WO1996002575A1

WO1996002575A1 - Articles of manufacture for removing hiv-1 from a sample, and methods of using same

Info

Publication number: WO1996002575A1
Application number: PCT/US1995/008805
Authority: WO
Inventors: Graham P. Allaway; Paul J. Maddon; John P. Moore
Original assignee: Progenics Pharmaceuticals, Inc.; Aaron Diamond Aids Research Centre (Adarc)
Priority date: 1994-07-13
Filing date: 1995-07-13
Publication date: 1996-02-01
Also published as: AU3195495A; CA2194784A1; AU706927B2; EP0771326A4; JPH10506613A; EP0771326A1

Abstract

This invention provides an article of manufacture comprising a solid support having operably affixed thereto an agent, or plurality of agents, capable of specifically forming a complex with a domain present on an HIV-1 envelope glycoprotein. This invention further provides an aqueous-soluble agent which is capable of spefically forming a complex with a domain present on an HIV-1 envelope glycoprotein, and comprises a moiety capable of specifically forming a complex with a known ligand, which moiety permits the removal of the agent from a sample via contact with an immobilized form of the known ligand. This invention further provides methods of treating a bodily fluid sample so as to remove therefrom HIV-1 if present in the sample using the articles of manufacture or agent of the subject invention. Finally, the subject invention provides kits for treating a bodily fluid sample so as to remove therefrom HIV-1 or HIV-1 gp120 envelope glycoprotein if present in the sample.

Description

ARTICLES OF MANUFACTURE FOR REMOVING HIV-1 FROM A SAMPLE. AND METHODS OF USING SAME

This application is a continuation-in-part of U.S. Serial No. 08/274,240, filed July 13, 1994, the contents of which are hereby incorporated by reference.

Throughout this application, various publications are referenced by Arabic numerals. Full citations for these references may be found at the end of the specification immediately preceding the claims. The disclosures of these publications are hereby incorporated by reference into this application to describe more fully the art to which this invention pertains.

Background of the Invention

Approximately 12 million units of blood are collected and distributed by the American Red Cross each year. Nearly all of these units are fractionated and transfusions are usually performed with blood components rather than whole blood. The majority of transfusions (60%) are performed with packed red blood cells (PRBC) . Platelets comprise 27% of transfused components and fresh-frozen plasma 11% (1) . Whole blood is stored at room temperature prior to fractionation, which must occur within 6 hours of collection. After fractionation, the PRBC fraction is refrigerated, platelets are stored at room temperature and plasma is frozen.

Despite rigorous pre-screening of donors and post-donation testing of blood for antibodies to HIV-1, approximately 50- 100 units of blood contaminated with HIV-l enter the U.S.blood supply each year. As primary HIV-l isolates are fairly robust, the infectious potential of contaminate blood survives storage of blood components (2-5) . Thus, administration of contaminated components to transfusio recipients often, perhaps invariably, leads to HIV- transmission. The subsequent infection is usually fatal and can be transmitted to others. Consequently, publi confidence in the safety of donated blood has been reduced A practical method for the efficient and safe eliminatio of HIV-l from donated blood would therefore be an importan contribution to public health.

Contamination of donated blood can arise as a result o several scenarios. Perhaps the most common source o contamination results from the donation of blood b individuals who have been infected with HIV-l, but who hav not yet developed antibodies detectable by standar screening assays. These pre-seroconversion cases can b highly viremic. During the acute phase of HIV-l infection virus titres in excess of 10* TCID₅₀/ml can be found i plasma in the absence of detectable HIV-l antibodies (6,7)

Studies have shown that it is possible to detect anti-gpl2 antibodies by ELISA 4 days before they register in conventional antibody-detection ELISA, and after plasm virus titres decline by more than 100-fold from their pea values. The infectious potential of such individuals i obvious. As the HIV epidemic spreads into the low-ris population, the efficacy of pre-donation screening ma diminish, allowing more such contaminated units into th blood supply. Another potential source of contamination ma arise from random error during screening procedures resulting in the use of contaminated samples from antibody positive, asymptomatic individuals. It is also possibl that some antibody-positive samples may not be detectabl by current screening tests, either because the antibod concentration in the plasma is extremely low, or because a immune response is not raised against those epitopes best represented in ELISA-based screening assays. Such cases have been documented (8, 9), and as HIV-l diversifies in sequence and more exotic strains are introduced into the U.S., the possibility must be considered that the number of non-detectable, antibody-positive cases might increase.

Another possibility is that a small number of HIV-infected individuals never develop an antibody response, and never develop clinical symptoms of HIV infection, but are able to transmit the virus to a recipient. Cases of individuals exposed to HIV, but apparently uninfected, have been documented (9-11) . Whether these individuals are truly uninfected is not yet certain, and there are no documented cases of transmission of HIV from such people. Nonetheless, the possibility exists.

A procedure to reduce the infectivity of contaminated blood should be effective irrespective of the source of contamination which might be one of the sources described above. Thus, the procedure must be able to deal with the very high titres of HIV-l found during the acute, antibody- negative phase of infection and with the lower titres of HIV-l present in antibody-positive samples that are not eliminated by conventional screening tests. The presence of anti-HIV antibodies should not compromise the procedure of choice. Chemical and detergent methods have been developed which are suitable for treating plasma to inactivate HIV-l but which are not suitable for treating whole blood or blood components containing cells or platelets.

CD4-based molecules include recombinant soluble CD4 (sCD4;

VI-V4 domains of CD4) and fusion proteins between portions of CD4 and other proteins including human immunoglobulins. In vitro experiments with CD4-based molecules demonstrate three important antiviral properties (12-15) : (1) binding to HIV gpl20 and competitively inhibiting viral attachment to and subsequent infection of human cells; (2) dissociation of gpl20 from the viral surface, thereby irreversibly inactivating HIV particles; and (3) inhibition of the intercellular spread of virus from infected cells to uninfected cells by inhibiting HIV-l envelope-mediated cell fusion.

Extensive in vitro experiments demonstrate that CD4-based molecules can neutralize the broadest range of both laboratory-adapted and primary isolates of HIV-l with the greatest overall potency when compared to monoclonal antibodies or HIV immune globulin (16) . This is not surprising since CD4 is the major receptor for HIV-l and it is unlikely that the virus can mutate to avoid binding to CD4 without also losing its ability to infect cells. Although laboratory-adapted strains of HIV-l exhibit greater sensitivity to neutralization by CD4-based molecules as compared to primary isolates in vitro, all clinical isolates of HIV-l tested are inhibited by CD4-based molecules at concentrations achievable in humans. Little or no toxicity or imrnunogenicity has been observed in clinical trials with CD4-based molecules (18) . In addition, administration of CD4-Ig to chimpanzees prior to challenge with HIV-l^ protects them from infection, documenting the protective effect of CD4-based molecules in vivo (19) .

