or CCR5) (red) (Upper). Colocalization of the two molecules is demonstrated by the yellow (red-green colocalization) staining, suggesting their clusterization. Correlation maps of these images (Bottom) show the regions of true overlap of green and red staining selected from the noncolocalized staining and the background fluorescence, represented as white dots.

DETAILED DESCRIPTION OF THE INVENTION

It must be noted that as used herein and in the appended claims, the singular forms "a," "and," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and reference to "the antibody" includes reference to one or more antibodies and equivalents thereof known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs. Although any methods, devices and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, the preferred methods, devices and materials are now described.

All publications mentioned herein are incorporated herein by reference in full for the purpose of describing and disclosing the cell lines, antibodies, and methodologies which are described in the publications which might be used in connection with the presently described invention. The publications discussed above and throughout the text are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention.

In one embodiment, the invention provides a method for identifying an agent that interacts with CD4 and CCR5, by incubating a cell expressing CD4 and CCR5 with the agent under conditions sufficient to allow the components to interact, and evaluating the interaction of CD4 and CCR5, (e.g. by comparing to a control or by evaluating the binding of an immunodeficiency virus envelope protein with the cell). The phrase "agent that interacts with a

CD4/CCR5 interaction" denotes agonists, antagonist such as antibodies, a synthetic ligand of CD4 or CCR5, an agent which increases the intramolecular distance between CD4 and CCR5, or appropriate fragments of the natural or synthetic ligands which bind to CD4 or CCR5 and block the interaction of CD4 or CCR5 or affect immunodeficiency virus (e.g., HIV) interaction with CD4 and CCR5. The term includes both biologic agents and chemical compounds. The determination and isolation of ligand/compositions is well described in the art. See, e.g., Lerner, Trends NeuroSci. 17:142-146, 1994, which is hereby incorporated by reference in its entirety. "Incubating" includes conditions which allow contact between the test composition and CD4, CCR5, or the

CD4/CCR5 complex. Contacting includes in solution and in solid phase.

The term "CD4" refers to a 55 kDa transmembrane glycoprotein belonging to the immunoglobulin superfamily. In humans CD4 is expressed on peripheral T- cells, thymocytes, on macrophages and granulocytes and in specific regions of the brain. CD4 has been cloned, sequenced (Littman, D.R., 1987, Annual Review of Immunology 5: 561-84, herein incorporated by reference), and the crystal structure has been analyzed. The term "CCR5" refers to a seven transmembrane-spanning G-protein-coupled receptor that binds MIP-1 -alpha and also MIP-1 -beta and RANTES with high affinity and generate inositol phosphates in response to these chemokines. CCR5 has been cloned and sequenced (Raport, C.J., et al., J. Biol. Chem. 271(29): 17161-17166, 1996; Samson et al., Biochemistry 35(11):3362-3367, 1996, both herein incoφorated by reference).

The interaction of CD4 and CCR5 may be assayed directly using assays well known to one of skill in the art. An example of such an assay is a coimmunoprecipitation assay described herein (see Examples). The interaction can be measured utilizing antibodies that bind either CD4 or

CCR5. The binding of the HIV envelope protein with the cell expressing CD4 and CCR5 can also be measured. In one embodiment, the binding of an envelope protein to CD4/ CCR5 in a cell contacted with a test agent is compared to the binding of an envelope protein to CD4/CCR5 in a cell not contact with the test agent.

Retroviruses are RNA viruses wherein the viral genome is RNA. When a host cell is infected with a retrovirus, the genomic RNA is reverse transcribed into a DNA intermediate which is integrated very efficiently into the chromosomal DNA of infected cells. The integrated DNA intermediate is referred to as a provirus. The term "lentivirus" is used in its conventional sense to describe a genus of viruses containing reverse transcriptase. The lentiviruses include the "immunodeficiency viruses" which include human immunodeficiency virus (HIV) type 1 and type 2 (HIV-1 and HIV-2) and simian immunodeficiency virus (SIV). In the absence of effective therapy, most individuals infected with a human immunodeficiency virus develop acquired immune deficiency syndrome (AIDS) and succumb to either opportunistic infections and malignancies resulting from either the deterioration of the immune system or the direct effects of the virus. The retroviral genome and the pro viral DNA have three genes: the Gag, the Pol, and the Env, which is flanked by two long terminal repeat (LTR) sequences. The Gag gene encodes the internal structural (matrix, capsid, and nucleocapsid) proteins; the Pol gene encodes the RNA directed DNA polymerase (reverse transcriptase), and the Env gene encodes viral envelope glycoproteins.

The test ligand(s)/composition can be a combinatorial library for screening a plurality of compositions. Compositions identified in the method of the invention can be further evaluated, detected, cloned, sequenced, and the like, either in solution or after binding to a solid support, by any method usually applied to the detection of a specific DNA sequence such as PCR, oligomer restriction (Saiki, et al., Bio/Technology, 3:1008-1012, 1985), allele-specific oligonucleotide (ASO) probe analysis (Conner, et al, Proc. Natl. Acad. Sci. USA, 80:278, 1983), oligonucleotide ligation assays (OLAs) (Landegren, et al, Science, 241 : 1077, 1988), and the like. Molecular techniques for DNA analysis have been reviewed (Landegren, et al., Science, 242:229-237, 1988).

Any of a variety of procedures may be used to clone the genes of use with the method of the present invention when the test composition is in a combinatorial library or is expressed as a gene product (as opposed to a chemical composition). One such method entails analyzing a shuttle vector library of DNA inserts (derived from a cell which expresses the composition) for the presence of an insert which contains the composition gene. Such an analysis may be conducted by transfecting cells with the vector and then assaying for expression of the composition binding activity. The preferred method for cloning these genes entails determining the amino acid sequence of the composition protein. Usually this task will be accomplished by purifying the desired composition protein and analyzing it with automated sequencers. Alternatively, each protein may be fragmented as with cyanogen bromide, or with proteases such as papain, chymotrypsin or trypsin (Oike, Y., et al., J. Biol. Chem., 257:9751-9758, 1982; Liu, C, et al., Int. J. Pept. Protein Res., 21:209- 215, 1983). Although it is possible to determine the entire amino acid sequence of these proteins, it is preferable to determine the sequence of peptide fragments of these molecules.

The compositions of the present invention can be extracted and purified from the culture media or a cell by using known protein purification techniques commonly employed, such as extraction, precipitation, ion exchange chromatography, affinity chromatography, gel filtration and the like.

Compositions can be isolated by affinity chromatography using the modified receptor protein extracellular domain bound to a column matrix or by heparin chromatography.

Also included in the screening method of the invention is a combinatorial chemistry method for identifying chemical compounds that bind to CD4 or CCR5 and inhibit the interaction of CD4 and CCR5. Ligands/compositions can be assayed in immunodeficiency virus replication assays, such as the assay described herein to determine whether the composition inhibits or blocks HIV replication, or inhibits the binding of an envelope protein to the CD4/CCR5 complex.

The interaction of the CD4/CCR5 complex with an immunodeficiency virus, such as HIV, can be measured. Specifically, the interaction can be measured by the interaction of the CD4/CCR5 complex with an immunodeficiency envelope protein. In HIV, the Env protein includes gpl20, a glycoprotein of 120 kDa. In one embodiment, the envelope protein is gpl20. Although the inventors are under no duty to explain how the invention works, the following theory is provided, but is not intended to limit the present invention. Agents inhibiting the CD4/CCR5 interaction could interfere with HIV entry and HIV Env-mediated fusion by at least three possible mechanisms: 1) the agent may increase the distance between CD4 and CCR5 thereby affecting the interaction between the env gpl20 and CCR5 or CD4 in the gpl20-CD4/CCR5 complex, 2) the existence of preformed CD4/CCR5 complexes increases the efficiency of the trimolecular complex (gpl20-CD4- CCR5) formation, thus the disruption of a CD4/CCR5 complex decreases the entry efficiency, or 3) the binding of CD4 to CCR5 induces conformational changes in either or both molecules which can be altered to affect the subsequent stages of virus entry.

In one embodiment, a method is provided for identifying an agent that interferes with the replication of an immunodeficiency virus (e.g., HIV), by incubating a cell expressing CD4 and CCR5 with the agent under conditions sufficient to allow the components to interact, and then contacting the cell with the immunodeficiency virus (e.g., HIV). The ability of the agent to block the binding of an envelope protein of the immunodeficiency virus or to block or inhibit replication of the immunodeficiency virus is then measured. One of ordinary skill in the art can readily measure binding of gpl20 or the replication of an immunodeficiency virus. For example, in order to measure HIV replication, the production of p24 protein can be evaluated, the presence of HIV nucleic acids within cells or envelope mediated cell fusion can be evaluated. The interaction of the immunodeficiency virus in a cell contacted with the agent is compared to a control cell, such as a cell not contacted with the agent. In one embodiment, the present invention relates to substantially purified CD4 or CCR5-binding and/or blocking agents that interfere with the interaction of CD4 and CCR5 and thereby inhibit immunodeficiency virus replication. Without being bound by theory, such agents bind CD4 or CCR5 on a target cell, and would prevent or inhibit the CD4/CCR5 complex from forming or prevent or inhibit the interaction of an immunodeficiency virus, such as HIV, with the CD4/CCR5 complex. These agents could represent research and diagnostic tools in the study of HIV infection and the development of more effective anti-HIV therapeutics. In addition, pharmaceutical compositions comprising isolated and purified agents that interfere with the interaction of

CD4/CCR5 interaction may represent effective anti-HIV therapeutics.

