WO1993004693A1 - Synergistic inhibition of hiv-1 - Google Patents

Abstract

The invention employs two agents which, individually, function imperfectly, but which, when combined, act synergistically to neutralize HIV-1 virus. The invention employs an anti-V3 loop antibody in combination with a second agent which is either: 1) an antibody that is capable of binding to the CD4 binding site of HIV gp120 or 2) a soluble CD4 polypeptide.

Description

SYNERGISTIC INHIBITION OF HIV-1

This application is a continuation-in-part of U.S. Serial No. 07/756,677 filed on September 9, 1991, entitled SYNERGISTIC INHIBITION OF HIV-1 by Barbara J. Potts et al.

Background of the Invention This invention relates to the treatment of Human Immunodeficiency Virus infection. Human immunodeficiency Virus (HIV) , the etiologic agent of Acquired Immunodeficiency Syndrome (AIDS) and related disorders, is a retrovirus which infects certain immune system cells, including T4 lymphocytes and CD4+ cells of the monocyte/macrophage lineage. HIV binds to the surface of these cells via a high affinity interaction between CD4 and the HIV outer envelope glycoprotein, gpl20, and is internalized by fusion of the virus and the cell membrane. It is likely that similar events cause the fusion of HIV-infected and uninfected CD4+ cells leading to the formation of syncytia ( ultinucleated giant cells) .

CD4, a member of the immunoglobulin (Ig) superfamily (Clark et al., Proc. Natl . Acad . Sci . USA 84:1649, 1987), consists of four extracellular Ig-like domains, a hydrophobic transmembrane segment, and a short cytoplas ic region. Soluble CD4 (sCD4) polypeptides lacking the transmembrane and cytoplasmic domains have been produced by recombinant techniques (Fisher et al., Nature 331:76, 1988) sCD4 polypeptides have been shown to inhibit HIV infection of CD4+ cells, possibly by competing with membrane bound-CD4 for gpl20 binding.

In addition to its activity as a receptor for HIV gpl20, Lenert et al. (Science 248:1649, 1990) report that recombinant CD4 has the capacity to bind to immunoglobulin molecules, and that CD4-complexed antibody binds antigen with higher affinity- than does antibody alone. Lenert et al. suggest that this mechanism may account for previous reports of antibody-mediated enhancement of HIV infection (Robinson et al. , Biochem and Biophys. Research Comm. 149:693 1987; Robinson et al._Λ Lancet 790, 1988; Takeda et al., Science 242:580, 1988; Montefiori et al., J. Virol . 64:113, 1990; Homsy et al. , Science 244:1357, 1989). Therapeutics based on the soluble CD4 protein, or fragments or derivatives of CD4, have been proposed for the prevention and treatment of HIV-related infections. Additionally, cytotoxic hybrid proteins composed of a toxin fused to all or part of the CD4 receptor have been proposed as a way to destroy cells expressing HIV gpl20. Berger et al. (PCT Publication No. WO 90/01035) describe the construction of a CD4- Pseudomonas exotoxin hybrid protein. Till et al. (Science 242:1166, 1988) report that a CD4-ricin A fusion protein decreases DNA synthesis in cultures of chronically infected H9 cells. In a variation of this strategy, Seed (PCT Publication No. WO 89/06690) and Capon et al. (Nature, 337:529, 1989) designed immunoglobulin-CD4 fusion proteins designed to direct immune system response to gpl20-bearing cells. Another molecule of this type has been shown to activate complement (Traunecker et al. Nature 339:78, 1989). Fanger et al. (PCT Publication No. WO 91/00360) suggest the use of a high affinity Fc gamma receptor-specific antibody fused to CD4 (or the CD4 binding domain of gpl20) for treatment of HIV infection.

Antibodies that have neutralizing activity against the.HIV virus have been proposed for treatment of HIV infection. The primary targets for neutralizing anti-HIV antibodies are within gpl20 and the loop structure within the third variable (V3) domain of gpl20, is believed to be the principal neutralization domain (PND) of gpl20. Because of the extreme sequence heterogeneity of gpl20 among HIV isolates, the V3 loop elicits predominately strain-specific neutralizing antibodies. Nevertheless, Scott et al. (PCT Publication No. WO 90/15078) have identified anti-V3 loop antibodies that recognize short, highly conserved sub-sequences of the loop and are capable of neutralizing a broad range of HIV isolates. Studies indicate that the conserved regions of gpl20 critical for CD4 binding are discontinuous, suggesting that the CD4-binding site is a conformationally restricted epitope (Kowalski et al., Science 237:1351, 1987; Olshevsky et al., J. Virol . 64:5701, 1990).

Fisher et al. (PCT Publication No. WO 89/11860) report synergistic effects of sT4 (sCD4) polypeptide and an anti-retroviral reagent such as the nucleoside derivative, AZT, or a glucoside inhibitor, agents that act at different sites of HIV virus replication, in inhibiting HIV infection. Johnson et al. (EP Publication No. 0 401 194) , Harmenberg et al. (PCT Publication No. WO 91/01137) and Brankowan et al. (PCT Publication No. WO 91/11081) report synergistic effects using combinations of nucleoside derivatives and other chemical compounds. Mittler (Science 245:1380, 1989) report synergism between gpl20 protein and an anti-gpl20 antibody in blocking T-cell activation.

Dhiver et al. (AIDS 3:835, 1989) studied the therapeutic potential of a combination of an anti-CD4 antibody with zidovudine (AZT) .

Summary of the Invention In general, the invention features methods and compositions for treatment of HIV-1 infection. The invention.employs two agents which, individually, function imperfectly, but which, when combined, act synergistically to neutralize HIV-1 virus. By "synergistic" action is meant that HIV neutralization effect of the combination is greater than the sum of the neutralization effects of the two agents when not combined.

The invention effects HIV-1 neutralization by employing an anti-V3 loop antibody in combination with a second agent which is either: 1) an antibody (preferably high affinity) that is capable of binding to the CD4 binding site of HIV gpl20 (anti-CD4 binding site antibody) or 2) a soluble CD4 polypeptide, or fragment or derivative thereof (collectively referred to as sCD4) which is capable of binding to either: 1) the Fd region (the variable, or V, region of an Ig heavy chain (VH) plus the first constant, or C, domain of the heavy chain (CHI)) of an anti-V3 loop antibody, or 2) gpl20. Suitable sCD4^rs are a ino acid sequences corresponding to all or a portion of the extracellular domain of the CD4 excluding the include the cytoplasmic and transmembrane regions. Suitable sCD4's can be of a variety of lengths and post-translational modifications (e.g., glycosylation) . Such polypeptides, when produced recombinantly in a host cell, are secreted freely into the medium, rather than anchored in the host cell membrane. An sCD4 consisting of all of CD4 save the transmembrane and cytoplasmic domains may be able to interact with the gpl20 CD4 binding site as well as with immunoglobulin molecules. sCD4's useful in the present invention may be smaller sCD4 polypeptides that have Ig- binding activity in the absence of gpl20 binding activity or that have gpl20 binding activity in the absence of Ig binding activity.

As used herein, an "anti-V3 loop antibody" or "antibody directed against the V3 loop" is an antibody which binds within a specific region of the gpl20 molecule referred to as the principal neutralization domain (PND) . The PND is an approximately 36 amino acid sequence within the third variable (V3) domain of HIV-1 gpl20 between conserved cysteine residues located at amino acid positions 303 and 338 (according to the numbering convention of Ratner) . The cysteine residues form a disulfide bond, defining a "loop" which contains the largely conserved Gly-Pro-Gly sequence in its center. As used herein, "neutralizing" refers to the ability of the antibody to reduce HIV infection of cells by cell- free virions, or fusion of infected cells, or both. Assays described herein are used to measure neutralization. The infectivity reduction assay is the most preferred assay. As used herein "anti-CD4 binding site antibody" or "antibody directed against the CD4 binding site of gpl20" is an antibody which recognizes and binds to a portion of the site on HIV-1 gpl20 where CD4 binds.