Serum neutralizing antibody activity against HIV is directed at the virus envelope (20-30) . Although there are neutralization sites in the ecto-domain of gp41 (the transmembrane glycoprotein of HIV-l) , most neutralizing antibodies in sera from naturally infected humans are directed against the outer envelope glycoprotein, gpl20 (20-30) . The V3 loop of HIV-l gpl20 has been designated as the "principal neutralizing determinant" as it is the site recognized by potent, often type-specific, neutralizing antibodies that arise early in infection (26, 31-39) . Antibodies to the V3 region also appear to correlate with protection in two successful vaccination and challenge experiments in the HIV-1-infected chimpanzee model (40, 41) . Although broadly-reactive anti-V3 MAbs are now well characterized, it seems reasonable to assume that some of the group-specific neutralizing antibody activity in sera from HIV-l-infected humans during the asymptomatic phase results from the induction of antibodies directed to discontinuous epitopes, at least a fraction of which antibodies act by blocking the binding of HIV to its CD4 receptor (42, 43) . However, none of these MAbs is as cross- reactive as CD4-based molecules, and none is as potent at neutralizing laboratory-adapted or primary HIV-l isolates in vitro (16) . Several studies have demonstrated that some combinations of CD4-based molecules and Mabs directed against gpl20 or gp41 are synergistic in neutralizing HIV-l (44-48) .

As noted above, primary HIV-l isolates are less sensitive to neutralization by CD4-based molecules when compared to their T-cell line-adapted counterparts. The mechanism for this difference appears to result from the fact that intact virions of primary isolates exhibit a reduced ability to bind sCD4 and to undergo subsequent sCD4-induced gpl20 dissociation or shedding (15, 49) .

Numerous studies have demonstrated that the envelope glycoprotein of HIV-l, gpl20, may have a direct role in the pathogenesis of HIV-l infection. Some potential mechanisms for gpl20 in immunopathogenesis of HIV-l infection are described by Fauci, A. and Rosenberg, Z. (In: Textbook of AIDS Medicine, Broder et al. eds. , 1994, Williams an Wilkins, Baltimore, MD, pp 55-75) . These mechanism include binding of gpl20 to CD4 on CD4⁺ cells resulting i immunosuppression or apoptosis. The removal of gpl20 fro the blood or other bodily fluids of HIV-1-infecte individuals would reduce these pathogenic effects an improve clinical outcome. Removal might be accomplished for example, by attaching CD4-based proteins or antibodie to a filter and circulating blood through this filte (plasmapheresis) . This procedure could also be used t remove HIV-l virions from the blood which would also b beneficial.

Summary of the Invention

This invention provides an article of manufacture comprising a solid support having operably affixed thereto an agent capable of specifically forming a complex with a domain present on an HIV-l envelope glycoprotein.

This invention further provides an article of manufacture comprising a solid support having operably affixed thereto a plurality of agents each capable of specifically forming a complex with a domain present on an HIV-l envelope glycoprotein.

This invention further provides an aqueous-soluble agent which (a) is capable of specifically forming a complex with a domain present on an HIV-l envelope glycoprotein, and (b) comprises a moiety capable of specifically forming a complex with a known ligand, which moiety permits the removal of the agent from a sample via contact with an immobilized form of the known ligand.

This invention further provides a method of treating a bodily fluid sample so as to remove therefrom HIV-l if present in the sample which comprises contacting the sample under suitable conditions with an article of manufacture comprising a solid support having operably affixed thereto an agent capable of specifically forming a complex with a domain present on an HIV-l envelope glycoprotein, thereby removing therefrom HIV-l if present in the sample.

This invention further provides a method of treating a bodily fluid sample so as to substantially reduce the likelihood of a subject's becoming infected with HIV-l as a result of contact with the sample which comprises contacting the sample with a suitable amount of an aqueous-soluble agent capable of specifically forming a complex with a domain present on an HIV-l envelope glycoprotein, so as to form a complex between the agent and HIV-l if present in the sample and thereby reduce the likelihood of a subject's becoming infected with HIV-l as a result of contact with the sample.

This invention further provides a method of treating a bodily fluid sample so as to substantially reduce the likelihood of a subject's becoming infected with HIV-l as a result of contact with the sample which comprises the steps of (a) contacting the sample with a suitable amount of an aqueous-soluble agent capable of specifically forming a complex with a domain present on an HIV-l envelope glycoprotein, thereby forming a complex between the agent and HIV-l if present in the sample; and (b) removing any complex so formed from the resulting sample, so as to thereby reduce the likelihood of a subject's becoming infected with HIV-l as a result of contact with the sample.

This invention further provides a method of treating a bodily fluid sample so as to remove therefrom HIV-l or HIV gpl20 envelope glycoprotein if present in the sample which comprises contacting the sample under suitable conditions with an article of manufacture comprising a solid support having operably affixed thereto an agent capable of specifically forming a complex with a domain present on an HIV-l envelope glycoprotein, thereby removing therefrom HIV-l or HIV-l gpl20 envelope glycoprotein if present in the sample.

This invention further provides a method of treating a bodily fluid sample so as to substantially reduce the likelihood of a subject's becoming infected with HIV-l as a result of contact with the sample which comprises the steps of (a) contacting the sample with a suitable amount of an aqueous-soluble agent which (i) is capable of specifically forming a complex with a domain present on an HIV-l envelope glycoprotein, and (ii) comprises a moiety capable of specifically forming a complex with a known ligand, which moiety permits the removal of the agent from a sample via contact with an immobilized form of the known ligand, thereby forming a complex between the agent and HIV-l if present in the sample; and (b) removing any complex so formed from the resulting sample by contacting the resulting sample with an immobilized form of the known ligand, so as to thereby reduce the likelihood of a subject's becoming infected with HIV-l as a result of contact with the sample.

This invention further provides a method of treating a bodily fluid sample so as to substantially reduce the likelihood of a subject's becoming infected with HIV-l as a result of contact with the sample which comprises the steps of (a) contacting the sample under suitable conditions with an article of manufacture comprising a solid support having operably affixed thereto an agent capable of specifically forming a complex with a domain present on an HIV-l envelope glycoprotein; and (b) contacting the sample with a suitable amount of an aqueous-soluble agent capable of specifically forming a complex with a domain present on an HIV-l envelope glycoprotein, so as to form a complex between the agent and HIV-l if present in the sample, with the proviso that step (a) may either precede or follow step (b) .

This invention further provides a method of treating a bodily fluid sample so as to substantially reduce the likelihood of a subject's becoming infected with HIV-l as a result of contact with the sample which comprises the steps of (a) contacting the sample under suitable conditions with an article of manufacture comprising a solid support having operably affixed thereto an agent capable of specifically forming a complex with a domain present on an HIV-l envelope glycoprotein; and (b) (i) contacting the sample with a suitable amount of an aqueous-soluble agent capable of specifically forming a complex with a domain present on an HIV-l envelope glycoprotein, thereby forming a complex between the agent and HIV-l if present in the sample, and (ii) removing an complex so formed from the resulting sample, with the proviso that step (a) may either precede or follow step

(b) .