One nonlimiting example of an agent that interferes with the interaction of CD4 and CCR5 that inhibit immunodeficiency virus replication or immunodeficiency virus induced cytokine production is an antibody which binds to either CD4 and CCR5 and thereby inhibits the CD4/CCR5 interaction.

In one embodiment, the invention provides a method for inhibiting the interaction of CD4 and CCR5 and inhibiting the interaction of CD4/CCR5 with an immunodeficiency virus (e.g., HIV) in a cell by contacting the cell with an effective amount of an antibody, or a biologically active fragment thereof, such that the antibody, or fragment thereof, which interferes with the interaction of CD4 and CCR5.

CD4 or CCR5 polypeptides can be used to produce antibodies which are immunoreactive or bind to epitopes of CD4 or CCR5 polypeptides. Pooled monoclonal antibodies with different epitopic specificities, as well as distinct monoclonal antibody preparations are included. The invention includes the use of commercially available monoclonal antibodies which recognize CD4. In one embodiment, the antibody specifically binds domain 1 and/or domain 2 of CD4. One specific nonlimiting example of an antibody of use with the method of the invention is mAb CG7 (see Examples). The invention also includes the use of antibodies which bind CCR5. In one embodiment, the antibody specifically binds the second extracellular loop (ecl-2) of CCR5. Specific nonlimiting examples of an antibodies of use with the method of the invention that bind CCR5 are 2D7 and ml 82 (see Examples section).

The preparation of additional monoclonal antibodies is conventional. See, for example, Kohler & Milstein, Nature 256:495, 1975; Coligan et al, sections

2.5.1-2.6.7; and Harlow et al, in: Antibodies: a Laboratory Manual, page 726, Cold Spring Harbor Pub., 1988, which are hereby incorporated by reference. Briefly, monoclonal antibodies can be obtained by injecting mice with a composition comprising an antigen, verifying the presence of antibody production by removing a serum sample, removing the spleen to obtain B lymphocytes, fusing the B lymphocytes with myeloma cells to produce hybridomas, cloning the hybridomas, selecting positive clones that produce antibodies to the antigen, and isolating the antibodies from the hybridoma cultures. Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of well-established techniques. Such isolation techniques include affinity chromatography with Protein-A Sepharose, size-exclusion chromatography, and ion-exchange chromatography. See, e.g., Coligan et al., sections 2.7.1-2.7.12 and sections 2.9.1-2.9.3; Barnes et al., "Purification of Immunoglobulin G (IgG)," in: Methods in Molecular Biology, Vol. 10, pages 79- 104, Humana Press, 1992.

Methods of in vitro and in vivo multiplication of monoclonal antibodies are well known to those skilled in the art. Multiplication in vitro may be carried out in suitable culture media such as Dulbecco's Modified Eagle Medium or RPMI 1640 medium, optionally supplemented by a mammalian serum such as fetal calf serum or trace elements and growth-sustaining supplements such as normal mouse peritoneal exudate cells, spleen cells, thymocytes or bone marrow macrophages. Production in vitro provides relatively pure antibody preparations and allows scale-up to yield large amounts of the desired antibodies. Large scale hybridoma cultivation can be carried out by homogenous suspension culture in an airlift reactor, in a continuous stirrer reactor, or in immobilized or entrapped cell culture. Multiplication in vivo may be carried out by injecting cell clones into mammals histocompatible with the parent cells, e.g., syngeneic mice, to cause growth of antibody-producing tumors. Optionally, the animals are primed with a hydrocarbon, especially oils such as pristane (tetramethylpentadecane) prior to injection. After one to three weeks, the desired monoclonal antibody is recovered from the body fluid of the animal.

Polyclonal antibodies can also be used in the method of the invention. The preparation of polyclonal antibodies is well-known to those skilled in the art. See, for example, Green et al., "Production of Polyclonal Antisera," in: Immunochemical Protocols, pages 1-5, Manson, ed., Humana Press, 1992; Coligan et al., "Production of Polyclonal Antisera in Rabbits, Rats, Mice and

Hamsters," in: Current Protocols in Immunology, section 2.4.1, 1992, which are hereby incorporated by reference.

Antibodies of the present invention may also be derived from subhuman primate antibody. General techniques for raising therapeutically useful antibodies in baboons can be found, for example, in Goldenberg et al., International Patent Publication WO 91/11465, 1991, and Losman et al., Int. J. Cancer 46:310, 1990, which are hereby incorporated by reference. Alternatively, a therapeutically useful antibody that blocks the interaction of CD4 and CCR5 may be derived from a "humanized" monoclonal antibody. Humanized monoclonal antibodies are produced by transferring mouse complementarity determining regions from heavy and light variable chains of the mouse immunoglobulin into a human variable domain, and then substituting human residues in the framework regions of the murine counterparts. The use of antibody components derived from humanized monoclonal antibodies obviates potential problems associated with the immunogenicity of murine constant regions. General techniques for cloning murine immunoglobulin variable domains are described, for example, by

Orlandi et al, Proc. Nat'l Acad. Sci. USA 86:3833, 1989, which is hereby incorporated in its entirety by reference. Techniques for producing humanized monoclonal antibodies are described, for example, by Jones et al., Nature 321:522, 1986; Riechmann et al, Nature 332:323, 1988; Verhoeyen et al., Science 239:1534, 1988; Carter et al., Proc. Nat'l Acad. Sci. USA 89:4285,

1992; Sandhu, Crit. Rev. Biotech. 12:437, 1992; and Singer et al., J. Immunol. 150:2844, 1993, which are hereby incorporated by reference.

Antibodies of the invention also may be derived from human antibody fragments isolated from a combinatorial immunoglobulin library. See, for example, Barbas et al., in: Methods: a Companion to Methods in Enzymology, Vol. 2, page 119, 1991; Winter et al., Ann. Rev. Immunol. 12:433, 1994, which are hereby incoφorated by reference. Cloning and expression vectors that are useful for producing a human immunoglobulin phage library can be obtained, for example, from STRATAGENE Cloning Systems (La Jolla, CA).

In addition, antibodies of the present invention may be derived from a human monoclonal antibody. Such antibodies are obtained from transgenic mice that have been "engineered" to produce specific human antibodies in response to antigenic challenge. In this technique, elements of the human heavy and light chain loci are introduced into strains of mice derived from embryonic stem cell lines that contain targeted disruptions of the endogenous heavy and light chain loci. The transgenic mice can synthesize human antibodies specific for human antigens, and the mice can be used to produce human antibody-secreting hybridomas. Methods for obtaining human antibodies from transgenic mice are described by Green et al., Nature Genet. 7:13, 1994; Lonberg et al., Nature 368:856, 1994; and Taylor et al., Int. Immunol.. 6:579, 1994, which are hereby incoφorated by reference.

The term "antibody" as used in this invention includes intact molecules as well as fragments thereof, such as Fab, F(ab')2, and Fv which are capable of binding the epitopic determinant. These antibody fragments retain some ability to selectively bind with its antigen or receptor and are defined as follows: (1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain;

(2) Fab', the fragment of an antibody molecule can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule;

(3) (Fab')2, the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab')2 is a dimer of two Fab' fragments held together by two disulfide bonds;

(4) Fv, defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and

- lϊ (5) Single chain antibody ("SCA"), defined as a genetically engineered molecule containing the variable region of the light chain, the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule.

Methods of making these fragments are known in the art. (See, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988, incoφorated herein by reference). As used in this invention, the term "epitope" means any antigenic determinant on an antigen to which the paratope of an antibody binds. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. An example of an epitopic determinant is domain 1 or domain 2 of CD4. A further example of an epitopic determinant is ecl2 of CCR5.

Antibody fragments of the present invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli of DNA encoding the fragment. Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab')2. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfliydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments. Alternatively, an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly. These methods are described, for example, by Goldenberg, U.S. patents No. 4,036,945 and No. 4,331,647, and references contained therein. These patents are hereby incoφorated in their entireties by reference. See also Nisonhoff et al., Arch. Biochem. Biophys. 89:230, 1960; Porter, Biochem. J. 73:119, 1959; Edelman et al., Methods in Enzymology, Vol. 1, page 422, Academic Press, 1967; and Coligan et al. at sections 2.8.1-2.8.10 and 2.10.1-2.10.4.

Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.

For example, Fv fragments comprise an association of VH and VL chains.

This association may be noncovalent, as described in Inbar et al., Proc. Nat'l Acad. Sci. USA 69:2659, 1972. Alternatively, the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde. See, e.g., Sandhu, supra. Preferably, the Fv fragments comprise VH and VL chains connected by a peptide linker. These single-chain antigen binding proteins (sFv) are prepared by constructing a structural gene comprising DNA sequences encoding the VH and VL domains connected by an oligonucleotide. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains. Methods for producing sFvs are described, for example, by Whitlow et al, Methods: a Companion to Methods in Enzymology, Vol. 2, page 97, 1991; Bird et al., Science 242:423-426, 1988; Ladner et al., U.S. patent No. 4,946,778; Pack et al., Bio/Technology 11:1271- 77, 1993; and Sandhu, supra.