Anti-V3 antibodies used in the invention are capable of binding to the PND epitope of HIV-1 gpl20.

Preferably, the anti-V3 antibody is capable of binding to a highly conserved epitope of the PND, and neutralizing at least two HIV strains. Most preferably, the anti-V3 antibody is capable of neutralizing a broad range of HIV variants. Examples of antibodies useful in the methods and compositions of the invention are monoclonal antibodies 58.2, 59.1 and 83.1 (described below).

According to the invention, the anti-V3 loop antibody and either the anti-CD4 binding site antibody or the sCD4 polypeptide can be administered simultaneously or near enough in time so that they provide a synergistic effect in neutralizing HIV. Most preferably, the combination with an anti-V3 antibody is capable of reducing virus infectivity by >3 logs in the infectivity reduction assay described below. Combination therapies containing an anti-V3 loop antibody and an anti-CD4 binding site antibody, or sCD4 polypeptide, may offer advantages over single agent therapeutic regimens, including synergistic interactions, more complete neutralization, reduced opportunity for emergence of drug-resistant HIV isolates and possible dose reductions of each agent below its toxic concentration.

Thus, in one aspect, the invention features a composition for treatment of HIV-1 infection, the composition includes an antibody directed against the V3 loop of gpl20 and an antibody directed against the CD4 binding site of gpl20, wherein the HIV-1 neutralization activity of the composition is greater than the sum of the HIV-1 neutralization activity of the V3 loop antibody in the absence of the CD4 binding site antibody and the neutralization activity of the CD4 binding site antibody in the absence of the V3 loop antibody.

In another aspect, the invention features a composition for treatment of HIV-1 infection, the composition includes an antibody directed against the V3 loop of gpl20 and an sCD4, wherein the HIV-1 neutralization activity of the composition is greater than the sum of the HIV-1 neutralization activity of the V3 loop antibody in the absence of sCD4 and the neutralization activity of sCD4 in the absence of the V3 loop antibody.

In preferred embodiments, the V3 loop antibody is a neutralizing antibody; the V3 loop antibody is capable of neutralizing two or more HIV-1 strains; the V3 loop antibody is 50.1; the V3 loop antibody is 59.1; the V3 loop antibody is 58.2; and the V3 loop antibody is 83.1.

In a related aspect, the invention features a method for treatment of HIV-1 infection in a human patient, the method includes administering to the patient a composition which includes an antibody directed against the V3 loop of gpl20 and an antibody directed against the CD4 binding site of gpl20, wherein the HIV-1 neutralization activity of the composition is greater than the sum of the HIV-1 neutralization activity of the V3 loop antibody in the absence of the CD4 binding site antibody and the neutralization activity of the CD4 binding site antibody in the absence of the V3 loop antibody. In a related aspect the invention features a method for treatment of HIV-1 infection in a human patient, the method includes administering to the patient a composition which includes an antibody directed against the V3 loop of gpl20 and sCD4, wherein the HIV-1 neutralization activity of the composition is greater than the sum of the HIV-1 neutralization activity of the V3 loop antibody in the absence of sCD4 and the neutralization activity of sCD4 in the absence of the V3 loop antibody. In a related aspect the invention includes a method for treatment of HIV-1 infection in a human patient, the method includes administering to the patient an effective amount of an antibody directed against the V3 loop of gpl20 and an antibody directed against the CD4 binding site of gpl20, wherein the HIV-1 neutralization activity of the V3 loop antibody in combination with the CD4 binding site antibody, is greater than the sum of the HIV-1 neutralization activity of the V3 loop antibody in the absence of the CD4 binding site antibody and the neutralization activity of the CD4 binding site antibody in the absence of the V3 loop antibody.

In a related aspect the invention features a method for treatment of HIV-1 infection in a human patient, the method includes administering to the patient an effective amount of an antibody directed against the V3 loop of gpl20 and sCD4, wherein the HIV-1 neutralization activity of the V3 loop antibody in combination with the sCD4, is greater than the sum of the HIV-1 neutralization activity of the V3 loop antibody in the absence of the sCD4 and the neutralization activity of the sCD4 in the absence of the V3 loop antibody.

Other features and advantages of the invention will be apparent from the following description of the preferred embodiments and from the claims. Detailed Description

The drawings are first briefly described. Drawings

Figure 1 is a graphical representation of the effect of antibody 59.1 alone (squares), antibody F105 alone (circles), and antibodies 59.1 and F105 combined (triangles) on the reverse transcriptase activity of HIV- MN infected cells.

Figure 2 is a pair of graphs depicting the effects of antibodies 59.1 and F105 on the reverse transcriptase activity of HIV-MN infected cells.

Figure 3 is a graph depicting the effects of antibodies 59.1 and F105 on the reverse transcriptase activity of HIV-MN infected cells.

Figure 4 is a graph depicting the effects of antibodies 59.1 and F105 on the reverse transcriptase activity of HIV-MN infected cells.

Figure 5 is a pair of graphs depicting the effects of antibodies 59.1 and 1.5E on the reverse transcriptase activity of HIV-MN infected cells. Figure 6 is a pair of graphs depicting the effects of antibody 59.1 and sCD4 on the reverse transcriptase activity of HIV-MN infected cells.

Figure 7 is a graph depicting the effects of antibodies 50.1 and F105 on the reverse transcriptase activity of HIV-MN infected cells. Figure 8 is a pair of graphs depicting the effects of antibodies 58.2 and F105 on the reverse transcriptase activity of HIV-MN infected cells.

Figure 9 is a pair of graphs depicting the effects of antibodies 83.1 and F105 on the reverse transcriptase activity of HIV-MN infected cells.

Figure 10 is a graph depicting the effects of antibodies 60.1 and F105 on the reverse transcriptase activity of HIV-MN infected cells. Synergistic Inhibition of HIV-1

The invention provides treatment of HIV infection using an anti-CD4 binding site antibody, or a sCD4 , in combination with an anti-V3 loop antibody. In combination, the two anti-HIV agents act synergistically to neutralize HIV-1.

Therapeutic administration of anti-V3 loop antibodies (antibodies which recognize an epitope within the V3 loop of gpl20) has previously been proposed. Many of these antibodies are capable of neutralizing HIV infection in vitro. Although certain of these antibodies recognize conserved sequences within the V3 loop of more than one HIV strain, they may lack desired potency and breadth of reactivity. Recombinant sCD4 has also exhibited activity against HIV in vitro; however, its clinical efficacy has not been clearly demonstrated.

Without being bound by theory, we believe that the synergistic effect provided by the claimed compositions may be explained by the following models. These models may be useful for predicting which combinations of anti- HIV agents will interact synergistically. According to one model, synergy between an anti-V3 loop antibody and an anti-CD4 binding site antibody or sCD4 occurs because the anti-CD4 binding site antibody or the sCD4 binds to the CD4 binding domain of gpl20 and alters the conformation of gpl20 such that the V3 loop is better exposed, giving the anti-V3 loop antibody increased access to its epitope. According to a second model, which applies to synergy between anti-V3 loop antibodies and a sCD4, the sCD4 may bind directly to the anti-V3 loop antibody, resulting in increased avidity of the anti-V3 antibody for its epitope.

Thus, enhanced neutralization of HIV virus may be accomplished by a process involving at least two steps: (1) increasing the accessibility of the V3 loop of the HIV envelope glycoprotein, gpl20, or increasing the affinity of the V3 loop antibody for its epitope, and (2) blocking the V3 loop with a V3 loop antibody. Preparation of Anti-V3 Antibodies

Anti-V3 loop antibodies may be generated and screened as described below. These antibodies may be directed against the V3 loop of any desired HIV-1 isolate.