This invention further provides a method of treating a bodily fluid sample so as to substantially reduce the likelihood of a subject's becoming infected with HIV-l as a result of contact with the sample which comprises the steps of (a) contacting the sample under suitable conditions with an article of manufacture comprising a solid support having operably affixed thereto an agent capable of specifically forming a complex with a domain present on an HIV-l envelope glycoprotein; and (b) (I) contacting the sample with a suitable amount of an aqueous-soluble agent which (1) is capable of specificall forming a complex with a domain present on an HIV-l envelope glycoprotein, and (2) comprises a moiety capable of specifically forming a complex with a known ligand, thereby forming a complex between the agent and HIV-l if present in the sample, and (II) removing any complex so formed from the resulting sample by contacting the resulting sample with an immobilized form of the known ligand, with the proviso that step (a) may either precede or follow step (b) . This invention further provides a kit for treating a bodily fluid sample so as to substantially reduce the likelihood of a subject's becoming infected with HIV-l as a result of contact with the sample which comprises, in separate compartments: (a) an article of manufacture comprising a solid support having operably affixed thereto an agent capable of specifically forming a complex with a domain present on an HIV-l envelope glycoprotein; and (b) an aqueous-soluble agent capable of specifically forming a complex with a domain present on an HIV-l envelope glycoprotein.

This invention further provides a kit for treating a bodily fluid sample so as to substantially reduce the likelihood of a subject's becoming infected with HIV-l as a result of contact with the sample which comprises, in separate compartments: (a) an article of manufacture comprising a solid support having operably affixed thereto an agent capable of specifically forming a complex with a domain present on an HIV-l envelope glycoprotein; (b) an aqueous- soluble agent which (1) is capable of specifically forming a complex with a domain present on an HIV-l envelope glycoprotein, and (2) comprises a moiety capable of specifically forming a complex with a known ligand, which moiety permits the removal of the agent from a sample via contact with an immobilized form of the known ligand; and (c) an article of manufacture comprising a solid support having operably affixed thereto the known ligand capable of specifically forming a complex with the moiety of the aqueous-soluble agent of step (b) .

Finally, this invention provides a kit for treating a bodily fluid sample so as to substantially reduce the likelihood of a subject's becoming infected with HIV-l as a result of contact with the sample which comprises, in separate compartments: (a) an aqueous-soluble agent which (i) is capable of specifically forming a complex with a domain present on an HIV-l envelope glycoprotein, and (ii) comprises a moiety capable of specifically forming a complex with a known ligand, which moiety permits the removal of the agent from a sample via contact with an immobilized form of the known ligand; and (b) an article of manufacture comprising a solid support having operably affixed thereto the known ligand capable of specifically forming a complex with the moiety of said aqueous-soluble agent.

This invention also provides a kit for reducing the amount of HIV-l or HIV-l gpl20 envelope glycoprotein present in a bodily fluid sample which comprises the above-described article of manufacture.

Brief Description of the Figure

Figure 1 CD4-IgG2 effectively neutralizes HIV-l in the plasma of HIV-l infected individuals.

Detailed Description of the Invention

The plasmids pT4B, CD4IgG2-pcDNAl, CD4-kLC-pRcCMV, CD4- IgG₂HC-pRcCMV were deposited pursuant to, and in satisfaction of, the requirements of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure with the American Type Culture Collection (ATCC) , 12301 Parklawn Drive, Rockville, Maryland 20852 under ATCC Accession Nos. 68389, 40952, 75194 and 75193, respectively.

Specifically, this invention provides an article of manufacture comprising a solid support having operably affixed thereto an agent capable of specifically forming a complex with a domain present on an HIV-l envelope glycoprotei .

The solid support may be any solid support known in the art to which the agent can be operably affixed. Solid supports include, by way of example, natural or synthetic polymers. Synthetic polymers include, by way of example, polystyrene, polyethylene and polypropylene. Natural polymers include, by way of example, latex. The solid support may be selected, for example, from the group consisting of a bead, a receptacle, and a filter.

Solid supports in the form of beads are widely used and readily available to those skilled in the art. Beads include, for example, latex and polystyrene beads.

The receptacle can be any receptacle in which a bodily fluid is stored, or with which such fluid comes into contact. For example, the receptacle may be in the form of a bag or tubing. In the preferred embodiment, the receptacle is a bag specifically intended for the collection and/or storage of blood or blood components.

Solid supports in the form of filters are widely used and readily available to those skilled in the art. Filters include, for example, polyester filters (e.g., polyester leukofiltration devices) and cellulose acetate filters.

The agent affixed to the solid support may either be a protein or a non-protein agent. In one embodiment, the agent is an antibody. As used herein, the term "antibody" includes, but is not limited to, both naturally occurring and non-naturally occurring antibodies. Specifically, the term "antibody" includes polyclonal and monoclonal antibodies, and antigen-binding fragments thereof. Furthermore, the term "antibody" includes chimeric antibodies, wholly synthetic antibodies, and antigen-binding fragments thereof.

In another embodiment, the agent is a CD4-based molecule. As used herein, CD4 means the mature, native, membrane-bound CD4 protein comprising a cytoplasmic domain, a hydrophobic transmembrane domain, and an extracellular domain which binds to the HIV-l gpl20 envelope glycoprotein.

As used herein, a CD4-based molecule is any molecule comprising at least one sequence of amino acid residues corresponding to that portion of CD4 which is required for CD4 to form a complex with the HIV-l gpl20 envelope glycoprotein. If the CD4-based molecule is sCD4, then the sequence of amino acid residues corresponding to that portion of CD4 which is required for CD4 to form a complex with the HIV-l gpl20 envelope glycoprotein is the amino acid sequence from +1 to about +106. As used herein, sCD4 means a water soluble, extracellular fragment of human CD4. If the CD4-based molecule comprises a portion of a non-CD4 protein, then the sequence of amino acid residues corresponding to that portion of CD4 which is required for CD4 to form a complex with the HIV-l gpl20 envelope glycoprotein is the amino acid sequence from +1 to about +179. Thus, a CD4-based molecule includes one or more of CD4's gpl20-binding sites.

Examples of CD4-based molecules include, but are in no way limited to, the CD4-based molecules discussed infra.

The CD4-based molecule may be a CD4-immunoconjugate. The CD4-immunoconjugate may be a CD4-gammal chimeric heavy chain homodimer, a CD4-gamma2 homodimer, or a CD4-IgGl heterotetramer.

In the preferred embodiment, the CD4-immunoconjugate is a heterotetramer comprising two heavy chains and two light chains, both heavy chains being either (a) IgG2 heavy chains or (b) chimeric CD4-IgG2 heavy chains, and both light chains being either (a) kappa light chains or (b) chimeric CD4-kappa light chains, with the proviso that either both heavy chains or both light chains or all four chains are CD4 chimeras. The chimeric CD4-IgG2 heavy chains may be the heavy chains encoded by the expression vector designated CD4-IgG2HC-pRcCMV (ATCC No. 75193) , and the chimeric CD4-kappa light chains may be the light chains encoded by the expression vector designated CD4-kLC-pRcCMV

(ATCC No. 75194) .

Methods of making CD4-based molecules are well known in the art and are exemplified in the Experimental Details section which follows.

As used herein, "operably affixed" means affixed in a manner permitting the formation of a complex between the affixed agent and the domain present on an HIV-l envelope glycoprotein. Methods of operably affixing an agent to a solid support are well known to those skilled in the art.

As used herein, "capable of specifically forming a complex with a domain present on an HIV-l envelope glycoprotein" means capable of forming a complex with a domain present on an HIV-l envelope glycoprotein but not capable of forming a complex with any other domain.