Another form of an antibody fragment is a peptide coding for a single complementarity-determining region (CDR). CDR peptides ("minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See, for example, Larrick et al., Methods: a Companion to Methods in Enzymology, Vol. 2, page 106, 1991.

Antibodies which inhibit the interaction of CD4 and CCR5 can be prepared using an intact polypeptide or fragments containing small peptides of interest as the immunizing antigen. The polypeptide or a peptide used to immunize an animal can be derived from translated cDNA or chemical synthesis which can be conjugated to a carrier protein, if desired. Such commonly used carriers which are chemically coupled to the peptide include keyhole limpet hemocyanin (KLH), thyroglobulin, bovine serum albumin (BSA), and tetanus toxoid. The coupled peptide is then used to immunize the animal (e.g., a mouse, a rat, or a rabbit).

If desired, polyclonal or monoclonal antibodies can be further purified, for example, by binding to and elution from a matrix to which the polypeptide or a peptide to which the antibodies were raised is bound. Those of skill in the art will know of various techniques common in the immunology arts for purification and/or concentration of polyclonal antibodies, as well as monoclonal antibodies (See for example, Coligan et al., Unit 9, Current Protocols in Immunology, Wiley Interscience, 1991, incoφorated by reference).

The monoclonal antibody which inhibits the interaction of CD4 and CCR5, or a biologically active fragment thereof can be administered alone, or in combination with another agent. One specific, nonlimiting, example of an agent of use with the invention is a fragment of CD4 or CCR5 that binds or blocks the interaction of an immunodeficiency envelope protein (e.g., gpl20) with the CD4/CCR5 complex. Derivatives, analogs, and mutants of CD4 or CCR5 are useful in the method of the subject invention.

As used in connection with the present invention the term "polypeptide" refers to a polymer in which the monomers are amino acid residues which are joined together through amide bonds. When the amino acids are alpha-amino acids, either the L-optical isomer or the D-optical isomer can be used, the L-isomers being preferred. The terms "polypeptide" or "protein" as used herein is intended to encompass any amino acid sequence and include modified sequences such as glycoproteins. The term "polypeptide" is specifically intended to cover naturally occurring proteins, as well as those which are recombinantly or synthetically synthesized. Polypeptides of interest with the method of the invention are for example CD4 fragments that bind CCR5. Also included are polypeptides such as CD4 fragments, that bind to CCR5 and inhibit the binding of an immunodeficiency envelope protein to CCR5-CD4. Similarly of use with the method of the invention are, for example, CCR5 fragments that bind CD4 but inhibit the binding of an immunodeficiency envelope protein. All functional fragments of CD4 and CCR5 are included so long as they have the ability to inhibit the binding of human immunodeficiency virus envelope protein to a cell, or to inhibit (HIV) replication. In one embodiment, an antibody that binds CD4 or CCR5 can be administered with a CD4 or CCR5 fragment.

Minor modifications of the CD4 or CCR5 primary amino acid sequence may result in proteins that can bind CCR5 or CD4, respectively, but prevent the binding of an immunodeficiency envelope protein, as compared to the unmodified counteφart polypeptide described herein. Such modifications may be deliberate, as by site-directed mutagenesis, or may be spontaneous. The term "conservative variation" as used herein denotes the replacement of an amino acid residue by another, biologically similar residue. Examples of conservative variations include the substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine, and the like. The term "conservative variation" also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid provided that antibodies raised to the substituted polypeptide also immunoreact with the unsubstituted polypeptide.

An "epitope" is a region of a polypeptide capable of binding an immunoglobulin generated in response to contact with an antigen. The term "soluble" refers to a form of CD4 or CCR5 that is not inserted into a cell membrane. The term "fragment" refers to a portion of a polypeptide which exhibits at least one useful epitope. The term "functional fragments of a polypeptide," refers to all fragments of a polypeptide that retain an activity of the polypeptide. Biologically functional fragments, for example, can vary in size from a polypeptide fragment as small as an epitope capable of binding an antibody molecule to a large polypeptide capable of participating in the characteristic induction or programming of phenotypic changes within a cell. In one embodiment, a peptide fragment of CD4 containing domain 1, domain 2, or both domains of CD4 or a fragment containing modified domain 1, domain 2, or both domains is useful in a method of the invention. In another embodiment, a peptide fragment of CCR5 including the second extracellular domain (ecl-2) or a modified ecl-2 is of use with the method of the invention. Additionally, other variants and fragments of CD4 or CCR5 can be used in the present invention. Variants include analogs, homologs, derivatives, muteins and mimetics of CD4 or CCR5 that have the ability to inhibit the interaction with an immunodeficiency virus envelope protein. The variants and fragments can be generated directly from CD4 and CCR5 itself by chemical modification, by proteolytic enzyme digestion, or by combinations thereof. Additionally, genetic engineering techniques, as well as methods of synthesizing polypeptides directly from amino acid residues, can be employed.

Peptide or compounds that mimic the binding and function of CD4 or CCR5

("mimetics") can be produced by the approach outlined in Saragovi et al., Science 253: 792-95, 1991. Mimetics are molecules which mimic elements of protein secondary structure. See, for example, Johnson et al., "Peptide Turn Mimetics," in Biotechnology And Pharmacy, Pezzuto et al., Eds., (Chapman and Hall, New York, 1993). The underlying rationale behind the use of peptide mimetics is that the peptide backbone of proteins exists chiefly to orient amino acid side chains in such a way as to facilitate molecular interactions.

Variants and fragments also can be created by recombinant techniques employing genomic or cDNA cloning methods. Site-specific and region- directed mutagenesis techniques can be employed. See Current Protocols in Molecular Biology vol. 1, ch. 8, Ausubel et al., eds., J. Wiley & Sons 1989 & Supp. 1990-93; Protein Engineering, Oxender & Fox eds., A. Liss, Inc., 1987. In addition, linker-scanning and PCR-mediated techniques can be employed for mutagenesis. See PCR Technology, Erlich ed., Stockton Press, 1989; Current

Protocols in Molecular Biology, vols. 1 & 2, supra. Protein sequencing, structure and modeling approaches for use with any of the above techniques are disclosed in Protein Engineering, loc. cit., and Current Protocols in Molecular Biology, vols. 1 & 2, supra. If the compounds described above are employed, the skilled artisan can routinely insure that such compounds are amenable for use with the present invention utilizing assays such as ELISA assays for cytokines (e.g., MIP-1 , and MIP-1 ) known in the art, or for example, the assays of CD4/CCR5 binding and the interaction with immunodeficiency virus described herein. If a compound blocks the binding of an immunodeficiency envelope protein to a CD4/CCR5 complex, the compounds polypeptides, peptides or mimetics are suitable according to the invention.

The term "substantially purified" as used herein refers to a polypeptide which is substantially free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated. One skilled in the art can purify CD4 or CCR5 or a fragment thereof using standard techniques for protein purification, and the purity of the polypeptides can be determined using standard methods including for example, polyacrylamide gel electrophoresis, (e.g., SDS-PAGE), column chromatography (e.g., high performance liquid chromatography (HPLC)), and amino-terminal sequence analysis. Isolation and purification of microbially expressed CD4 or CCR5 polypeptide, or fragments thereof, provided by the invention, may be carried out by conventional means including preparative chromatography and immunological separations involving monoclonal or polyclonal antibodies.

The CD4 or CCR5 mutant or fragment of use with the subject invention can be administered alone, or can be administered in conjunction with another agent that affects the interaction of CD4/CCR5 with an immunodeficiency envelope protein. An "agent" is any polypeptide, peptidomimetic, chemical compound, biological agent, or molecule with a desired function. The present invention also provides gene therapy for the administration of fragments of CD4 or CCR5 of use with the method of the invention. Such therapy would achieve its therapeutic effect by introduction of a therapeutic polynucleotide into cells at risk of infection with an immunodeficiency virus. The "therapeutic polynucleotide" may be polynucleotide sequences encoding a fragment or mutant of CD4 or CCR5 or encode a polypeptide that binds to CD4 or CCR5 capable of inhibition CD4-CCR5 interaction or envelope binding. These polynucleotides include DNA, cDNA and RNA sequences. Such polynucleotides include naturally occurring, synthetic, and intentionally manipulated polynucleotides. For example, a CD4 or CCR5 encoding polynucleotide may be subjected to site-directed mutagenesis. The polynucleotides of the invention include sequences that are degenerate as a result of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences are included in the invention. The term "polynucleotide" or "nucleic acid sequence" refers to a polymeric form of nucleotides at least 10 bases in length. The nucleotides of the invention can be ribonucleotides, deoxyribonucleotides, or modified forms of either nucleotide. The term includes single and double stranded forms of DNA. By "isolated polynucleotide" is meant a polynucleotide that is not immediately contiguous with both of the coding sequences with which it is immediately contiguous (one on the 5' end and one on the 3' end) in the naturally occurring genome of the organism from which it is derived.