Methods for preparing and analyzing antibodies directed towards the V3 loop of a particular HIV-1 isolate (HIV-MN or an HIV-MN viral variant) , are also described in U.S. Application No. 07/665,306, filed March 6, 1991 and in PCT Publication No. WO 91/15078, assigned to the same assignee and hereby incorporated by reference. These antibodies are preferred because of their potential to neutralize two or more HIV isolates. Preparation of the Immunogen

The immunogen can be any molecule that includes a portion of the V3 loop of any HIV-1 isolate.

Preferred anti-V3 loop antibodies recognize epitopes within a highly conserved sequence of the V3 loop. LaRosa et al. (Science 249:932, 1990) describes a particular amino acid subsequence within the center of the V3 loop that is present in a majority of the HIV isolates. The conserved subsequence is: Ile-Gly-Pro-Gly- Arg. Anti-V3 loop antibodies which recognize an epitope within this subsequence are preferred because they are likely to be broadly neutralizing, i.e., they will neutralize virus of more than one HIV strain.

The immunogen used to generate anti-V3 loop antibodies can include gpl60, gpl20, fragments of gpl20 or gpl60 which include all or part of the V3 loop, or synthetic peptides which include all or part of the V3 loop. In order to generate antibodies having broad neutralizing capability, the V3 loop sequences should contain a highly conserved V3 loop subsequence, Ile-Gly- Pro-Gly-Arg.

The immunizing peptide, polypeptide or protein may be in linear form or alternatively may contain the V3 loop formed into a closed loop by creation of a disulfide bond between cysteine residues at the termini of the V3 loop sequence. If the immunizing peptide contains more than one V3 loop, each may be separately formed into a loop through disulfide bonding. Preferred immunogens for generating anti-V3 loop antibodies include a RP70 peptide formed into a closed loop (described below) .

Synthetic peptides containing the desired sequences can be synthesized using an automated peptide synthesizer. Intact recombinant gpl60 envelope polypeptide can be produced in insect cells using a baculovirus expression system and purified as described in Rusche et al., U.S. Application No. 091,481, filed August 31, 1987, assigned to the same assignee as the present invention, hereby incorporated by reference.

Synthetic peptides or protein fragments to be used as immunogens can be either unconjugated or conjugated to an immunogenic carrier, e.g., keyhole limpet hemocyanin (KLH) or ovalbumin, using succinyl maleimidomethyl σyclohexanylcarboxylate (SMCC) as a conjugation agent (Yoshitake et al., J. Biochem . 92:1413, 1982), as follows. Briefly, 1 mg of SMCC dissolved in 50 μl of dimethylformamide is added to 6 mg of carrier (at a concentration of 10-20 mg/ml in 0.1M NaPO., pH 6.5) and incubated at room temperature for 0.5 h. The solution is then passed through a Sephadex G-25 column to remove excess unreacted SMCC and 2 mg of peptide is added (suspended in a degassed solution of 0.1M NaPO., pH 8, ImM EDTA at a concentration of 10 mg/ml) .- The solution is mixed by N_ gas and incubated at 4°C overnight. The sample is then dialyzed in 6M urea, 0.1M NaPO., pH 7 until the precipitate dissolves. The sample is next eluted through a BioGel P-10 column equilibrated in 6M urea, 0.1M NaPO.. The voided protein is collected and dialyzed in distilled H_0. The sequences of several peptides (RP142, RP70,

RP342, RPlOO, RP102, RP108, RP123C, and RP174C) useful in immunogens are shown in Table 1. This list is not meant to be exhaustive; it merely lists a few of the peptides which may be used as immunogens. Table 1: Examples of Peptides Useful as Immunogens

RP142 Y N K R rC R l H I G P G R A F Y T T K N I I G (C)

RP32 I H I G P G R A F Y T

RP70 r N C T R P N Y N K R K R I H I G P G R A F Y TT K N I I G T I R Q A H C N I S RP100 (S G G) T R tC G . rl I G P G R A . Y CG G S C)

RP102 (S G G) T R r S I S I G P G R A F (G G S C)

RP108 (S G G) H I G P G R A F Y A T G (G G S C)

RP123c CO H I G P G R A F (C)

RP135 (III_B) H N T R K S I R I Q R G P G R A F VT I G K I G (C) RP174c (C) N N T R K S I R I Q R G P G R A F V T I G K r G (C)

RP339 (RF) I T K G P G R V I Y CC)

Note: Amino acids in parentheses are not in the natural sequence of the indicated isolate

Of the peptides listed in Table 1, RP70, RP123c, and RP174c can be formed into closed loops by creation of a disulfide bond between the two cysteine residues near the ends of the amino acid sequence. A method for creating such a bond is described in Zhang et al. (Biochemistry 27:3785, 1988). The peptides can be prepared for immunization by emulsification in complete Freund's adjuvant according to standard techniques.

Generation of Antibodies Anti-V3 loop antibodies were prepared by intraperitoneal immunization of mouse strains (Balb/c, C57BL/6, A.SW, B10.BR, or BIO.A, Jackson Labs., Bar Harbor, ME) with 10-50 μg per mouse of circularized RP70 (Table 1) or recombinant gpl60. The mice were given booster immunizations of the immunogen, either in an emulsification of incomplete Freund's adjuvant or in soluble form, two to three times at two to four week intervals following the initial immunization. Mice were bled and the sera assayed for the presence of antibodies reactive with the immunogen. Mice showing a strong serological response were boosted, and (3-5 days later) spleen cells from these mice were fused with NS-1 (American Type Culture Collection, Rockville, MD, Accession No. TIB18) , SP2-0 (ATCC No. CRL8287, CRL8006) , or P3.X63.AG8.653 myeloma cells incapable of secreting both heavy and light immunoglobulin chains (Kearney et al., J. Immunol . 123:1548, 1979) by standard procedures based on the method of Kohler and Milstein, (Nature 256:495, 1975). Supernatants from hybridomas which appeared 6-21 days after fusion were screened for production of antibodies by an ELISA screening assay using the immunizing peptide.

Each well of a 96-well Costar flat-bottom microtiter plate was coated with the peptide by placing a 50 μl aliquot of a PBS solution containing the peptide at a final concentration of 0.1-10 μg/ml in each well. The peptide solution was aspirated and replaced with PBS + 0.5% BSA. Following incubation, the wells were aspirated, washed, and 50 ul of hybridoma supernatant was added. Following incubation, the wells were washed 3 times with PBS, and then incubated with 50 μl of an appropriate dilution of goat anti-mouse immunoglobulin conjugated with horseradish peroxidase (HRP, Zymed Laboratories, San Francisco, CA) . The wells were washed again 3 times with PBS and 50 μl of ImM ABTS (2,2 azino- bis (3-ethylbenzthiazoline-6-sulfonic acid) in 0.1M Na- Citrate, pH 4.2, to which a 1:1000 dilution of 30% H₂0_ had been added) , the substrate for HRP, was added to detect bound antibody. HRP activity was monitored by measuring the absorbance at 410nm.

Hybridomas that test positive by the ELISA method can be tested for their ability to bind to cells which express the HIV envelope protein. In one such assay recombinant vaccinia virus expressing the env gene of a particular HIV strain are used to infect cells of the CD4+ human T-lymphoma line, CEM-ss (AIDS Research and Reference Reagent Program, Rockville, MD, catalog #776) . Hybridoma supernatant (or purified antibodies) are incubated with the infected cells, and antibody binding is detected by indirect immune florescence using a secondary antibody and a florescence activated cell sorter. As a control, binding to otherwise identical cells which do not express an HIV env gene is measured. Hybridomas producing antibodies which bind to env expressing cells (but not to non-expressing cells) are then selected for further characterization. Cells expressing the env gene of any HIV strain may be prepared as described below. Antibody Purification and Amplification

Hybridomas that tested positive for peptide binding in the ELISA assay were subcloned by the limiting dilution method. Hybridoma cells and irradiated splenocytes from nonimmunized syngeneic mice (final concentration 5 cells/ml and 2.5 x 10 cells/ml, respectively) were mixed and 200 ul of the mixed suspension were plated in microtiter wells to give 1 hybridoma cell per well. Subclones which appeared 7-14 days later were assayed again by the ELISA procedure described above. Representative positive subclones were subcloned a second time.