As used herein, "HIV-l" is synonymous with the terms "HIV-l particle," "HIV-l virion" or "HIV-l virus."

In one embodiment, the HIV-l envelope glycoprotein is the HIV-l gpl20 envelope glycoprotein. In another embodiment, the HIV-l envelope glycoprotein is the HIV-l gp41 envelope glycoprotein.

In one embodiment, the domain present on the HIV-l envelope glycoprotein is a conserved domain. As used herein, a "conserved domain" is an envelope glycoprotein domain which is present on, and whose structure is invariant among, at least 90% of all strains of HIV-l. In the preferred embodiment, the conserved domain present on the HIV-l envelope glycoprotein is the CD4-binding domain of the HIV-l gpl20 envelope glycoprotein.

In another embodiment, the domain present on the HIV-l envelope glycoprotein is a non-conserved domain. For example, the non-conserved domain may be a domain present on the V3 loop of the HIV-l gpl20 envelope glycoprotein.

As used herein, a "plurality of agents" means at least two agents. In one embodiment, the plurality of agents consists of a plurality of CD4-based molecules. In another embodiment, the plurality of agents consists of a plurality of antibodies. In a further embodiment, the plurality of agents comprises an antibody and a CD4-based molecule.

As used herein, "aqueous-soluble" means capable of existing in soluble form in water at 4°C at a concentration of at least lpM.

The use of a moiety capable of specifically forming a complex with a known ligand is commonly referred to in the art as "molecular tagging." The moiety may be selected, for example, from the group consisting of a small molecule and a protein. The ligand may be selected, for example, from the group consisting of a metal ion, a small molecule and a protein. Specific examples of moiety/ligand combinations include, but are not limited to, (a) oligohistidine/nickel ion, (b) glutathione S-transferase/glutathione, and (c) biotin/streptavidin.

As used herein, "treating a bodily fluid sample so as to remove therefrom HIV-l" means either (a) rendering the HIV-l in the bodily fluid sample unable to invade CD4⁺ cells, (b) physically separating HIV-l from the bodily fluid sample, or (c) a combination of (a) and (b) , with the proviso that the HIV-l present in the resulting sample and capable of invading CD4* cells does not exceed 50% of the amount of such HIV-l present in the sample prior to removing HIV-l. As used herein, a CD4⁺ cell is a cell having CD4 present on its surface, wherein the CD4^* cell is capable of specifically binding to and fusing with HIV-l contacted therewith.

Suitable conditions for contacting the sample with the subject article of manufacture are conditions which would permit the formation of a complex between the agent and HIV-l. Such conditions are known to those skilled in the art.

This invention provides a method of substantially reducing the amount of HIV-l gpl20 envelope glycoprotein in a bodily fluid sample which comprises contacting the sample with a suitable amount of an aqueous-soluble agent capable of specifically forming a complex with a domain present on an HIV-l envelope glycoprotein, so as to form a complex between the agent and HIV-l if present in the sample and thereby reduce the amount of HIV-l gpl20 envelope glycoprotein in the sample.

In an embodiment, the blood of HIV-infected individuals will be passed through filters on which CD4-based proteins or antibodies have been immobilized. This would allow the removal of HIV-l virions and/or HIV-l gpl20 from the blood. The presence of gpl20 in the blood may be pathogenic for example by binding to CD4-expressing cells and inhibiting the immune response or by initiating apoptosis of these cells.

In the preferred embodiment, the subject is a human. As used herein, substantially reducing the likelihood of the subject's becoming infected with HIV-l means reducing the likelihood of the subject's becoming infected with HIV-l by at least two-fold. For example, if a subject has a 1% chance of becoming infected with HIV-l, a two-fold reduction in the likelihood of the subject's becoming infected with HIV-l would result in the subject's having a 0.5% chance of becoming infected with HIV-l. In one embodiment, substantially reducing the likelihood of the subject's becoming infected with HIV-l means reducing the likelihood by at least ten-fold. In the preferred embodiment, substantially reducing the likelihood of a subject's becoming infected with HIV-l means reducing the likelihood by at least 100-fold.

As used herein, "the subject's becoming infected with HIV-l" means the invasion of the subject's own cells by HIV-l.

As used herein, contact with a bodily fluid sample is any contact sufficient to cause HIV-l in the sample to be transmitted to the subject's body, and thereby infect the subject with HIV-l.

The amount of aqueous-soluble agent suitable to substantially reduce the likelihood of a subject's becoming infected with HIV-l may be determined according to methods known to those skilled in the art. In one embodiment, the suitable amount of aqueous-soluble agent is an amount between about lpM and about lOmM. In the preferred embodiment, the suitable amount of aqueous-soluble agent is an amount between about lpM and about lOμM.

In one embodiment, the agent is an antibody. In another embodiment, the agent is a CD4-based molecule. The CD4based molecule may be a CD4-immunoconjugate.

In the preferred embodiment, the CD4-immunoconjugate is a heterotetramer comprising two heavy chains and two light chains, both heavy chains being either (a) IgG2 heavy chains or (b) chimeric CD4-IgG2 heavy chains, and both light chains being either (a) kappa light chains or (b) chimeric CD4-kappa light chains, with the proviso that either both heavy chains or both light chains or all four chains are CD4 chimeras.

The chimeric CD4-IgG2 heavy chains may be the heavy chains encoded by the expression vector designated CD4-IgG2HC- pRcCMV (ATCC No. 75193) , and the chimeric CD -kappa light chains may be the light chains encoded by the expression vector designated CD4-kLC-pRcCMV (ATCC No. 75194) .

Removing complex from the resulting sample may be accomplished according to methods well known to those skilled in the art. Such methods include, for example, affinity chromatography.

The subject method may further comprise the step of removing uncomplexed agent from the sample should such removal be desirable (e.g., when the agent would cause undesirable effects in a subject to whom it is administered) .

Methods of immobilizing a ligand are well known to those skilled in the art. As used herein, a ligand in its "immobilized form" is capable of forming a complex with the moiety specifically recognized by the ligand in its free form.

This invention further provides a method of treating a bodily fluid sample so as to substantially reduce the likelihood of a subject's becoming infected with HIV-l as a result of contact with the sample which comprises the steps of (a) contacting the sample under suitable conditions with an article of manufacture comprising a solid support having operably affixed thereto an agent capable of specifically forming a complex with a domain present on an HIV-l envelope glycoprotein; and (b) (i) contacting the sample with a suitable amount of an aqueous-soluble agent capable of specifically forming a complex with a domain present on an HIV-l envelope glycoprotein, thereby forming a complex between the agent and HIV-l if present in the sample, and (ii) removing any complex so formed from the resulting sample, with the proviso that step (a) may either precede or follow step (b) .

This invention further provides a method of treating a bodily fluid sample so as to substantially reduce the likelihood of a subject's becoming infected with HIV-l as a result of contact with the sample which comprises the steps of (a) contacting the sample under suitable conditions with an article of manufacture comprising a solid support having operably affixed thereto an agent capable of specifically forming a complex with a domain present on an HIV-l envelope glycoprotein; and (b) (I) contacting the sample with a suitable amount of an aqueous-soluble agent which (1) is capable of specifically forming a complex with a domain present on an HIV-l envelope glycoprotein, and (2) comprises a moiety capable of specifically forming a complex with a known ligand, thereby forming a complex between the agent and HIV-l if present in the sample, and (II) removing any complex so formed from the resulting sample by contacting the resulting sample with an immobilized form of the known ligand, with the proviso that step (a) may either precede or follow step (b) .