The invention includes signal sequences, such as expression control sequences required for secretion of a polypepetide, operatively linked to sequences encoding a CD4 or CCR5 polypeptide of use with the subject invention. "Operatively linked" refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. An expression control sequence operatively linked to a coding sequence is ligated such that expression of the coding sequence is achieved under conditions compatible with the expression control sequences. As used herein, the term "expression control sequences" refers to nucleic acid sequences that regulate the expression of a nucleic acid sequence to which it is operatively linked. Expression control sequences are operatively linked to a nucleic acid sequence when the expression control sequences control and regulate the transcription and, as appropriate, translation of the nucleic acid sequence. Thus expression control sequences can include appropriate promoters, enhancers, transcription terminators, a start codon (i.e., ATG) in front of a protein-encoding gene, splicing signal for introns, maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and secretion signals, and stop codons. The term "control sequences" is intended to included, at a minimum, components whose presence can influence expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences. Expression control sequences can include a promoter.

By "promoter" is meant minimal sequence sufficient to direct transcription. Also included in the invention are those promoter elements which are sufficient to render promoter-dependent gene expression controllable for cell-type specific, tissue-specific, or inducible by external signals or agents; such elements may be located in the 5' or 3' regions of the gene. Both constitutive and inducible promoters, are included in the invention (see e.g., Bitter et al., Methods in Enzymology 153:516-544, 1987). For example, when cloning in bacterial systems, inducible promoters such as pL of bacteriophage , plac, ptφ, ptac (ptφ-lac hybrid promoter) and the like may be used. When cloning in mammalian cell systems, promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the retrovirus long terminal repeat; the adenovirus late promoter; the vaccinia virus 7.5K promoter) may be used. Promoters produced by recombinant DNA or synthetic techniques may also be used to provide for transcription of the nucleic acid sequences of the invention.

In the present invention, the polynucleotide encoding a CD4 or CCR5 polypeptide or polypeptide fragment of use with the subject invention may be inserted into an expression vector which contains a promoter sequence which facilitates the efficient transcription of the inserted genetic sequence of the host. The expression vector typically contains an origin of replication, a promoter, as well as specific genes which allow phenotypic selection of the transformed cells. Vectors suitable for use in the present invention include, but are not limited to the pMSXND expression vector for expression in mammalian cells (Lee and Nathans, J. Biol. Chem. 263:3521, 1988) and retrovirus derived vectors. The DNA segment can be present in the vector operably linked to regulatory elements, for example, a promoter (e.g., immunoglobulin, T7, metallothionein I, or polyhedron promoters). The term therefore includes, for example, a recombinant DNA which is composed of a promotor and a secretion sequence operably linked to a nucleic acid sequence encoding a CD4 or CCR5 polypeptide or fragment thereof.

Delivery of the therapeutic polynucleotide can be achieved using a recombinant expression vector such as a chimeric virus or a colloidal dispersion system. Especially preferred for therapeutic delivery of nucleic acid sequences is the use of targeted liposomes.

Various viral vectors which can be utilized for gene therapy as taught herein include adenovirus, heφes virus, vaccinia, or, preferably, an RNA virus such as a retrovirus. Preferably, the retroviral vector is a derivative of a murine or avian retrovirus. Examples of retroviral vectors in which a single foreign gene can be inserted include, but are not limited to: Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV). Preferably, when the subject is a human, a vector such as the gibbon ape leukemia virus (GaLV) is utilized. A number of additional retroviral vectors can incoφorate multiple genes. All of these vectors can transfer or incoφorate a gene for a selectable marker so that transduced cells can be identified and generated. By inserting a sequence encoding a fragment or mutant CD4 or CCR5 polypeptide into the viral vector, along with another gene which encodes the ligand for a receptor on a specific target cell, for example, the vector is now target specific. Retroviral vectors can be made target specific by attaching, for example, a sugar, a glycolipid, or a protein. Preferred targeting is accomplished by using an antibody to target the retroviral vector. Those of skill in the art will know of, or can readily ascertain without undue experimentation, specific polynucleotide sequences which can be inserted into the retroviral genome or attached to a viral envelope to allow target specific delivery of the retroviral vector containing a the CD4 or CCR5 sequences of use with the method of the invention.

Since recombinant retroviruses are defective, they require assistance in order to produce infectious vector particles. This assistance can be provided, for example, by using helper cell lines that contain plasmids encoding all of the structural genes of the retrovirus under the control of regulatory sequences within the LTR. These plasmids are missing a nucleotide sequence which enables the packaging mechanism to recognize an RNA transcript for encapsidation. Helper cell lines which have deletions of the packaging signal include, but are not limited to Q2, PA317, and PA12, for example. These cell lines produce empty virions, since no genome is packaged. If a retroviral vector is introduced into such cells in which the packaging signal is intact, but the structural genes are replaced by other genes of interest, the vector can be packaged and vector virion produced.

Alternatively, NIH 3T3 or other tissue culture cells can be directly transfected with plasmids encoding the retroviral structural genes Gag, Pol and Env, by conventional calcium phosphate transfection. These cells are then transfected with the vector plasmid containing the genes of interest. The resulting cells release the retroviral vector into the culture medium.

Another targeted delivery system for the therapeutic polynucleotides is a colloidal dispersion system. Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. The preferred colloidal system of this invention is a liposome.

Liposomes are artificial membrane vesicles which are useful as delivery vehicles in vitro and in vivo. It has been shown that large unilamellar vesicles (LUV), which range in size from 0.2-4.0 m can encapsulate a substantial percentage of an aqueous buffer containing large macromolecules. RNA, DNA and intact virions can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form (Fraley et al., Trends Biochem. Sci. 6:77, 1981). In addition to mammalian cells, liposomes have been used for delivery of polynucleotides in plant, yeast and bacterial cells. In order for a liposome to be an efficient gene transfer vehicle, the following characteristics should be present: (1) encapsulation of the genes of interest at high efficiency while not compromising their biological activity; (2) preferential and substantial binding to a target cell in comparison to non-target cells; (3) delivery of the aqueous contents of the vesicle to the target cell cytoplasm at high efficiency; and (4) accurate and effective expression of genetic information (Mannino et al., Biotechniques 6:682, 1988).

The composition of the liposome is usually a combination of phospholipids, particularly high-phase-transition-temperature phospholipids, usually in combination with steroids, especially cholesterol. Other phospholipids or other lipids may also be used. The physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations.

Examples of lipids useful in liposome production include phosphatidyl compounds, such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides. Particularly useful are diacylphosphatidyl-glycerols, where the lipid moiety contains from 14-18 carbon atoms, particularly from 16-18 carbon atoms, and is saturated. Illustrative phospholipids include egg phosphatidylcholine, dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.

The targeting of liposomes can be classified based on anatomical and mechanistic factors. Anatomical classification is based on the level of selectivity, for example, organ-specific, cell-specific, and organelle-specific. Mechanistic targeting can be distinguished based upon whether it is passive or active. Passive targeting utilizes the natural tendency of liposomes to distribute to cells of the reticulo-endothelial system (RES) in organs which contain sinusoidal capillaries. Active targeting, on the other hand, involves alteration of the liposome by coupling the liposome to a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein, or by changing the composition or size of the liposome in order to achieve targeting to organs and cell types other than the naturally occurring sites of localization. The surface of the targeted delivery system may be modified in a variety of ways. In the case of a liposomal targeted delivery system, lipid groups can be incoφorated into the lipid bilayer of the liposome in order to maintain the targeting ligand in stable association with the liposomal bilayer. Various linking groups can be used for joining the lipid chains to the targeting ligand.

The mvention also contemplates various pharmaceutical compositions that inhibit or block immunodeficiency virus infection. The pharmaceutical compositions according to the invention are prepared by bringing an agent that blocks the interaction of CD4 and CCR5 (e.g., an antibody against CD4 or

CCR5, an isolated and purified peptide fragment of CD4 or CCR5, a nucleic acid sequence encoding a fragment of CD4 or CCR5, or an isolated agent according to the present invention) into a form suitable for administration (e.g., a pharmaceutically acceptable carrier) to a subject using carriers, excipients and additives or auxiliaries. Frequently used carriers or auxiliaries include magnesium carbonate, titanium dioxide, lactose, mannitol and other sugars, talc, milk protein, gelatin, starch, vitamins, cellulose and its derivatives, animal and vegetable oils, polyethylene glycols and solvents, such as sterile water, alcohols, glycerol and polyhydric alcohols. Intravenous vehicles include fluid and nutrient replenishers. Preservatives include antimicrobial, anti-oxidants, chelating agents and inert gases. Other pharmaceutically acceptable carriers include aqueous solutions, nontoxic excipients, including salts, preservatives, buffers and the like, as described, for instance, in Remington's Pharmaceutical Sciences, 15th ed., Easton: Mack Publishing Co., 1405-1412, 1461-1487, 1975, and The National Formulary XIV., 14th ed., Washington: American

Pharmaceutical Association, 1975, the contents of which are hereby incoφorated by reference. The pH and exact concentration of the various components of the pharmaceutical composition are adjusted according to routine skills in the art. See Goodman and Gilman's The Pharmacological Basis for Therapeutics, 7th ed.

In another embodiment, the invention relates to a method of blocking infection with immunodeficiency virus. This method involves administering to a subject a therapeutically effective dose of a pharmaceutical composition containing the compounds of the present invention and a pharmaceutically acceptable carrier. "Administering" the pharmaceutical composition of the present invention may be accomplished by any means known to the skilled artisan. The term "ameliorate" refers to a decrease or lessening of the symptoms of a disorder being treated, or a lessening of the susceptible to an immunodeficiency virus infection. By "subject" is meant any mammal, preferably a human.