The isotypes of the antibodies were determined by the ELISA method using goat anti-mouse-HRP preparations which corresponded to each of the five major mouse immunoglobulin isotypes (IgM, IgGl, IgG2A, IgG2B and IgG3) .

Purified antibodies were prepared by injecting hybridoma subclones that repeatedly tested positive by ELISA and/or syncytium inhibition assays (described below) were injected intraperitoneally into pristane- primed syngeneic mice. The ascites which developed were recovered two to three weeks after injection and the monoclonal antibodies were purified as follows, using procedures which were dependent on the isotype of the antibody. Following elution, all IgG antibodies were dialyzed against PBS.

IgM antibodies were purified by 50% NH_SO precipitation of ascites fluid from mice injected with the corresponding hybridoma cells, and then dialysis of the precipitate against 4X PBS. The dialyzed antibody was then passed over an Ultrogel A-6 column (Biotechnics, Villeneuve-La-Garenne, France) pre- equilibrated with 4X PBS. The antibody-containing fraction was identified using ELISA. Ascites fluid containing IgGl antibodies was diluted 4-fold in 0.1M Tris-HCl, 3M NaCl, pH 8.9, and isolated by passage through a Protein A-Sepharose affinity column equilibrated with the same Tris-NaCl buffer. The antibody was eluted using 0.1M Na-Citrate, pH 6.0. Ascites fluid containing IgG2 antibodies was diluted two-fold in PBS, and then bound to a Protein-A- Sepharose affinity column equilibrated with PBS. It was then eluted from the column with 0.15M NaCl, 0.1M acetic acid, pH 3.0. Following elution, the antibody was immediately neutralized by the addition of 1M Na₂HC0₃.

Ascites fluid containing IgG3 antibodies was diluted 4-fold in 0.1M Tris-HCl, 3M NaCl, pH 8.9, passed over a Protein-A-Sepharose affinity column, and antibody was eluted from the Protein A column with 0.15M NaCl, 0.1M acetic acid.

Alternatively, all IgG subclasses can be purified by the following procedure. Ascites fluid is diluted 2- fold in 0.1M Tris-HCl, 3M NaCl pH 8.9, passed over Protein A Sepharose affinity column, and eluted with 0.15M NaCl, 0.1M acetic acid, pH 3.0. Engineered Antibodies

Since, for the most part, monoclonal antibodies are produced in species other than humans, they are often immunogenic to humans. In order to successfully use antibodies in the treatment of humans, it may be necessary to create chimeric antibody molecules wherein the antigen binding portion (the variable region) is derived from one species, and the portion involved with providing structural stability and other biological functions (the constant region) is derived from a human antibody. Methods for producing chimeric antibodies in which the variable domain is derived from one species and the constant domain is derived from a second species are well known to those skilled in the art. See, for example, Neuberger et al., WO Publication No. 86/01533, priority September 3, 1984; Morrison et al., EP Publication No. 0,173,494, priority August 27, 1984. An alternative method, in which an antibody is produced by replacing only the complementarity determining regions (CDRs) of the variable region with the CDRs from an immunoglobulin of the desired antigenic specificity, is described by Winter (GB Publication No. 2,188,638, priority March 27, 1986). Murine monoclonals can be made compatible with human therapeutic use by producing an antibody containing a human Fc portion (Morrison, Science 229:1202, 1985). Single polypeptide chain antibodies are also more easily produced by recombinant means than are conventional antibodies. Ladner et al. (U.S. Patent No. 4,946,778) describes methods for producing single polypeptide chain antibodies and these methods may be adapted to produce antibodies useful in the methods and compositions of the invention. Established procedures would allow construction, expression, and purification of such a hybrid monoclonal antibody. Quadromas can be used to generate bispecific antibodies (Reading et al., U.S. Patent Nos. 4,474,893 and 4,714,681, , hereby incorporated by reference) . Determination of Antibody Specificity The peptide competition assay described below can be used to determine the strain specificity of anti-V3 loop antibodies. In addition, assay for antibody binding to cells expressing an HIV env gene (described above) may also be used to assess antibody specificity. Finally, the epitope mapping assays described in Higgins et al. (U.S. Application No. 07/699,773 filed May 14, 1991, assigned to the same assignee as the present invention and hereby incorporated by reference) may also be used to identify antibodies which recognize a particular V3 loop epitope. Some or all of these assays may be used to select anti-V3 loop antibodies. Anti-V3 Loop Antibodies

Described below are several antibodies which recognize sequences within the V3 loop. Using assays described below these, and other anti-V3 loop antibodies, can be tested for their synergistic potential in combination with the second agent of the invention.

Hybridomas F50, F58, F59 and F83 were generated from immunization of BALB/C mice with the closed loop immunogen RP70 as described above. Antibodies designated 50.1 (formerly F50/P8D10) , 58.2 (formerly F58/P6F2: ATCC Accession No. HB10688) , 59.1 (formerly F59/P5B3) and 83.1 (formerly F83/P6F12) were identified as antibodies which are not HIV variant-specific (i.e. they are broadly neutralizing) . Assays described above demonstrated that antibody 50.1 shows specificity towards the left side of the V3 loop (Arg-Ile-His-Ile-Gly) ; antibody 59.1 recognizes the epitope Gly-Pro-Gly-Arg-Ala-Phe, and was capable of neutralizing strains HIV-MN, HIV-SF2, HIV-WMJ2 and HIV-III; antibody 83.1 recognizes the Ile-Xxx-Ile- Gly-Pro-Gly-Arg epitope (where Xxx is any amino acid) , and was capable of neutralizing strains HIV-MN, HIV- Alabama, HIV-SF2, HIV-WMJ2 and HIV-Duke 6587-5; antibody 58.2 recognizes the epitope Ile-Gly-Pro-Gly-Arg-Ala-Phe and was capable of neutralizing HIV variants HIV-MN, HIV- SF2, HIV-Ala, HIV-Duke 6587-5 and the macrophage variants grown in human peripheral blood lymphocytes, AD-87, JL-FL and Bal (AIDS Research and Reference Reagent Program) . Preparation of sCD4 Polypeptides sCD4 polypeptide includes all proteins, polypeptides, and peptides which are natural or recombinant sCD4 polypeptides (rsCD4) , or soluble derivatives thereof. Such polypeptides can be produced by standard techniques well known to those skilled in the art. See Fisher et al. (Nature 331:76, 1988) for details of CD4 production. Preparation of anti-CD4 Binding Site Antibodies

The CD4 binding site on HIV gpl20 is a conformationally determined epitope that is required for attachment of the virus to CD4+ cells. Generally, immunization of mammals with intact envelope protein or oligopeptide fragments that contain the sequences involved in CD4 binding do not yield suitable anti-CD4 binding site antibodies due to the conformational constraints of this epitope required to elicit a neutralizing antibody. Therefore, it is preferred that the anti-CD4 binding site antibodies of the present invention be isolated from an HIV-infected mammal, most preferably a human. Methods for isolating human anti-CD4 binding site monoclonal antibodies are described by

Posner et al. (J. Immunol . 164:4325, 1991) and Robinson et al. (AIDS Res . Human Retroviruses 6:567, 1990). Resulting antibodies are screened for neutralizing activity and for the ability to inhibit gpl20/CD4 interactions.