The methods of the subject invention may further comprise the step of removing CD4* cells from the bodily fluid sample. In the preferred embodiment, the CD4⁺ cells are leukocytes. Methods of removing leukocytes from a bodily fluid sample are well known to those skilled in the art and include, for example, leukofiltration.

As used herein, a bodily fluid is any fluid which is present in a subject's body and is capable of containing

HIV-l in an HIV-l-infected subject. Bodily fluids include, but are not limited to, whole blood or derivatives thereof

(e.g., red blood cell and platelet preparations), saliva, cerebrospinal fluid, tears, vaginal secretions, urine, alveolar fluid, synovial fluid, semen, pleural fluid and bone marrow. In the preferred embodiment, the bodily fluid is a fluid which is to be administered to a subject. Also in the preferred embodiment, the bodily fluid sample is selected from the group consisting of whole blood, a red blood cell preparation, a platelet preparation and semen.

The bodily fluid samples such as whole blood may further comprise exogenous substances added thereto for clinical or storage purposes. Such exogenous substances include, by way of example, anticoagulants (e.g., citrate) and preservatives (e.g., dextrose).

In one embodiment, the contacting steps of the methods of the subject invention are performed at about 4°C. In another embodiment, the contacting steps of the methods of the subject invention are performed at about 20°C. In still another embodiment, the contacting steps of the methods of the subject invention are performed at about 37°C.

The invention also provides a kit for treating a bodily fluid sample so as to substantially reduce the likelihood of a subject's becoming infected with HIV-l as a result of contact with the sample which comprises the above-described article of manufacture.

This invention further provides a kit for treating a bodily fluid sample so as to substantially reduce the likelihood of a subject's becoming infected with HIV-l as a result of contact with the sample which comprises, in separate compartments: (a) an article of manufacture comprising a solid support having operably affixed thereto an agent capable of specifically forming a complex with a domain present on an HIV-l envelope glycoprotein; and (b) an aqueous-soluble agent capable of specifically forming a complex with a domain present on an HIV-l envelope glycoprotein.

This invention provides a kit for treating a bodily fluid sample so as to substantially reduce the likelihood of a subject's becoming infected with HIV-l as a result of contact with the sample which comprises, in separate compartments: (a) an aqueous-soluble agent which (i) is capable of specifically forming a complex with a domain present on an HIV-l envelope glycoprotein, and (ii) comprises a moiety capable of specifically forming a complex with a known ligand, which moiety permits the removal of the agent from a sample via contact with an immobilized form of the known ligand; and (b) an article of manufacture comprising a solid support having operably affixed thereto the known ligand capable of specifically forming a complex with the moiety of said aqueous-soluble agent.

This invention also provides a kit for reducing the amount of HIV-l or HIV-l gpl20 envelope glycoprotein present in a bodily fluid sample which comprises the above-described article of manufacture. In an embodiment, the bodily fluid is blood.

The kits of the subject invention may further comprise suitable buffers.

In order to facilitate an understanding of the following examples, certain frequently occurring methods and/or terms are best described in Sambrook, et al. (17) . This invention will be better understood by reference to the Experimental Details section which follows, but those skilled in the art will readily appreciate that the specific experiments detailed are only illustrative of the invention as described more fully in the claims which follow thereafter.

Experimental Details

A. Introduction

Primary viruses appear to become more sensitive to sCD4 if incubated therewith for prolonged periods at 4°C rather than at 37°C. Preliminary results indicate that neutralization by anti-V3-loop HuMAbs is also more sensitive at 4°C than at 37°C, implying that the resistance mechanism of HIV to neutralization by sCD4 applies also to MAbs. Advantage of this phenomenon is taken in the subject invention, since the routine storage of whole blood and PRBC, for example, occurs at 4°C.

In the subject invention, particular advantages are found in using a tetrameric CD4-immunoglobulin protein (CD4-IgG2) formed by the fusion of the VI and V2 domains of CD4 with the constant regions of the heavy and light chains of human IgG2. This molecule is even more effective than monomeric or dimeric CD4-based molecules in neutralizing HIV-l, including primary isolates of the virus. Of additional importance in the subject invention is the observation that CD4-IgG2 effectively neutralizes HIV-l in the plasma of HIV-l-infected patients when tested in an ex vivo assay, where it was more effective than any of the neutralizing monoclonal antibodies tested.

B. Materials and Methods

1. Reagents

(a) General sCD4, CD4-gammal chimeric heavy chain homodimers,

CD4-gamma2 chimeric heavy chain homodimers and CD4-IgG2 chimeric heterotetramers may be obtained from Progenies Pharmaceuticals, Inc. (Tarrytown, New York) . Mous antibody [9205] to the V3 loop of gpl20, derived from th HIV-I_HTLVUΪB isolate, may be obtained from DuPont NE Research Products (Wilmington, Delaware) . Human antibod 2F5 to a conserved epitope of HIV-l gp41 may be obtaine from Viral Testing Systems Corporation (Houston, Texas) an from Waldhei Pharmazeutika (Vienna, Austria) .

(b) sCD4 Soluble CD4 (a genetically-engineered, water-solubl extracellular fragment of human CD4) is disclosed, fo example, in Patent Cooperation Treaty Internationa Publication No. WO 88/01304. Soluble CD4 is als commercially available.

Soluble CD4, also designated sCD4, may be produced b truncating pT4B (ATCC No. 68389) after the V4J4 domain. Such DNA fragments terminate before the transmembran segment, which begins at approximately nucleotide positio 1264.

Purification and characterization of soluble CD4 fragment is greatly enhanced by constructing a cell line (preferabl mammalian) which overexpresses the secreted protei fragment. Strategies which allow the overexpression o proteins may be employed in bacteria, yeast, insect an mammalian systems. Inducible expression systems may also b employed in bacteria and yeast to overproduce protein which may be toxic if constitutively expressed. Overexpression of soluble CD4 fragments may be accomplishe by amplifying a soluble CD4 expression vector, resulting i constitutive overexpression. The amplification o dihydrofolate reductase (dhfr) genes by growth i progressively increased concentrations of the dru methotrexate, an antagonist of dhfr, is widely employed. Since the amplified unit is not limited to dhfr coding sequences, this approach results in the coamplification of sequences adjacent to them. Therefore, dhfr may be used as a selectable marker and as a means of coamplifying newly introduced sequences. This strategy may be successfully employed to increase the expression of several different genes cotransformed with dhfr plasmids.