The pharmaceutical compositions are preferably prepared and administered in dose units. Solid dose units are tablets, capsules and suppositories. For treatment of a patient, depending on activity of the compound, manner of administration, nature and severity of the disorder, age and body weight of the patient, different daily doses are necessary. Under certain circumstances, however, higher or lower daily doses may be appropriate. The administration of the daily dose can be carried out both by single administration in the form of an individual dose unit or else several smaller dose units and also by multiple administration of subdivided doses at specific intervals.

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 the disease in the patient and can be determined by one skilled in the art. The dosage can be adjusted by the individual physician in the event of any contraindications and can be readily ascertained without resort to undue experimentation. In any event, the effectiveness of treatment can be determined by monitoring the level of CD4+ T-cells in a patient infected with an immunodeficiency virus. An increase or stabilization in the relative number of CD4+ cells should correlate with recovery of the patient's immune system.

The pharmaceutical compositions according to the invention are in general administered topically, intravenously, orally or parenterally or as implants, but even rectal use is possible in principle. Suitable solid or liquid pharmaceutical preparation forms are, for example, granules, powders, tablets, coated tablets,

(micro)capsules, suppositories, syrups, emulsions, suspensions, creams, aerosols, drops or injectable solution in ampule form and also preparations with protracted release of active compounds, in whose preparation excipients and additives and/or auxiliaries such as disintegrants, binders, coating agents, swelling agents, lubricants, flavorings, sweeteners or solubilizers are customarily used as described above. The pharmaceutical compositions are suitable for use in a variety of drug delivery systems. For a brief review of present methods for drug delivery, see Langer, Science, 249:1527-1533, 1990, which is incoφorated herein by reference.

The pharmaceutical compositions according to the invention may be administered locally or systemically. By "therapeutically effective dose" is meant the quantity of a compound according to the invention necessary to prevent, to cure or at least partially arrest the symptoms of a disease and its complications or to decrease the ability of an immunodeficiency virus to infect or replicate in a cell. Amounts effective for this use will, of course, depend on the severity of the disease and the weight and general state of the patient. Typically, dosages used in vitro may provide useful guidance in the amounts useful for in situ administration of the pharmaceutical composition, and animal models may be used to determine effective dosages for treatment of particular disorders. Various considerations are described, e.g., in Gilman et al., eds., Goodman and Gilman's: the Pharmacological Bases of Therapeutics, 8th ed., Pergamon Press, 1990; and Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Co., Easton, Pa., 1990, each of which is herein incoφorated by reference. Effectiveness of the dosage can be monitored by CD4+ count using methods well known to one of ordinary skill in the art.

The pharmaceutical compositions of the invention, including antibodies, peptides, peptidomimetics, chemical compositions, etc., are all useful for treating subjects either having or at risk of having an immunodeficiency virus (e.g., HIV) related disorder. AIDS and ARC are preferred examples of such disorders. HIV-associated disorders have been recognized primarily in "at risk" groups, including homosexually active males, intravenous drug users, recipients of blood or blood products, and certain populations from Central

Africa and the Caribbean. The syndrome has also been recognized in heterosexual partners of individuals in all "at risk" groups and in infants of affected mothers.

The immunotherapeutic method of the invention includes a prophylactic method directed to those hosts at risk for the immunodeficiency virus infection. For example, the method is useful for humans at risk for HIV infection. A "prophylactically effective" amount of antibody or peptide, for example, refers to that amount which is capable of inhibiting HIV replication transmission, and infertility of cells. Transmission of HIV occurs by at least three known routes: sexual contact, blood (or blood product) transfusion and via the placenta. Infection via blood includes transmission among intravenous drug users. Since contact with HIV does not necessarily result in symptomatic infection, as determined by seroconversion, all humans may be potentially at risk and, therefore, should be considered for prophylactic treatment by the therapeutic method of the invention.

The compositions described herein and useful in the method of the invention can be administered to a patient prior to infection with an immunodeficiency virus (i.e., prophylactically) or at any of the stages described below, after initial infection. For example, HIV infection may run any of the following courses: 1) approximately 15% of infected individuals have an acute illness, characterized by fever, rash, and enlarged lymph nodes and meningitis within six weeks of contact with HIV. Following this acute infection, these individuals become asymptomatic. 2) The remaining individuals with HIV infection are not symptomatic for years. 3) Some individuals develop persistent generalized lymphadenopathy (PGL), characterized by swollen lymph nodes in the neck, groin and axilla. Five to ten percent of individuals with PGL revert to an asymptomatic state. 4) Any of these individuals may develop AIDS-related complex (ARC); patients with ARC do not revert to an asymptomatic state. 5) Individuals with ARC and PGL, as well as asymptomatic individuals, eventually (months to years later) develop AIDS which inexorably leads to death.

EXAMPLES Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following examples are illustrative only, and not limiting of the remainder of the disclosure in any way whatsoever. EXAMPLE 1 EXPERIMENTAL PROCEDURES Cells, viruses, chemokines, soluble CD4 fragments and mAbs 3T3 cells expressing CD4, CD4 and CCR5 or CXCR4 (Deng et al, Nature 381 :661-666, 1996), and A2.01.T4.T8 cells expressing hybrid CD4-CD8 molecules were provided by D. Littman. Primary human CD4 T cells were purified from an apheresis of a healthy donor by negative selection to >95% purity (June et al., Mol. Cell Biol. 7:4472-4481 (1987)). Purified cells were cultured at 10⁶ cells per ml in PRMI with 10% FCS (HyClone), 2 mM glutamine, and 50 mg/ml gentamicin (Bio fluids, Rockville, MD). These cells were stimulated with either 5 mg/ml Con A (Calbiochem) and 100 units/ml IL- 2 (Boehringer Mannheim) (+ cells) or 5 x 10⁵ cells per ml L cells stably transfected with the human CD32 gene (received from G.Delespesse, University of Montreal, Canada) and 200 ng/ml anti-CD3 antibody UCHT1 (Beverley & Callard, Eur, J. Immunol. 11 :329-334 (1981)) (++ cells). After 3 days of culture, CD4 T cells were removed from the L cells, and 100 units/ml IL-2 was added. Human macrophages and monocytes were obtained from peripheral blood. Monocytes were allowed to differentiate for 14 days in the presence of human macrophage colony-stimulating factor. (Recombinant vaccinia viruses required for the reporter gene fusion assay (Nussbaum et al.,

J.Virol. 68:5411-5422, 1994) and for expression of the R5 HIV Env (Bal, JRFL) (Broder and Berger, Proc. Natl. Acad. Sci. U.S.A. 92:9004-9008, 1995; Feng et al, Science 272:872-877, 1996) and CD4 (Broder et al., Virology 193:483-491, 1993) were previously described. The vaccinia virus containing the gene for CCR5 (vvCCR5-l 107) was developed by using the CCR5 cDNA from pCDNA3 (provided by M. Parmentier, Universite Libre de Bruxelles, Belgium) which was subcloned into the Smal site of pMC1107 (Carroll, 1993) by BamHl-Xbal restriction and blunt-end cloning into the Smal site. The recombinant vaccinia virus was then obtained using standard techniques employing Ecogpt selection (Broder and Earl, Methods Mol. Biol. 62:173-197, 1997) The 1251-labeled human MIP-1 was purchased from DuPont NEN (Boston, MA), and unlabeled chemokines were from R&D systems (Minneapolis, MN). The soluble D1D2CD4 fragment was a gift from J. Sodroski, and/or the anti-CD4 mAbs (CG7, CG10 or CGI) a gift from J.

Gershoni and G. Denisova. The anti-CD4 mAb Q4120 was previously described (Healey et al., J.Exp.Med. 172:1233-1242, 1990). The anti-CD4 polyclonal antibody T4-4 was obtained through the AIDS Research and Reference Reagent Program from R. Sweet (SmithKline Beechman Pharmaceuticals). The anti-CCR5 mAbs ml 80, ml 81, ml 82 and ml 83 were purchased from R&D Systems (Minneapolis, MN), 2D7 and 5C7 were previously described (Wu et al, J. Exp. Med. 185:1681-1691, 1997). The goat polyclonal anti-CCR5 antibody CKR5(C20) was purchased from Santa Cruse Biotechnology, Inc. (Santa Cruz, CA).