Examples of anti-CD4 binding site antibodies are the F105 human monoclonal antibody (Posner, supra) and the 1.5E antibody (Robinson et al., supra) . Peptide Titration Assay A peptide titration assay can be used as an initial screen to predict if a given anti-V3 loop antibody will have strong neutralization activity by itself, and if it has potential to act synergistically with a second agent. In this assay, the antibody is tested for its ability to prevent syncytia formation among gpl60 expressing CD4+ cells in the presence of competitor peptide whose sequence is derived from a V3 loop sequence. This assay can be used to test for potential neutralization activity of any anti-V3 loop antibody towards any HIV isolate by using a peptide derived from the V3 loop from the HIV isolate of interest as the competitor.

Syncytia formation was measured in the presence of an anti-V3 monoclonal antibody mixed with one or more test peptides representing V3 loop sequences of a variety of HIV isolates. A partial list of V3-derived peptide sequences from HIV isolates is presented in Table 2. These sequences represent V3 loop epitopes of laboratory- adapted HIV strains as well as field isolates.

Table 2: Peptides Used in Competition Assays

In the peptide titration assay, the test peptide, at a series of concentrations ranging from lOuM to O.Ol M, was added to anti-V3 loop antibody (at 5 times the .concentration required for the 90% endpoint in an Std. SN assay, described below) , incubated for 30*" at 37° and then added to CEM-ss CD4+ cells expressing HIV-MN gpl60. These cells express gpl60 because they are infected with a recombinant vaccinia virus that encodes the HIV-MN env gene.

The HIV gpl20 envelope protein produced and presented on the surface of these cells enables them to bind to the CD4 receptor on other cells, resulting in cell fusion and the formation of syncytia. Antibodies that bind to the V3 loop of the gpl60-expressing cells (anti-V3 loop antibodies) can inhibit syncytia formation. If the test peptide competes with the gpl60 epitope recognized by the antibody for binding with the anti-V3 loop antibody, syncytia formation occurs. Conversely, if the test peptide does not compete with the cell surface epitope recognized by the antibody for binding with the anti-V3 loop antibody, syncytia formation in the presence of peptide is inhibited relative to syncytia formation in the absence of the peptide.

Using the peptide titration assay, we have defined the parameters to predict whether a particular anti-V3 antibody is likely to neutralize a particular virus isolate, and whether the neutralizing activity is likely to be synergistically enhanced in combination with a second anti-HIV agent, such as sCD4 or anti-CD4 binding site antibody. Accordingly, if the peptide competition is positive (i.e., >50% increase in syncytia formation in the presence of a peptide) at a concentration of ≤luM peptide, neutralization by the anti-V3 antibody is likely to be synergistically enhanced in the presence of the second agent. If peptide competition is negative (<50% increase in syncytia inhibition in the presence of a peptide) at a peptide concentration of < luM peptide, but positive at concentrations between luM and lOuM, the antibody alone is likely to weakly neutralize the virus isolate, but may act synergistically with the second anti-HIV agent to provide strong neutralization. Results of the peptide titration assays (employing some of the peptides listed in Table 2) for anti-V3 monoclonal antibodies 58.2, 59.1, 50.1 and 83.1 are presented in Table 3. In this table a "+" indicates positive competition and suggests that the virus from which the competing peptide was derived can be neutralized by the indicated anti-V3 loop antibody and that the antibody could act synergistically with an appropriate second anti-HIV agent. A "+/-" indicates somewhat less positive competition and suggests that the virus from which the competing peptide was derived can be weakly neutralized by the indicated anti-V3 loop antibody and that the antibody could act synergistically with an appropriate second anti-HIV agent. A "-" indicates that the peptide did not compete; therefore the corresponding antibody is not expected to neutralize the virus from which that peptide was derived nor could the antibody be expected to act synergistically with a second anti-HIV agent.

Table 3: Peptide Competition Assay Results

50.1 59.1

+

+

+/"

+/- +/-

+/-

+/"

Standard and Extended Serum Neutralization Assays Two biological assays, the standard serial neutralization assay (Std. SN) and the expanded serial neutralization assay (Ex. SN) , were used to predict the potential synergistic activity of various combinations of anti-V3 loop antibodies and second anti-HIV agents (CD4 binding site antibody or sCD4) . These assays use reverse transcriptase (RT) activity as a measurement of viral activity. The reduction in reverse transcriptase activity under a given set of conditions is a measure of viral neutralization. These assays can be used to determine both the optimal ratio of the two anti-HIV agents and the optimal absolute concentration of each anti-HIV agent. As described in more detail below, the Std. SN assay measures RT activity at a single time point 7 days post-infection. As a result, it is possible to compare a number of conditions with relatively few assays. However, since each viral isolate has a characteristic time course of infection, the 7 day time point used in the Std. SN assay may not include the period of optimal viral replication. As a result, in some instances, the Std. SN assay will not permit accurate determination of the effectiveness of the added anti-HIV agents. The Ex. SN assay measures RT activity at several timepoints out to 15 (or 20) days post-infection and thus is more likely to include the period of optimal viral activity for any given viral isolate. Therefore, the Ex. SN assay is preferred for assessing synergy.

When considering the results of both Std. SN and Ex. SN assays it should be understood that the antibody dilution that reduces RT activity by 90% after 7 days is defined as the 90% endpoint dilution. Further, for each antibody or polypeptide used, results are reported in μg/ l. In each case 100 ul of virus was added to 100 ul of the anti-HIV agent at the indicated concentration.

Three HIV isolates were used in the Std. SN and Ex. SN assays: HIV-MN, HIV-IIIB and HIV-Ala. HIV-Ala is considered a relevant field isolate because it has had a low number of passages in CEM cells. (The sequence of the HIV-Ala V3 loop has been reported to be the most representative of North American HIV isolates.) The viruses used in this assay were propagated in H9 cells (ATCC, Rockville, MD; or AIDS Research and Reference Reagent Program, Rockville, MD) for 15-30 days to establish a chronic cell line. Newly formed virions were harvested from the supernatant of infected cells and used to infect test cultures as described below. Serial two-fold dilutions of anti-V3 loop antibody and serial two-fold dilutions of sCD4 or anti-CD4 binding site antibody, (alone or in combination) were incubated with 64 infectious units of HIV virus for 30 minutes at 37°, then added to CEM-ss cells (50,000 cells per well in 96-well plates) . After 7 days, cell free supernatants were harvested and assayed for RT activity using the method of Willey et al. (J. Virol . 62:139, 1988). For determination of 90% and 50% endpoints (i.e., reduction in RT activity by 90% or 50%) , densitometry readings of autoradiographs were generated at 410 nm using a Molecular Devices microplate reader.