Using recombinant DNA technology, a vector expressing a secreted, soluble, extracellular fragment of CD4 encoded by the human cDNA clone pT4B may be generated. Base pairs 1 1252 of pT4B encode the leader peptide of CD4 needed for the synthesis of secreted protein, as well as the extracellular portion of CD4 encompassing the four VJ-like domains (V1J1V4J4) , but not the transmembrane and cytoplasmic regions which anchor the protein in the membrane. This vector contains sequences encoding the extracellular portion of the CD4 protein which contains the HIV binding domain. These sequences are placed downstream from the SV40 early region promoter. In addition, a TAA termination codon followed by the polyadenylation region of the bovine growth hormone gene is placed downstream from the truncated CD4 cDNA to provide the signals necessary for the termination of protein synthesis, transcription termination, and polyadenylation of the RNA transcript. The resulting soluble CD4 minigene is then ligated to the mouse dihydrofolate reductase (dhfr) gene to generate a plasmid capable of being amplified after introduction into dhfr-deficient (dhfr-) Chinese hamster ovary (CHO) cells.

For example, the 1.8 kb EcoRI-BamHI fragment of pT4B, which contains the entire CD4 coding sequence, is inserted between the StuI and Bell sites of the mammalian expression vector DSP modified to contain the SV-40 early promoter and the bovine growth hormone polyadenylation sequence. Through the use of synthetic linkers, the Haell (bp 124) - Hpall (bp 1252) fragment of pT4B is inserted between the Kpnl an Xbal sites of the plasmid pUC18. A soluble CD4 expressio vector 5 is created by ligating:

1. a 0.95 kb Bglll - Sad fragment of modified DS which contains the 1.8 kb EcoRI-BamHI fragment o pT4B (this segment contains the SV40 early 10 promoter, the CD4 leader sequence, and the amin terminal portion of the extracellular CD sequence) ;

2. the 0.66 kb Sad - Xbal fragment of the pUC1 plasmid containing the Haell-Hpall fragment o pT4B (this segment contains the carboxy termina portion of the extracellular CD4 sequenc followed by a TAA termination codon inserte after valine 371) ; and

3. the 2.48 kb Bglll - Xbal fragment of modified DS which contains the bovine growth hormone poly adenylation sequence.

Finally, the 2.2 kb Bglll - BamHI fragment from anothe modified DSP containing a mouse dhfr expression cassett (beta-globin promoter - mouse dhfr coding region - SV4 polyadenylation region) flanked by Bglll and BamHI sites, is inserted into the BamHI site of a plasmid to create soluble CD4 expression plasmid.

DXB-ll, a clone of Chinese hamster ovary cells deficient i dhfr, is transfected with the soluble CD4 expressio plasmid. The DXB-ll transformants are then grown in F1 medium, without hypoxanthine or thymidine, containing 10 dialyzed fetal bovine serum. Clones are selected an subjected to stepwise increasing concentrations of methotrexate (mtx) , an antagonist of dhfr, to select for stable transformants which have amplified the newly introduced dhfr gene and adjacent soluble CD4 sequences.

Purification of the sCD4 protein was performed using ion exchange chromatography. Ion exchange chromatography is well known to those killed in the art.

(c) CD4-IqG2 Chimeras

(i) Stable expression

Dhfr- Chinese hamster ovary cells (CHO) were transfected with 20 micrograms of CsCl-purified DNA in a 1000:1 molar ratio of CD4IgG2-pcDNAl:p410 (p410 is an expression plasmid containing the dhfr gene) , although other ratios may also be used. Approximately 3-5 days post-transfection, cells were placed in selective medium (nucleoside-free alpha MEM containing 5% dialyzed fetal calf serum) . Approximately 10-15 days post-selection, individual cell clones were picked and analyzed for stable expression of CD4-gamma2 chimeric heavy chain homodimer by several screening techniques, such as ELISA and precipitation with Protein A- sepharose beads followed by SDS-PAGE under reducing and nonreducing conditions. Clones expressing the highest levels were subjected to successive rounds of amplification of the newly introduced DNA sequences in increasing concentrations of methotrexate. Stable CHO cell lines were thus generated which secrete between 10-100 micrograms/milliliter of CD4-gamma2 chimeric heavy chain homodimer.

(ii) Purification of CD4-qamma2 chimeric heavy chain homodimer from CHO conditioned media CD4-gamma2 chimeric heavy chain homodimer was purified by column chromatography. CHO cells secreting CD4-gamma2 chimeric heavy chain homodimer were grown to high density in roller bottles in medium containing alpha MEM with 5% IgG-free fetal calf serum. Conditioned media was collected, clarified by centrifugation, and diluted 1:1 with PBS either with or without detergent (i.e. Tween) in this and subsequent buffers. The diluted media was then applied to a 5ml column of Protein A-Sepharose fast flow previously equilibrated with PBS, at a flow rate of 60ml/hour. After extensive washing, the specifically bound material was eluted with lOOmM glycine/HCl, pH 3.5, directly into an aliquot of 1M Tris.HCl pH 8.0 to immediately neutralize the eluted fractions. The fractions were then analyzed by SDS- PAGE under reducing and non-reducing conditions followed by silver staining and pooled.

The pooled fractions were then applied to a 10 ml column of S-sepharose fast flow previously equilibrated with 50mM BES pH 7.0 at a flow rate of 120ml/hr. After application of the sample, a step elution gradient (consisting of the following 4 steps: 5 column volumes of 50mM BES pH 7.0, 4 column volumes of 50mM BES pH 7.0, lOOmM NaCl, 6 column volumes of 50mM BES pH 7.0 225mM NaCl, followed by 8 column volumes of 50mM BES pH 7.0, 500mM NaCl) was employed for specific solution of the CD4-gamma2 chimeric heavy chain homodimer. The CD4-gamma2 chimeric heavy chain homodimer was eluted from the column in 50mM BES pH 7.0, 500mM NaCl. The peak fractions were then pooled and concentrated to yield a final protein concentration of at least 1 mg/ml. The pooled and concentrated fractions were then applied to a 120 ml column of Sephacryl S-300HR previously equilibrated with PBS, at a flow rate of 8ml/hr. The CD4-gamma2 chimeric heavy chain homodimer fraction was specifically eluted in PBS, and concentrated to at least lmg/ml. (d) Co-expression of CD4-IqG2HC-pRcCMV and CD4-kLC-pRcCMV in mammalian cells to produce CD4-IqG2 chimeric heterotetramer

(i) Stable expression

Dhfr- Chinese hamster ovary cells (CHO) are transfected with 20 micrograms of CsCl-purified DNA in a ratio of 1000:1000:1 CD4-IgG2HC-pRcCMV:CD4-kLC-pRcCMV:p410 (p410 is an expression plasmid containing the dhfr gene) , although other ratios may also be used. At approximately 3-5 days post-transfection, cells are placed in selective medium

(nucleoside-free alpha MEM containing 5% dialyzed fetal calf serum) . At approximately 10-15 days post-selection, individual cell clones are picked. The clones are then analyzed for stable expression of. CD4-IgG2 chimeric heterotetramers by several screening techniques, such as ELISA and precipitation with Protein A-sepharose beads followed by SDS-PAGE under reducing or non-reducing conditions. Clones expressing the highest levels are subjected to successive rounds of amplification of the newly introduced DNA sequences in increasing concentrations of methotrexate. Stable CHO cell lines are thus generated which secrete high levels of CD4-IgG2 chimeric heterotetramer.