Immunoprecipitation

Cells (typically 5-10 x 10 per sample) were washed once with phosphate- buffered saline (PBS), labeled with biotin if needed, and then resuspended in cold (4oC) PBS at a final density of 10 cells/ml. Immunoprecipitating antibodies at the required concentration, typically 1.5-3 μg/ml, were added to the cell suspension and incubated with gentle mixing for 4 hours at 4°C. Cells were then pelleted by centrifugation and resuspended in lysis buffer (1% Brij97, 5 mM iodoacetamide (added immediately before use), 150 mM NaCl, 20 mM Tris (pH 8.2), 20 mM EDTA, and protease inhibitors) at 4°C for 40 min with gentle mixing. In an alternative protocol the cells were first lysed and then incubated with the immunoprecipitating antibody with essentially the same results. The nuclei were pelleted by centrifugation at about 17,000 φm for 25 min in a refrigerated Eppendorf centrifuge. Protein G-Sepharose beads (Sigma, St. Louis, MO) prewashed with PBS were added to the samples and incubated at 4°C for 14 hours. The beads were then washed four times with one ml of ice cold lysis buffer. Samples were then eluted by adding 4X sample buffer for SDS-PAGE gel and boiled for 5 min or kept overnight at 37 °C with essentially the same result. They were run on a 10% SDS-PAGE gel and were electrophoretically transferred to nitrocellulose membranes. The membranes were blocked with 20 mM tris-HCI (pH 7.6) buffer containing 140 mM NaCl, 0.1% Tween-20 and 5% nonfat powdered milk. For Western blotting these membranes were incubated with the respective antibodies, then washed and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies. For detection of cell surface biotinylated proteins the nitrocellulose membranes were incubated with streptavidin-conjugated HRP. In both cases they were developed by using the supersignal chemiluminescent substrate from Pierce (Rockford, II). The images were acquired by a BioRad phosphoimager (BioRad, Hercules, CA) at the highest resolution (0.1 mm) or by using sensitive film, and printed by a laser printer (Lexmark Optra S 1650) at the highest resolution (1200x1200). Silver staining was performed by using the Silver Stain Plus kit following the company protocol (Bio-Rad).

Confocal Laser Scanning Microscopy HeLa Cd4 cells (Maddon et al, Cell 47:333-348 ( 1996)) maintained in DMEM supplemented with 10 % FCS and 500 ug G418 (Gibco/BRL) were transiently transfected with an expression vector containing myc-tagged CCR5 (Ugolini et al., J. Immunol. 159, 3000-3008 (1997)). Transfected cells were evaluated for expression of CCR5 at the cell surface by using indirect immunofluorescent staining with the anti-myc mAb 9E10 (Evan et al, Mol. Cell Biol. 5:3610-3616

(1985)) followed by anti-Mo-phycoerythrin and flow cytometric analysis (Ugolini et al., supra) by using a Leica TCS 4D instrument (Leica, Heidelberg, Germany) interfaced with an argon krypton ion laser and with fluorescence filters and detectors allowing detection of FITC and Texas Red markers. To identify fluorescence localization, correlation maps were calculated by using a local statistical method (Demandolx & Davoust, J. Microsc. (Oxford) 185 (1997)).

Cell-cell fusion assay

The cell-cell fusion assay was previously described (Nussbaum et al., J.Virol. 68:5411-5422, 1994). Briefly, recombinant vaccinia viruses at multiplicity of infection 10 were used to infect the target (vCB21R) and effector cells (vTF 7.3 plus virus expressing the Bal Env). The -gal fusion assay was performed two hours after mixing the cells. The extent of fusion was quantitated colorimetrically.

EXAMPLE 2 MEMBRANE- ASSOCIATED CD4 CAN BE CO-IMMUNOPRECIPITATED BY ANTIBODIES TO CCR5 AND VICE VERSA

It was previously found that the soluble D1D2 domain fragment but not the entire extracellular portion of CD4 competes with the chemokine MIP-1 for binding to CCR5 (Wu et al., 1996, supra). Although the differences between the two-domain and the four-domain fragments of CD4 could be attributed to a better exposure of conformational epitopes in the two-domain CD4 fragment and orientation effects, another possibility is that the CD4D1D2-CCR5 interaction does not reflect the properties of the wild type (membrane- associated) CD4 binding to CCR5. Therefore, an immunoprecipitation assay was used to directly detect the interactions between the native membrane- associated CD4 and CCR5 (see Example 1).

To demonstrate coimmunoprecipitation of cell surface-associated CD4 and CCR5, 3T3 cell lines transfected to express CD4 (3T3, CD4), CD4 and CXCR4 (3T3, CD4.CxCR4) or CD4 and CCR5 (3T3, CD4.CCR5) were used. The CD4 concentrations at the surface of these cells were very similar: their ratios were 1.26:1.2:1, respectively, as measured by using flow cytometry (see FIG 1A, lane II) The surface concentrations of CCR5 and CXCR4 were also very similar. Because CD4 is easier to detect by Western blotting and biotinylation that CCR5, in most cases anti-CCR5 antibodies were used to coimmunoprecipitate CD4. It was found that the CCR5 N-terminus-specific mAb 5C7 (Wu et al. J. Exp. Med. 186:1373-1381 (1997))(commercially available from Leukocyte, Inc.) was the most efficient antibody in immunoprecipitating CCR5 compared with a battery of other anti-CCR5 antibodies. This mAb coimmunoprecipitated surface-associated CD4 in 3T3 lines coexpressing CD4 and CCR5 (Fig. 1 A, lane IV) The band was CD4 because it aligns with the CD4 band obtained by direct immunoprecipitation using an anti-CD4mab (OCT4) (Fig. 1 A, lane II), has the expected molecular weight (55 kDa), and reacts specifically in the Western blot assay (Fig. 1 A, lane V). CD4 was highly and specifically enriched in the coimmunoprecipitates as was observed by comparing of cell-surface biotinylated lysates that were not subjected to immunoprecipitation (Fig. 1A, lane I) with the co immunoprecipitates (Fig. 1 A, lane IV). In these experiments, CCR5 was not detected because of the low efficiency of biotinylation; however, it was observed by using Western blotting (Fig. 1 A, lane VI) Similar results were obtained for a number of cell lines, including PM1 and LI.2 transfectants that co express CCR5 and CD4.

The specificity of the CD4 coimmunoprecipitation was demonstrated through several control experiments. Then anti-CCR5 mAb 5C7 did not coimmunoprecipitate CD4 from 3T3.CD44 cells that do not express CCR5 (Fig. 1 A, lanes IV and V). In another experiment, an antibody (4G10) to a related chemokine receptor CCXCR4 was used. This antibody is able to efficiently immunoprecipitate CXCR4 and coimmunoprecipitate CD4 and CXCR4 in cells expressing these two molecules (Fig. 3A) but did not coimmunoprecipitate CD4 in cells expressing CD4 and CCR5 (Fig. 1A, lane III). In addition, the amount of coimmunoprecipitated CD4 was proportional to the amount of immunoprecipitated CCR5, demonstrated by using two anit- CCR5 mAbs with different immunoprecipitating activities (Fig. IB), further suggesting a specific CD4-CCR5 interaction. CCR5 was coimmunoprecipitated with an anti-CD4 antibody (OKT4) (Fig. 1C, lane 2), but not with a control antibody (CG10), which does not bind to CD4 (lane 4), in cells coexpressing these two molecules (lane 2), but not in CCR5 negative cells (lane 3).

To further evaluate the specificity of the CD4-CCR5 interaction and to address the question of whether other proteins interact with CCR5 and potentially influence the CD4-CCR5 association, silver staining of proteins immunoprecipitated by the anti-CCR5 mAb 5C7 in parallel with biotinylation

(some proteins, particularly chemokine receptors are poorly labeled by biotin and at low concentration may not be detected) was used. Apart from the bands corresponding to CCR5 and CD4, the only other major bands are those representing the heavy and light chains of the precipitating mAb 5C7 and the bands that are apparently not specific to CCR5, because they were immunoprecipitated in CCR5-negative cells (Fig. ID). These data not only show that the interaction between CD4 and CCR5 is not mediated by another molecule but also indicate that the coimmunoprecipitation of CD4 and CCT5 is unlikely to be due to compartmentalization of these two molecules within defined membrane microdomains, which would lead to coimmunoprecipitation of a greater number of proteins. Another experiment supporting this notion shows that cell lysis with two different detergents (Brij97 and NP40) did not disrupt the CD4-CCR5 interaction. Furthermore, depletion of cholesterol from the cell membrane with methyl- cyclodextrin, a procedure that was shown to disrupt microdomain structure (Friedrichson & Kurzchalia, Nature (London) 394:802-805 (1998), did not block the coimmunoprecipitation of CD4 and CCR5 (Fig. IE).

Results similar to those described above for cell lines were also obtained with primary human cells susceptible to HIV-1 entry. Initial attempts to coimmunoprecipitate CD4 and CCR5 from the surface of primary T lymphocytes resulted in very weak bands that were at the limits of assay sensitivity, because of the low level expression of CCT5 at the surface of these cells and the relatively small percentage of cells expression it as evaluated by flow cytometry. By using two alternative procedures for activating the CD4+ T cells expression of CCR5 was significantly increased to high(+) and very high (++) levels, corresponding on average to approximately 2-4 x 10 and 3-5 x 10⁴ molecules, respectively, as estimated by using quantitative flow cytometry. In these cells, CD4 levels were similar, and the amount of CD4 coimmunoprecipitated with the anti-CCR5 mAb 5C7 correlated with their cell fusion efficiency (Fig. 2A). The amount of coimmunoprecipitated CD4 in human macrophages and monocytes also correlated with the efficiency of their fusion with cells expression the HIV-1 JRFL Env (Fig. 2B) but not with the surface concentration of CD4. Indeed, the monocytes expressed similar or higher levels of CD4, but the amount of coimmunoprecipitated CD4 was undetectable or barely detectable(Fig. 2b). The larger amount of coimmunoprecipitated CD4 in macrophages is likely related to the higher levels of CCR5 compared with monocytes - on average approximately 5-10 x 10³ vs. <2 x 10³ molecules per cell as estimated by using quantitative flow cytometry. However, the CCR5 levels in macrophages were lower compared with the ++ CD4+ T cells. It was found that CD4 could associate weakly with CXCR5 even in the absence of gpl20. However, the results varied in a cell line- and assay condition- dependent manner. To evaluate the strength of the CD4-CCR5 association relative to the CD4-CXCR5 interaction, 3T3 cell lines expressing CD4 and either CCR5 or CXCR5 at approximately the same surface concentration were used. In the 3T3.CD4.CXCR4 cells, CD4 associated weakly with CXCR4, but he association was dramatically increased by addition of X4 HIV-1 Env gpl20 (IIIB) (Fig 3 A). In contrast, the amount of CD4 coimmunoprecipitated by the anti-CCR5 mAb 5C7 in the 3T3.CD4.CCR5 cells was high even in the absence of gp 120, and the addition of X4R5 (89.6) or R5 (JRFL) HIV- 1 Env gp 120 did not significantly increase the CD4-CCR5 coimmunoprecipitation (Fig. 3B and C). The quantity of CD4 coimmunoprecipitated by anti-CCR5 mAbs in the absence of gpl20 was about the same as the quantity of CD4 coimmunoprecipitated by anti-CXCR4 mAbs in the presence of gpl20. These results indicate that the CD4-CXCR4 association is weaker than the CD4-