The Ex. SN assay is identical to the Std. SN assay except that media was replenished twice during the course of the assay (at day 7 post-infection and at day 12 post- infection) . Aliquots were assayed for RT activity (as described above) at 7, 12 and 15 or 20 days post- infection. Synergistic Neutralization of HIV-1 Figures 1-10 present the results of Std. SN and Ex SN assays illustrating the synergistic action of certain anti-V3 loop antibodies and CD4 binding site antibodies or sCD4 polypeptides. In all of the experiments whose results are presented in these figures, HIV-MN was used. FIG 1 presents the results of a Std. SN assay using antibody 59.1, an anti-V3 loop antibody, and F105, an anti-CD4 binding site antibody. RT activity, plotted as a function of total antibody concentration was measured at various concentrations of antibody 59.1 alone (squares) , antibody F105 alone (circles) and antibodies 59.1 and F105 added simultaneously (triangles). The results demonstrate that when both antibodies are present, the 90% endpoint is reached at a nearly 10-fold lower antibody concentration. FIGS 2-10 present the results of a number of Std. SN assays and Ex. SN assays using several different combinations of an anti-V3 antibody and a second anti-HIV agent. These results demonstrate that the degree of synergy observed can depend on the absolute amount of each anti-HIV agent (i.e. anti-V3 loop antibody and anti- CD4 binding site antibody or sCD4 polypeptide) as well as on the ratio of the two anti-HIV agents. For each of the combinations described below a Std. SN assay was used to determine the 90% endpoint for each anti-HIV agent of the combination acting alone as well as the 90% endpoint for the combination of anti-HIV agents. For each combination these results were then used to design Ex. SN assays in which each of the anti-HIV agents in the combination was added to HIV-MN infected cells, alone or in combination, at that agent's 90% endpoint dilution. Finally, in most cases, a similar Ex. SN assay was carried out in which each agent in a given combination was added to HIV-MN infected cells at the endpoint dilution that results in the greatest amount of synergy. Antibodies 59.1 and F105

For the anti-V3 loop antibody 59.1 a Std. SN assay indicated that the 90% endpoint dilution occurred at > 100 μg/ml meaning that even undiluted antibody did not reduce RT activity by 90%. For F105 the 90% endpoint was at 100 μg/ml. When the two antibodies were used together in a 1:1 ratio, the 90% endpoint was at a 12.5 dilution of each antibody.

FIG 2 illustrates the results of Ex. SN assays designed using the results of the Std. SN assays of antibodies 59.1 and F105 described above. FIG 2A illustrates the effect of the 59.1 antibody alone (squares) , the F105 antibody alone (triangles) and the two antibodies in combination (circles) . In each case the antibodies were added at 12.5 μg/ml. FIG 2B illustrates the effect of the 59.1 antibody alone (squares) , the F105 antibody alone (triangles) and the two antibodies in combination (circles) . In this case both antibodies (when added) were added at 6.25 μg/ml, the concentration which results in the greatest degree of synergy.

The above experiment was repeated. In this case the anti-V3 loop antibody 59.1 a Std. SN assay indicated that the 90% endpoint dilution occurred at >100 μg/ml meaning that even undiluted antibody did not reduce RT activity by 90%. For F105 the 90% endpoint was at 25 μg/ml. When the two antibodies were used together in a 1:1 ratio, the 90% endpoint was at 6.25 μg/ml of each antibody. FIG 3 illustrates the results of Ex. SN assays designed using the results of the Std. SN assays of antibodies 59.1 and F105 described above. FIG 4 illustrates the effect of the 59.1 antibody alone (squares) , the F105 antibody alone (triangles) and the two antibodies in combination (circles) . In each case the antibodies were added at a 3.13 μg/ml for 59.1 and 12/5 μg/ml for F105.

The above experiment was repeated with a different antibody ratio. In this case the anti-V3 loop antibody 59.1 a Std. SN assay indicated that the 90% endpoint dilution occurred at 25 μg/ml. For F105 the 90% endpoint was at 25 μg/ml. When the two antibodies were used together, this time in a 1:4 ratio, the 90% endpoint was at 3.13 μg/ml of the 59.1 antibody and 12.5 μg/ml of the F105 antibody

FIG 4 illustrates the results of Ex. SN assays designed using the results of the Std. SN assays of antibodies 59.1 and F105 described above. FIG 4 illustrates the effect of the 59.1 antibody alone (squares) , the F105 antibody alone (triangles) and the two antibodies in combination (circles) . In this case the 59.1 antibody was at 3.13 μg/ml and the F105 antibody was at 12.5 μg/ml. These results illustrate the importance of the ratio of agents. No synergy was seen here at a 1:4 ratio while good synergy was seen for the same combination at a 1:1 ratio (FIG 3). Antibodies 59.1 and 1.5E

For the anti-V3 loop antibody 59.1, a Std. SN assay indicated that the 90% endpoint dilution was at a >100 μg/ml. For 1.5E the 90% endpoint was at a >42 μg/ml. When the two antibodies were used together in a 2.4:1 ratio (59.1 antibody:1.5E antibody), the 90% - endpoint was at 25 μg/ml of 59.1 and a 10.50 μg/ml of 1.5E. FIG 5 illustrates the results of Ex. SN assays designed using the results of the Std. SN assays of antibodies 59.1 and 1.5E. FIG 5A illustrates the effect of the 59.1 antibody alone (squares), the 1.5E antibody alone (triangles) and the two antibodies in combination (circles). In these assays 59.1 antibody (when added) was at 25 μg/ml and the 1.5E antibody (when added) was at 10.5 μg/ml. FIG 5B illustrates the effect of the 59.1 antibody alone (squares), the 1.5E antibody alone (triangles) and the two antibodies in combination (circles). In this case the 59.1 antibody (when added) was at 6.25 μg/ml and the 1.5E antibody (when added) was at 2.63 μg/ml. These were the concentrations which resulted in the greatest degree of synergy for this antibody combination. Antibody 59.1 and Recombinant sCD4

For the anti-V3 loop antibody 59.1, a Std. SN assay indicated that the 90% endpoint dilution was at >100 μg/ml. For recombinant sCD4 the 90% endpoint was at 0.2 μg/ml. When the two anti-HIV agents were combined in a 8:1 ratio (59.1 antibody:recombinant sCD4) , the 90% endpoint was at 1.56 μg/ml of 59.1 and 0.2 μg/ml of recombinant sCD4.

FIG 6 illustrates the results of Ex. SN assays designed using the results of the Std. SN assays of antibody 59.1 and recombinant sCD4. FIG 5A illustrates the effect of the 59.1 antibody alone (squares), recombinant sCD4 alone (triangles) and the two anti-HIV agnents in combination (circles). In these assays 59.1 antibody (when added) was at a 1.56 μg/ml and the recombinant sCD4 (when added) was at a 0.2 μg/ml. FIG 5B illustrates the effect of the 59.1 antibody alone (squares) , the recombinant sCD4 alone (triangles) and the two anti-HIV agents in combination (circles) . In this case the 59.1 antibody (when added) was at a 0.78 μg/ml and the recombinant sCD4 (when added) was at a 0.1 μg/ml. These were the concentrations which resulted in the greatest degree of synergy for this combination of anti- HIV agents. Antibodies 50.1 and F105 For the anti-V3 loop antibody 50.1 a Std. SN assay indicated that the 90% endpoint dilution occurred at 0.78 μg/ml. For F105 the 90% endpoint was at 25 μg/ml. When the two antibodies were used together in a 1:4 ratio (50.1 antibody:F105 antibody), the 90% endpoint was at 0.39 μg/ml of 50.1 and 1.56 μg/ml of F105.

FIG 7 illustrates the results of Ex. SN assays designed using the results of the Std. SN assays of antibodies 50.1 and F105. FIG 7 illustrates the effect of the 50.1 antibody alone (squares), the F105 antibody alone (triangles) and the two antibodies in combination (circles). In these assays 50.1 antibody (when added) was at 0.78 μg/ml and the F105 antibody (when added) was at 1.56 μg/ml. Very little synergy was seen using this ratio of these agents; however the below-described infectivity reduction assay shows good synergy at a different ratio. Antibodies 58.2 and F105

For the anti-V3 loop antibody 58.2 a Std. SN assay indicated that the 90% endpoint dilution occurred at 0.31 μg/ml. For F105 the 90% endpoint was at 25 μg/ml. When the two antibodies were used together in a 1:10 ratio (58.2 antibody:F105 antibody), the 90% endpoint was at 0.16 μg/ml of 58.2 and a 1.56 μg/ml of F105. FIG 8 illustrates the results of Ex. SN assays designed using the results of the Std. SN assays of antibodies 58.2 and F105. FIG 8A illustrates the effect of the 58.2 antibody alone (squares), the F105 antibody alone (triangles) and the two antibodies in combination (circles) . In these assays 58.2 antibody (when added) was at 0.16 μg/ml and the F105 antibody (when added) was at 1.56 μg/ml. FIG 8B illustrates the effect of the 58.2 antibody alone (squares) , the F105 antibody alone (triangles) and the two antibodies in combination (circles). In this case the 58.2 antibody (when added) was at 0.08 μg/ml and the F105 antibody (when added) was at 0.78 μg/ml. These results demonstrate the dilutions which resulted in the greatest degree of synergy for this antibody combination. Antibodies 83.1 and F105

For the anti-V3 loop antibody 83.1 a Std. SN assay indicated that the 90% endpoint dilution occurred at 6.25 μg/ml. For F105 the 90% endpoint was at 25 μg/ml. When the two antibodies were used together in a 1:1 ratio (83.1 antibody:F105 antibody), the 90% endpoint was at 3.13 μg/ml of 83.1 and a 3.13 μg/ml of F105.