(ii) Purification of CD4-IqG2 chimeric heterotetramers from

CHO conditioned media CD4-IgG2 chimeric heterotetramers are purified using Protein A-Sepharose column chromatography. CHO cells secreting CD4IgG2 chimeric heterotetramers are grown to high density in roller bottles in medium containing alpha MEM with 5% IgG-free fetal calf serum. Conditioned media is collected, clarified by centrifugation, and diluted 1:1 with PBS either with or without detergent (i.e. Tween) in this and subsequent buffers. The diluted media is then applied to a 5ml column of Protein A-Sepharose fast flow previously equilibrated with PBS, at a flow rate of 60ml/hour. After extensive washing, the bound material is eluted with lOOmM glycine/HCl, pH 3.5, directly into a aliquot of 1M Tris.HCl pH 8.0 to immediately neutralize the eluted fractions. Fractions are then analyzed by SDS-PAG under reducing and non-reducing conditions followed b silver staining and pooled.

(e) Production of monoclonal anti-qpl20 and anti-αp4 antibodies

The anti-gpl20 and anti-gp41 monoclonal antibodies used i the subject invention are commercially available. It i also possible for one skilled in the art to make human, murine, or humanized murine anti-gpl20 or anti-gp4 antibodies by a variety of techniques.

For example it is possible to make human monoclonal anti gp41 antibodies as described infra. Briefly, periphera blood mononuclear cells (PBMCs) are isolated from the bloo of HIV-1-infected individuals who exhibit anti-gp4 antibodies in their serum. Epstein-Barr Virus (EBV, obtained, for example, from B95-8 cell supernatants) is added to the PBMC preparation which is then plated out i 96-well tissue culture plates at limiting dilution. Colonies of EBV-immortalized B lymphocytes grow out an those colonies producing anti-gp41 antibodies are identified by methods well known to those skilled in the art. For example, the media from these cells is used t immunoprecipitate gp41 from metabolically radiolabelle cells expressing gpl20/gp41. Also, colonies producing anti- gp41 antibodies may be identified by western blotting. Colonies producing monoclonal antibodies specific for the gp41 sequence ELDKWA may be identified by an assay such as the enzyme-linked immunosorbent assay. Briefly, the ELDKWA peptide is synthesized by methods well known to those skilled in the art, or obtained commercially. The ELDKWA peptide is used to coat the wells of a plastic 96 well microtiter plate, and the wells are incubated with dilutions of culture media from individual B lymphocyte colonies. Antibodies which bind to the peptide are identified using, for example, horseradish peroxidase-linked rabbit anti-human immunoglobulin antibodies, followed by peroxidase substrate.

Colonies making the antibodies of interest are expanded and fused with a suitable partner cell line, for example, a mouse/human heteromyeloma. Hybrids are selected by culture in selective medium in the presence of feeder cells, and stable antibody-secreting hybrids are cloned and expanded.

2. Methods of immobilizing agents on solid supports such as beads and filters

Numerous methods exist to immobilize protein-based agents to various types of solid supports including, but not limited to, plastic, latex and agarose. These methods include covalent and non-covalent methods. In the subject invention, it is important to use methods which fulfill the following requirements.

First, the methods should not significantly affect the properties of the agent, such as its binding affinity for the HIV-l envelope glycoprotein, or its ability to neutralize HIV-l. These parameters can be tested following immobilization by testing the ability of the immobilized agent to bind gpl20 and neutralize HIV-l in plasma. Second, the bound agent should be stable when stored or exposed to blood or blood products under the appropriate conditions for normal use (e.g. storage for several days at 4°C with PRBC) . The activity of the immobilized agents can be determined following storage/exposure to blood components using methods known to those skilled in the art. Loss (leakage) of the immobilized agent from the solid support over time can be determined by analyzing the mass of agent bound to unit mass of solid support using methods well known to those skilled in the art. Such methods include for example, enzyme-linked immunosorbent assays, where an antibody specific for the agent is used to assay for the quantity of the agent present.

Second Series of Experiments

1. Binding and neutralization of HIV-l by CD4-based molecules and antibodies Applicants have demonstrated that CD4-based proteins and antibodies to HIV-l bind gpl20 from different strains of the virus and neutralize many strains of HIV-l, including strains from different genetic clades. In these studies, the CD4-based proteins were more broadly reactive with gpl20 from different HIV-l variants than were the antibodies. The CD4-based molecules were also more broadly neutralizing than the antibodies. CD4-IgG2 was particularly potent and broad in its HIV-l neutralizing properties. Moreover, CD4-IgG2 was capable of neutralizing a primary HIV-l isolate obtained from an individual who presented in New York with symptoms of primary HIV-l infection, prior to seroconversion. This virus is representative of viruses that may enter the blood supply from antigen-positive, antibody-negative donors. The fact that CD4-IgG2 neutralizes this isolate is significant in the context of developing this agent to neutralize HIV-l in contaminated blood.

The above data support the concept that CD4-based molecules and antibodies to HIV-l could be used as soluble or immobilized reagents to reduce the infectivity of HIV-l contaminated blood either by binding to and removing HIV-l virions, or by neutralizing the virus. The CD4-based molecules and antibodies to HIV-l could also be used to bind and remove HIV-l and/or HIV-l gpl20 from the blood of HIV-l-infected individuals by plasmapheresis. Moreover, these agents are reactive against many different strains of HIV-l obtained from around the world.

2. CD4-based proteins effectively neutralize HIV-l in the plasma of HIV-l infected individuals. Applicants have demonstrated that CD4-IgG2 potentl neutralizes clinical isolates of HIV-l in plasma form HIV- infected individuals using an "ex-vivo" neutralizatio assay . The ex vivo neutralization assay procedure wa similar to that described by Daar et al. , 1990, Proc Nat Acad Sci USA 87:6574-6578. Briefly, dilutions of viremi plasma from HIV-l infected individuals were incubated wit PHA-activated normal PBMC. 25ug CD4-IgG2 (or antibody) wa added to each culture, with a final culture volume o approximately 1 ml. After 7 days, a p24 antigen assay wa performed on the culture supernatant to assess the degre of HIV-l replication. Several broadly neutralizing huma monoclonal antibodies were tested in parallel with CD4-IgG in these assays (all proteins at 25ug/ml) , including IgG12 a broadly neutralizing MAb directed to the CD4-bindin site, 19b-a cross-reactive V3 loop MAb; A-32-a broadl neutralizing MAb directed at a discontinuous epitope o gpl20 (described below) ; 21h-aCD4 binding site MAb. Th results obtained with two patients are illustrated i Figure 1. In this figure, a measure of the amount o infectious HIV-l in the plasma samples is given by th results in control cultures (medium alone or control IgG) , where virus replication could be easily detected i cultures exposed to a 125-fold or greater dilution o plasma. CD4-IgG2 potently neutralized HIV-l in thes samples, reducing virus replication to background level even in undiluted viremic plasma. The monoclona antibodies tested also neutralized HIV-l, but were les effective when compared to CD4-IgG2 in undiluted viremi plasma.

Similar data were obtained using viremic plasma sample from four other donors. This study demonstrates that CD4- IgG2 potently neutralizes HIV-l present in the plasma o HIV-infected individuals, supporting the concept that CD4- IgG2 could be used to remove or inactivate HIV-l in whole blood and blood components.