CCR5 interaction an d that the preformed complexes between CD4 and CCR5 are close to a saturation level, where the addition of gp20 cannot further increase their complex formation.

EXAMPLE 3

THE FIRST TWO DOMAINS OF CD4 ARE INVOLVED IN THE INTERACTION WITH CCR5 To identify possible regions of CD4 which are responsible for the interaction with CCR5 a cell line (A2.01.T4.T8) was used which expresses a hybrid CD4- CD8 molecule containing the first two domains of CD4 which was previously shown to support HIV-1 Env-mediated fusion (Poulin et al., J.Virol. 65:4893- 4901, 1991) although at a lower rate than the wild type CD4 (Golding et al., J.Virol. 67:6469-6475, 1993). In order to produce CCR5 expression in these cells, the CD4-CD8 cells were infected with a recombinant vaccinia virus encoding the CCR5 gene. The cells, coexpressing CCR5 and the CD4-CD8 hybrid molecule, fused with cells expressing the R5 HIV-1 Env Bal, although at somewhat lower efficiency compared to cells expressing wild type CD4, similarly to previously reported observations of fusion mediated by the X4 HIV-1 Env IIIB (Golding et al., 1993, supra). The CD4-CD8 molecules were coimmunoprecipitated by an anti-CCR5 mAb from the A2.01.T4.T8 cells expressing CCR5 but not from those infected with control wild type vaccinia virus (FIG. 4A). The surface levels of CD4-CD8 molecules in the cells infected with the CCR5 and wild type vaccinia viruses were not significantly different as quantified by flow cytometry and Western blotting (FIG. 4B). To establish that the CD8 portion of the CD4-CD8 hybrid molecule was not involved in the interaction with CCR5, HeLa cells expressing either CD4 or CD8 were used. CCR5 was again expressed in these cells by recombinant vaccinia. In those cells only CD4, but not CD8, was coimmunoprecipitated with an anti-CCR5 mAb (5C7), demonstrating that the CD8 portion of the hybrid CD4-CD8 molecule was not involved in the interaction with CCR5. Together these results suggest that the first two domains of CD4 associate with CCR5.

The previous observations that a soluble fragment of CD4 consisting of the first two domains (sCD4DlD2) competes with macrophage inflammatory proteins (MΙP)l-alpha for CCR5 was confirmed (Wu et al, Nature (London) 384:179- 183 (1996)). Interestingly, it was found that the HIV-1 infection inhibiting mAb Cg7 at 60 nm almost completely blocked the ability of sCD4DlD2 to displace the chemokine MIP-1 alpha (Table 1). The same antibody significantly inhibited the coimmunoprecipitation of CCR5 by the anti- CD4mAb (CGI) and a control mAb (CG10) were not effective in preventing the displacement of MIP1 -alpha by SCD4D1D2 (Table 1). These results suggest that specific regions of CD4, potentially those overlapping the epitope of CG7, probably located within the first CD4 domain (Denisova et al., J. Immunol. 158:1157-1164 (1997)), are involved in the association with CCR5.

Table 1: CG7 displacement inhibition of MIP-1 alpha

EXAMPLE 4 THE SECOND EXTRACELLULAR LOOP OF CCR5 PLAYS A ROLE IN THE INTERACTION WITH CD4 To localize possible CCR5 sites interacting with CD4 mixtures of mAbs recognizing the N-terminus, first (ecl-1) and second (ecl-2) extracellular loops of CCR5 were used. Increasing the concentration of a mAb against the ecl-2 of CCR5 (2D7) in a mixture with another anti-CCR5 mAb (5C7), recognizing its N-terminus, reduced the total amount of coimmunoprecipitated CD4 (FIG. 4D). The amount of immunoprecipitated CCR5 was not significantly affected, and perhaps was even slightly increased (FIG. 4D). This is likely due to the inefficient immunoprecipitation of CCR5 by 2D7 (FIG. 4E). Another HIV-1- blocking mAb specific for the CCR5 ecl-2 (ml 82, R&D systems, Minneapolis, MN) showed similar inhibitory effects although at lower efficiency (Fig. 4F) in contrast to the non-HIVl -blocking anti-CCR5 mAb ml83, which did not interfere with the CD4-CCR5 coimmunoprecipitation (Fig. 4G). Other mAbs to the N-terminus and mAbs to ecl-1 of CCR5 did not decrease the amount of coimmunoprecipitated CD4 when used in combination with 5C7 . These results strongly suggest that the ecl-2 of CCR5 is involved in the interaction with CD4. However, even at the highest mAb (2D7) concentration used (50 g/ml) the inhibition of the CD4-CCR5 coimmunoprecipitation was not complete, indicating that regions additional to the second extracellular loop of CCR5 are probably involved in the interaction with CD4.

Colocalization of the two molecules was observed as demonstrated by the yellow (red-green colocalization) staining, suggesting formation of large multimolecular complexes between these two molecules (Fig. 5). A correlation map was prepared. The regions of true overlap of green and red staining were selected from the noncolocalized staining and the background fluorescence and represented as white dots. A relatively high degree of colocalization is evident from this analysis (Fig. 5, lower). Less colocalization was observed between CD4 and CXCR5 (fig. 5, left) than with CCR5. The extent of this increase appears to be comparable with the colocalization between CD4 and CCT5 in the absence of gp 120 in a manner reminiscent of the immunoprecipitation data shown in Fig. 3. No significant colocalization was observed between CD45 and CCR5 or HLA class I and CCr5, suggesting specificity in the interaction between CD4 and CCR5. These results suggest that CCR5 (and to a lesser extent CXCR4) not only associates with native membrane-associated CD4 but that the two molecules form large multimolecular complexes possibly because of their dimeric structures.

EXAMPLE 5

THE CD4-CCR5 INTERACTION MAY PLAY A ROLE IN

HIV ENV-MEDIATED FUSION AND ENTRY INTO CELLS It has been previously demonstrated that the anti-CCR5 mAb 2D7 (Wu et al.,

Nature 387:527-530, 1997) inhibits entry of R5 and dual-tropic HIV-1 into U87MG-CD4 cells expressing transfected CCR5 (Hill et al., J. Virol. 71 :6296- 6304, 1997). To find possible correlations between inhibition of the CCR5- CD4 interaction and HIV-1 Env-mediated fusion by this antibody in 3T3 cells expressing CD4 and CCR5 (Deng et al., Nature 381:661-666, 1996) (used in many of the coimmunoprecipitation experiments), a recombinant vaccinia virus-based reporter gene assay was employed to quantitate cell-cell fusion (Nussbaum et al., J. Virol. 68:5411-5422, 1994). The mAb 2D7 inhibited fusion mediated by R5 (Bal and JRFL) and R5X4 (89.6) HIV Envs. The inhibition was concentration dependent, and cell-cell fusion was significantly decreased at concentrations in the range of 0.5 to 50 g/ml, a concentration range similar to that observed for inhibition of the CD4-CCR5 interaction. For several other anti-CCR5 mAbs there was correlation (r=0.98, p=0.001) between inhibition of fusion and inhibition of the CD4-CCR5 interaction (Table 2). These results indicate that the CD4-CCR5 interaction may play a role in membrane fusion mediated by the HIV-1 Env. However, similarly to the inhibition of the CD4-CCR5 interaction, even at the highest concentration used (50 g/ml) the inhibition of fusion was not complete.