FIG 9 illustrates the results of Ex. SN assays designed using the results of the Std. SN assays of antibodies 83.1 and F105. FIG 9A illustrates the effect of the 83.1 antibody alone (squares), the F105 antibody alone (triangles) and the two antibodies in combination (circles). In these assays 83.1 antibody (when added) was at 3.13 μg/ml and the F105 antibody (when added) was at 3.13 μg/ml. FIG 9B illustrates the effect of the 83.1 antibody alone (squares) , the F105 antibody alone (triangles) and the two antibodies in combination (circles). In this case the 83.1 antibody (when added) was at 0.78 μg/ml and the F105 antibody (when added) was at 0.78 μg/ml. These are the concentrations which resulted in the greatest degree of synergy for this antibody combination. Antibodies 60.1 and F105

For the anti-CDllb loop antibody 60.1 a Std. SN assay indicated that the 90% endpoint dilution occurred at >100 μg/ml. For F105 the 90% endpoint was at 25 μg/ml. When the two antibodies were used together in a 1:1 ratio (60.1 antibody:F105 antibody), the 90% endpoint was at 25 μg/ml of both antibodies.

FIG 10 illustrates the results of Ex. SN assays designed using the results of the Std. SN assays of antibodies 60.1 and F105. FIG 9 illustrates the effect of the 60.1 antibody alone (squares), the F105 antibody alone (triangles) and the two antibodies in combination (circles). In these assays 60.1 antibody (when added) was at 25 μg/ml and the F105 antibody (when added) was at 25 μg/ml.

Table 4 summarizes the results of these assays. In this table 'fold increase in potency' refers to the fold difference in the concentration of anti-HIV agents required to reach the 90% endpoint when both are present and the concentration of the more potent of the two anti- HIV agents required to reach the 90% endpoint in the absence of the other anti-HIV agent. Thus, if 6.25 ug/ml of each of the anti-HIV agents is required to reach the 90% endpoint when both are present and 50 ug/ml of the more potent anti-HIV agent or 100 ug/ml of the less potent anti-HIV agent are required when they are used seperately, the fold-increase in potency would be 50/6.25 or 8.

Table 4 : Synergy Studies Using Std. SN and Ex. SN Assays

The control for these experiments was a combination of an anti-CD4 binding site antibody, F105, with an unrelated antibody, 60.1, which binds to the CDllb receptor on neutrophils. Infectivity Reduction Assay The potential synergistic combinations predicted in the Std. SN and Ex. SN assays were tested in an Infectivity Reduction Assay (IRA) which measures the difference between the infectious dose of a virus in the presence and absence of a standard dilution of an anti- HIV agent (or combination of anti-HIV agents) . The potency of each of the anti-HIV agents (or combination of agents) is measured by the amount of reduction of total virus titer. In contrast to the Std. SN and Ex. SN assays described above, IRA conditions promote cell division and virus replication, and thus it is apt to more closely predict neutralization potential in vivo .

Both laboratory HIV strains and HIV field isolates are tested in the IRA using either CEM-ss cells or PBMC (peripheral blood mononuclear cells, prepared by standard methods) , which are more susceptible to infection and thought to be more like in vivo situations. Briefly, the IRA is performed as follows. Viral isolates are serially diluted 10-fold in RPMI containing 10% fetal bovine serum. For each dilution of virus, 20 μ.g of anti-V3 loop antibody and 10 μg of the second anti-HIV agent are incubated together for 1 hour at 37°. Each combination is then inoculated onto 1 x 10⁶ CEM-ss or PBMC in a 24- well plate. The cultures are maintained for the appropriate number of days (calibrated for the virus used, for example, HIV-MN is 21 days and 14 days for Duke 6587-5. Cultures are split 3 times a week (if PBMCs are used, they are supplemented with IL-2 three times a week to maintain optimal virus replication conditions) . In some cases, agents are added again on day 3 post- infection at the same concentration as the first addition. Virus replication (infectious units) was monitored by reverse transcriptase activity as described above. The infectious titer of virus plus the anti-HIV agents is compared to the infectious titer of virus plus media.

Table 5 shows results of IRA assays. Results are expressed as [X] Y/Z; [X] indicates the logs of virus present in the assay at full-time in the absence of antibody (note that full-time depends upon the virus strain used, and may be approximately 14-22 days) , Y indicates the logs of virus blocked at one-half time, and Z indicates the logs of virus blocked at the time of completion of the assay (full-time) . Total infectious units blocked are listed in the last column of the table.

For example referring to the first set of MN results presented in Table 5, 3 logs of HIV-MN virus were present at full-time in the absence of anti-HIV agents. Antibody 59.1 by itself blocked 0 logs of virus at both half-time and full-time, whether added to the infected cells once or twice in the course of the assay. Recombinant sCD4 by itself blocked 1.5 logs of virus at half-time and full-time. In contrast, when antibody 59.1 and recombinant sCD4 were added in combination, 3 logs of virus were blocked at half-time and 2.5 logs were blocked at full-time. When antibody 59.1 and recombinant sCD4 were added twice during the course of the assay, the virus was completely blocked at half-time and full-time.

Table 5 Results of Infectivity Reduction Assays VIRUS AGENTS TIMES

Additional Virus Neutralization Assays

In additional experiments, four anti-V3 monoclonal antibodies, 59.1, 50.1, 83.1 and 58.2 (all described above) were evaluated for their ability to neutralize HIV in the presence of sCD4 (Biogen, Cambridge, MA) or F105 (described above and PCT/US91/01394) . The assays used were: short term 90% neutralization (Std.SN) of constant viral load and long term 100% neutralization (IRA) of varying viral load. Two HIV laboratory isolates (MN and IIIB) , two field isolates (D6587-5 and D7887-7) , and one monotropic field isolate (AD-87) were used in these experiments. Both primary peripheral blood lymphocytes (PBL) and CEM-SS were used in these experiments. The results of these additional assays are reported in Tables 6, 7 and 8.

In the Std. SN assays presented in Table 6, 64 infectious units of virus was preincubated with 2-fold dilutions of the indicated anti-HIV agents individually or together, then inoculated onto CEM-SS cells. At 7 and 15 days post-infection virus replication was measured using a reverse transcriptase assay. For each anti-HIV agent combination the results are presented in four ways: the concentration (μg/ml) of each anti-HIV agent required to reach the 90% endpoint titer in the absence of the other anti-HIV agent (1st Agent and 2nd Agent) ; the concentration (μg/ml) of each anti-HIV agent of required to reach the 90% endpoint when both are present (Both) ; and the fold increase in neutralization observed when the anti-HIV agents are combined compared to the neutralization observed when the more potent anti-HIV agent of the pair is used alone. In addition, a combination index (CI) was calculated by the multiple drug-effect analysis program (Biosoft, Cambridge, UK) . Using this program a CI value of <1 is considered significant. In these experiments, the experimental error of the endpoint titer in the Std. SN assay is 2- fold. Controls for the Std. SN assays included 83.1 and 58.2 as well as a non-HIV IgG₂ monoclonal antibody (60.1). Antibodies 59.1, 83.1, 58.2, and F105 are IgG_x;

50.1 is IgG_2a. The binding affinities for 59.1, 83.1,

58.2 and 50.1 are 9.3 X 10^"5M, 1.25 X 10^"8M, 3.3 x 10^"7M and 2.2 x 10~⁷M respectively. The binding affinity for

F105 is 8.3 x 10~⁹M, for sCD4 the binding affinity is 4.0 x 10~⁹M. The epitope for F105 and sCD4 is the CD4 binding domain of gpl20.

Table 6: Additional Std. SN Results Fold

Increase in

The IRA assays were performed as follows. Anti-V3 loop monoclonal antibody (59.1, 58.2 or 83.1) at 200 μg/ml and sCD4 or F105 at 100 μg/ml were preincubated with serial 10-fold dilutions of the indicated virus for 1 hr at 37°C before plating on CEM-SS cells or peripheral blood lymphocytes in 2 ml cultures. PBL cultures were stimulated with PHA, or not, and then supplemented with IL-2 every 3 days; all cultures were split at 3 days post-infection and every 2-3 days thereafter. In most cases, both anti-HIV agents were added a second time at the original concentration 3 days post-infection. In one case, 59.1 was added 10 times (every time the culture was split) . All assays were performed in duplicate. Viral replication was assessed by measuring the reverse transcriptase activity of cell-free supernatants at two post-infection time points. The results of these assays are presented in Table 7. In this table the results are presented as [x] Y/Z. [x] indicates the logs of virus present in the assay at full time in the absence of antibody or sCD4; Y indicates the logs of virus blocked at half-time in the presence of the indicated anti-HIV agent(s) ; and Z indicates the logs of virus blocked at full-time in the presence of the indicated anti-HIV agent(s) . The total infectious units blocked are reported in the last column. The half- and full-time points were 10 and 20 days respectively for MN and 7 and 14 days respectively for the other strains. Table 7: Additional IRA Results

Significantly, these experiments not only demonstrated that anti-V3 loop antibodies can synergize with sCD4 or F105 to neutralize HIV when added to cells along with virus, but also that a second addition of the two anti-HIV agents several days post-infection can substantially increase HIV neutralization.

Referring to Table 7, 59.1 combined with sCD4 or F105 substantially increased (10-fold) neutralization of MN when added a second time (3 days) post-infection. Antibody 58.2 combined with sCD4 had a similar effect on D6587-5. The fact that addition of a combination of anti-HIV agents several day post-infection, when it would be expected that the virus has substantially spread, suggests that the combination may be capable of initiating a catalytic cascade of cell killing by cross- linking infected cells.

Although F105 and sCD4 apparently recognize overlapping binding sites on gpl20, sCD4 synergized with 59.1, 58.2, and 83.1, while F105 synergized only with 59.1.

Importantly, sCD4 combined with 83.1 completely blocked field isolate D7887-7, increasing neutralization 1000-fold. This strain has previously been considered neutralization resistant because even polyclonal sera raised against the V3 loop of this strain would not neutralize it.

Anti-V3 loop antibodies combined with sCD4 neutralized a monotropic isolate AD-87 propagated in PBL. Referring to Table 7, sCD4 combined with 83.1 or 58.2 increase neutralization 10-fold. Further, sCD4 combined with 58.2 increased neutralization of AD-87 propagated in non-PHA stimulated, IL-2 supplemented PBL 10-fold. This cell culture system includes 10% stimulated PBL, activated macrophages and natural killer cells.

It should be noted that sCD4 combined with anti-V3 loop antibodies did not demonstrate significant synergy in 7 day Std. SN assays but by 15 days these assays demonstrated synergy (Table 6) . This difference may be apparent potency of CD4 in short term virology assays (7 days) which drops dramatically if the assay is maintained for a longer time (15 days) . This apparent dichotomy between the short and long term ability to neutralize by sCD4 illustrated by an IRA analysis for virus D7887-7. At 7 days post infection sCD4 appears to block 3 logs of virus but by 14 days neutralization has dropped dramatically. In the IRA virus remains infectious for up to 5 days and sCD4 remains stable for 14 days suggesting that the reason for the drop in neutralization may be due to the inability of sCD4 to remain bound to the virus. This decrease in neutralization is not observed when 83.1 is combined with sCD4. This suggests that the mechanism of neutralization for the combined anti-HIV agents and the individual anti-HIV agents is different. This is consistent with studies demonstrating that conformational changes induced in the viral envelope by sCD4 binding are important for subsequent increased exposure of the V3 loop for p3t:otease cleavage and fusion of virus and cell membrane or for binding of anti-V3 loop antibodies "and neutralization (Sattentau et al., J. Exp. Med. 174:407, 1991; Scott et al. , Proc. Nat^rl . Acad. Sci . 87:8597, 1990) . This would suggest that sCD4, which has a short biological half-life, need only be present transiently to increase substantially the effectiveness of anti-V3 loop antibodies.

Use

For treatment of HIV infection, anti-HIV agents need not be administered simultaneously, but instead may be administered sequentially. It is only required that both agents be present for some period in a therapeutically effective amount. The anti-HIV agents may be administered in any pharmaceutically acceptable composition. It can be advantageous to re-administer the anti-HIV agents. In the re-administration, both anti-HIV agents need not be added simultaneously. It is only required that both be present for some period in a therapeutically effective amount.

What is claimed is:

Claims

Claims 1. A composition for treatment of HIV-l infection, said composition comprising an antibody directed against the V3 loop of gpl20 and an antibody directed against the CD4 binding site of gpl20, wherein the HIV-l neutralization activity of said composition is greater than the sum of the HIV-l neutralization activity of said V3 loop antibody in the absence of said CD4 binding site antibody and the neutralization activity of said CD4 binding site antibody in the absence of said V3 loop antibody.

2. A composition for treatment of HIV-l infection, said composition comprising an antibody directed against the V3 loop of gpl20 and sCD4, wherein the HIV-l neutralization activity of said composition is greater than the sum of the HIV-l neutralization activity of said V3 loop antibody in the absence of said sCD4 and the neutralization activity of said sCD4 in the absence of said V3 loop antibody.

3. The composition of claim 1 or claim 2 wherein said V3 loop antibody is neutralizing.

4. The composition of claim 3 wherein said V3 loop antibody is capable of neutralizing two or more HIV- 1 strains.

5. The composition of claim 3 wherein said V3 loop antibody is 50.1.

6. The composition of claim 3 wherein said V3 loop antibody is 59.1.

7. The composition of claim 3 wherein said V3 loop antibody is 58.2.

8. The composition of claim 3 wherein said V3 loop antibody is 83.1.

9. A method for treatment of HIV-l infection in a human patient, said method comprising administering to said patient the composition of claim 1 or 2.

10. A method for treatment of HIV-l infection in a human patient, said method comprising administering to said patient an effective amount of an antibody directed against the V3 loop of gpl20 and an antibody directed against the CD4 binding site of gpl20, wherein the HIV-l neutralization activity of said V3 loop antibody in combination with said CD4 binding site antibody, is greater than the sum of the HIV-l neutralization activity of said V3 loop antibody in the absence of said CD4 binding site antibody and the neutralization activity of said CD4 binding site antibody in the absence of said V3 loop antibody.

11. A method for treatment of HIV-l infection in a human patient, said method comprising administering to said patient an effective amount of an antibody directed against the V3 loop of gpl20 and sCD4, wherein the HIV-l neutralization activity of said V3 loop antibody in combination with said sCD4, is greater than the sum of the HIV-l neutralization activity of said V3 loop antibody in the absence of said sCD4 and the neutralization activity of said sCD4 in the absence of said V3 loop antibody.

12. The method of claim 10 or 11 wherein said administration occurs more than once.