Table 2. Correlation between inhibition of HIV-1 (Bal)-mediated fusion and CD4-CCR5 interactions by anti-CCR5 mAbs. The inhibition of the CD4- CCR5 interaction was evaluated by using a mixture of the anti-CCR5 mAb 5C7 (which binds the N-terminus of CCR5) with 2D7 (which binds to the second extracellular loop of CCR5), or ml 80 with ml 81 , ml 82 (which binds to the N-terminus of CCR5) or ml 83 (R&D systems (Minneapolis, MN) at 3 g/ml for each antibody. The signal intensity for each CD4 band was measured by a phosphoimager (BioRad, Hercules, CA) and is presented as a percentage of the signal produced in the absence of inhibiting antibody.

mAb Fusion CD4-CCR5 inhibition (%) inhibition (%)

None 0 0

5C7 0 0

5C7+2D7 49 40 ml80 0 0 ml80+ml81 7 12 ml80+ml82 16 10 ml80+ml83 2 0

The results of the work described herein suggest that CD4 is constitutively associated with CCR5 in the absence of gpl20. This is the first demonstration that a seven-transmembrane-domain G-protein-coupled receptor can associate with another cell surface receptor in the absence of stimuli, suggesting new possibilities for cross-talk between plasma membrane receptors. Recent reports demonstrated signal transduction through CCR5 (Davis et al., J. Microscopy 185:21-36, 1997; Weissman et al, Nature 389:981-985, 1997). However, it is not known whether the physiological ligand of CD4 can induce signaling through CCR5 and vice versa.

The physiological role of the CD4-CCR5 may have played a critical role in the evolution of HIV-1 and the development of viral immunopathogenesis. Based on the observation that some strains of HIV-2 use the HIV coreceptor CXCR4 as a primary receptor (Endres et al., Cell 87:745-756, 1996), it is possible that CCR5 was initially used as a primary receptor by a predecessor of HIV (see Dimitrov and Broder, 1997. HIV and Membrane Receptors, Austin, TX:

Landes Biosciences; Dimitrov, Cell 91 :721-730, 1997). In support of this notion are the observations that a number of SIV strains can enter CD4 negative cells by using CCR5 (Edinger et al, Proc. Natl. Acad. Sci. U.S.A. 94:14742-14747, 1997) and that SIV gpl20 can bind CCR5 (Martin et al, Science 278: 1470-1473, 1997). The work described herein is the first demonstration of a strong association between CD4 and CCR5 even in the absence of gpl20. This suggests a possible evolutionary pathway involving virus variants which can use simultaneously CD4 and CCR5. The close proximity of the CCR5 and CD4 molecules probably enhanced adaptation of the virus to the new receptor molecule CD4. HIV may have further evolved to use CD4 for binding (Martin et al., Science 278:1470-1473, 1997). The adaptation to the CD4 molecule as a primary receptor was probably a critical event for development of the specific characteristics of HIV disease in humans characterized by depletion of the CD4+ T lymphocytes.

Prior to the results presented in this application, a paradigm of the initial stages of HIV-1 entry was proposed in which an initial high affinity binding of gpl20 to CD4 leading to conformational changes in both molecules which results in an enhanced binding of gpl20 to CCR5 and exposure of the fusion peptide (reviewed in Dimitrov and Broder, 1997, supra; Dimitrov, 1997, supra). The recent elucidation of the crystal structure of a CD4-gpl20 complex supports this paradigm (Kwong et al., Nature 393:648-659, 1998). Although the inhibition of the CD4-gpl20 interaction with CCR5 by HIV-1 neutralizing anti- gpl20 mAbs which do not interfere with binding to CD4 (Wu et al, Nature

384:179-183, 1996; Trkola et al., Nature 384:184-187, 1996) suggest that CD4- induced conformational changes in gpl20 result in direct interaction of gpl20 with CCR5, the possibility that simultaneously CD4 interacts with CCR5 can not be excluded by the previously published experiments. The finding that an inhibitory anti-CCR5 mAb also inhibits the CD4-CCR5 interaction at about the same concentration as that required for inhibition of HIV-1 Env-mediated fusion, suggests that the interaction of CD4 with CCR5 is important for the initial stages of the HIV-1 entry.

Agents inhibiting the CD4-CCR5 interaction could interfere with HIV entry and HIV Env-mediate fusion by at least three possible mechanisms: 1) an increase in the distance between CD4 and CCR5 may affect the interaction between the Env gpl20 and CCR5 in the gpl20-CD4-CCR5 complex thus inhibiting the proper configuration needed for the subsequent steps in the entry process, 2) the existence of preformed CD4-CCR5 complexes increases the efficiency of the trimolecular complex (gpl20-CD4-CCR5) formation and therefore their disruption should decrease the entry efficiency, 3) the binding of CD4 to CCR5 induces conformational changes in either or both molecules which are needed for the subsequent stages of virus entry. Therefore, the CD4- CCR5 interaction could serve as a novel target for development of anti-HIV- 1 agents which may not be toxic because the physiological role, if any, of the CD4-CCR5 interaction is not important for survival, as indicated by the existence of healthy people who are homozygous for the CCR5 deletion mutant (reviewed in Dimitrov and Broder, HIV and Membrane Receptors, Austin, TX: Landes Biosciences, 1997) which is not expressed at the cell surface (Benkirane et al., J. Biol. Chem. 272:30603-3060, 1997). Interestingly, the ecl- 2 of CCR5, is important for the interaction with CD4, is also critical for chemokine binding as demonstrated by using CCR2/CCR5 chimera (Samson et al., J. Biol .Chem. 272:24934-24941, 1997) and competition with the anti- CCR5 mAb 2D7 (Wu et al, Nature 387:527-530, 1997). This suggests that this region is a good target for generating therapeutic agents not only against HIV-1 but also against inflammatory responses. The first two domains of CD4 have been identified herein as the major site of interaction with CCR5.

Although the invention has been described with reference to the presently preferred embodiment, it should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims.

Claims

What is claimed is:

1. A method for identifying an agent which inhibits the interaction of CD4 and CCR5 in a cell, comprising: contacting a cell which expresses CD4 and CCR5 with an agent under conditions sufficient to allow interaction between said cell and said agent; evaluating the interaction of CD4 and CCR5; comparing the interaction of CD4 and CCR5 in said cell contacted with said agent to the interaction of CD4 and CCR5 in a control cell; wherein a decreased interaction of CD4 and CCR5 in the cell contacted with the agent indicates the ability of the agent to inhibit infection with an immunodeficiency lentivirus.

2. The method of claim 1 , wherein said agent is an antibody.

3. The method of claim 2, wherein said antibody is a monoclonal antibody.

4. The method of claim 2, wherein said antibody binds to the N-terminus of CCR5 (5C7 and Ml 82).

5. The method of claim 2, wherein said antibody is a polyclonal antibody.

6. The method of claim 1 , wherein said agent is a polypeptide fragment of CCR5.

7. The method of claim 5, wherein the polypeptide fragment of CCR5 comprises the second extracellular loop of CCR5.

8. The method of claim 1, wherein said agent is a polypeptide fragment of CD4.

9. The method of claim 1 , wherein said polypeptide fragment of CD4 comprises a member of the group consisting of domain 1 of CD4 and domain 2 of CD4.

10. The method of claim 1, further comprising comparing said interaction of CD4 and CCR5 in said cell contacted with said agent with an interaction of CD4 and CCR5 in a control cell not contacted with said agent.

11. A method for preventing or inhibiting infection of a cell expressing CCR5 and CD4 with an immunodeficiency lentivirus, comprising contacting said cell with an agent, wherein said agent inhibits an interaction of CCR5 and CD4.

12. The method of claim 11, wherein said agent is an antibody.

13. The method of claim 12, wherein said antibody is a monoclonal antibody.

14. The method of claim 13, wherein said antibody binds to the N-terminus of CCR5 (5C7 and M182).

15. The method of claim 11 , wherein said antibody is a polyclonal antibody.

16. The method of claim 10, wherein said agent is a polypeptide fragment of CCR5.

17. The method of claim 16, wherein the polypeptide fragment of CCR5 comprises the second extracellular loop of CCR5.

18. The method of claim 11, wherein said agent is a polypeptide fragment of CD4.

19. The method of claim 18, wherein said polypeptide fragment of CD4 comprises a member of the group consisting of domain 1 of CD4 and domain 2 of CD4.

20. The method of claim 11, wherein said immunodeficiency lentivirus is a human immunodeficiency lentivirus.

21. The method of claim 20, wherein said human immunodeficiency lentivirus is a human immunodeficiency virus type 1 (HIV-1).

22. A method of treating a subject, comprising administering to the subject having or at risk of having an immunodeficiency virus infection a therapeutically effective amount of an agent, wherein said agent inhibits an interaction of CD4 and CCR5.

23. The method of claim 22, wherein said agent is an antibody.

24. The method of claim 23, wherein said antibody is a monoclonal antibody.

25. The method of claim 24, wherein said antibody binds to the N-terminus of CCR5 (5C7 and M182).

26. The method of claim 23, wherein said antibody is a polyclonal antibody.

27. The method of claim 22, wherein said agent is a polypeptide fragment of CCR5.

28. The method of claim 27, wherein the polypeptide fragment of CCR5 comprises the second extracellular loop of CCR5.

29. The method of claim 22, wherein said agent is a polypeptide fragment of CD4.

30. The method of claim 29, wherein said polypeptide fragment of CD4 comprises a member of the group consisting of domain 1 of CD4 and domain 2 of CD4.

31. A pharmaceutical composition comprising at least one dose of an agent which inhibits an interaction of CD4 and CCR5 in a pharmaceutically acceptable carrier.

32. The pharmaceutical composition of claim 31 , wherein said agent is an antibody.

33. The pharmaceutical composition of claim 30, wherein said antibody is a monoclonal antibody.

34. The pharmaceutical composition of claim 33, wherein said antibody binds to the N-terminus of CCR5 (5C7 and Ml 82).