3. CD4-IqG2 covalently immobilized to aqarose beads binds crpl20 CD4-IgG2 has been immobilized onto agarose beads using the hydrazide coupling chemistry previously used to immobilize antibodies through their carbohydrate moieties with high recovery of antigen binding activity (Hermanson GT et al. 1992 "Immobilized Affinity Ligand Techniques", pp 226-230. Academic Press, San Diego, CA)

CD4-IgG2 was oxidized with 15mM sodium m-periodate and immobilized overnight at room temperature onto Agarose Adipic Acid Hydrazide (Pharmacia, Piscataway, NJ) in pH 5.1 acetate buffer. The immobilization efficiency was approximately 50%, and the resulting affinity support contained approximately lmg CD4-IgG2/ml gel.

This CD4-IgG2 affinity gel was used to remove recombinant HIV-lu gpl20 from the culture medium of gpl20-secreting Chinese hamster ovary cells. An enzyme-linked immunosorbent assay specific for gpl20 was used to analyze the treated and untreated cell culture supernatants. It was demonstrated that immobilized CD4-IgG2 removed >90% of the gpl20 at a capacity of approximately 0.6 mg gpl20/ml gel.

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Claims

What is claimed is:

1. An article of manufacture comprising a solid support having operably affixed thereto an agent capable of specifically forming a complex with a domain present on an HIV-l envelope glycoprotein.

2. The article of manufacture of claim 1, wherein the domain present on the HIV-l envelope glycoprotein is a conserved domain.

3. The article of manufacture of claim 2, wherein the conserved domain present on the HIV-l envelope glycoprotein is the CP4-binding domain of the HIV-l gpl20 envelope glycoprotein.

4. The article of manufacture of claim 1, wherein the solid support is selected from the group consisting of a bead, a receptacle, and a filter.

5. The article of manufacture of claim 1, wherein the agent is an antibody.

6. The article of manufacture of claim 1, wherein the agent is a CD4-based molecule.

7. The article of manufacture of claim 6, wherein the CP4-based molecule is a CD -immunoconjugate.

8. The article of manufacture of claim 7, wherein the CD4-immunoco jugate is a heterotetramer comprising two heavy chains and two light chains, both heavy chains being either (a) IgG2 heavy chains or (b) chimeric CD4-IgG2 heavy chains, and both light chains being either (a) kappa light chains or (b) chimeric CD4-kappa light chains, with the proviso that either both heavy chains or both light chains or all four chains are CD4 chimeras.

9. The article of manufacture of claim 8, wherein the chimeric CD4-IgG2 heavy chains are encoded by the expression vector designated CD4-IgG2HC-pRcCMV (ATCC No. 75193) , and the chimeric CD4-kappa light chains are encoded by the expression vector designated CD4-kLC-pRcCMV (ATCC No. 75194) .

10. An article of manufacture comprising a solid support having operably affixed thereto a plurality of agents each capable of specifically forming a complex with a domain present on an HIV-l envelope glycoprotein.

11. An aqueous-soluble agent which (a) is capable of specifically forming a complex with a domain present on an HIV-l envelope glycoprotein, and (b) comprises a moiety capable of specifically forming a complex with a known ligand, which moiety permits the removal of the agent from a sample via contact with an immobilized form of the known ligand.

12. A method of treating a bodily fluid sample so as to remove therefrom HIV-l or HIV-l gpl20 envelope glycoprotein if present in the sample which comprises contacting the sample under suitable conditions with the article of manufacture of claim 1, t h e r e b y removing therefrom HIV-l or HIV-l gpl20 envelope glycoprotein if present in the sample.

13. A method of treating a bodily fluid sample so as to substantially reduce the likelihood of a subject's becoming infected with HIV-l as a result of contact with the sample which comprises contacting the sample with a suitable amount of an aqueous-soluble agent capable of specifically forming a complex with a domain present on an HIV-l envelope glycoprotein, so as to form a complex between the agent and HIV-l if present in the sample and thereby reduce the likelihood of a subject's becoming infected with HIV-l as a result of contact with the sample.

14. A method of substantially reducing the amount of HIV-l gpl20 envelope glycoprotein in a bodily fluid sample which comprises contacting the sample with a suitable amount of an aqueous-soluble agent capable of specifically forming a complex with a domain present on an HIV-l envelope glycoprotein, so as to form a complex between the agent and HIV-l if present in the sample and thereby reduce the amount of HIV-l gpl20 envelope glycoprotein in the sample.

15. The method of claim 13, or 14, wherein the agent is an antibody.

16. The method of claim 13, or 14, wherein the agent is a CD4-based molecule.

17. The method of claim 16, wherein the CD4-based molecule is a CD4-immunoconjugate.

18. The method of claim 17, wherein the CD4-immunoconjugate is a heterotetramer comprising two heavy chains and two light chains, both heavy chains being either (a) IgG2 heavy chains or (b) chimeric CD4-IgG2 heavy chains, and both light chains being either (a) kappa light chains or (b) chimeric CD4-kappa light chains, with the proviso that either both heavy chains or both light chains or all four chains are CD4 chimeras.

19. The method of claim 18, wherein the chimeric CD4-IgG2 heavy chains are encoded by the expression vector designated CD4-IgG2HC-pRcCMV (ATCC No. 75193) , and the chimeric CD4-kappa light chains are encoded by the expression vector designated CD4-kLC-pRcCMV (ATCC No. 75194) .

20. The method of claim 12, 13, or 14, wherein the bodily fluid is blood.

21. A method of treating a bodily fluid sample so as to substantially reduce the likelihood of a subject's becoming infected with HIV-l as a result of contact with the sample which comprises the steps of

(a) contacting the sample with a suitable amount of an aqueous-soluble agent capable of specifically forming a complex with a domain present on an HIV-l envelope glycoprotein, thereby forming a complex between the agent and HIV-l if present in the sample; and

(b) removing any complex so formed from the resulting sample, so as to thereby reduce the likelihood of a subject's becoming infected with HIV-l as a result of contact with the sample.

22. A method of treating a bodily fluid sample so as to substantially reduce the likelihood of a subject's becoming infected with HIV-l as a result of contact with the sample which comprises the steps of (a) contacting the sample with a suitable amount of the aqueous-soluble agent of claim 11, thereby forming a complex between the agent and HIV-l if present in the sample; and

(b) removing any complex so formed from the resulting sample by contacting the resulting sample with an immobilized form of the known ligand, so as to thereby reduce the likelihood of a subject's becoming infected with HIV-l as a result of contact with the sample.

23. A method of treating a bodily fluid sample so as to substantially reduce the likelihood of a subject's becoming infected with HIV-l as a result of contact with the sample which comprises the steps of

(a) contacting the sample under suitable conditions with the article of manufacture of claim 1; and

(b) contacting the sample with a suitable amount of an aqueous-soluble agent capable of specifically forming a complex with a domain present on an HIV-l envelope glycoprotein, so as to form a complex between the agent and HIV-l if present in the sample, with the proviso that step (a) may either precede or follow step (b) .

24. A method of treating a bodily fluid sample so as to substantially reduce the likelihood of a subject's becoming infected with HIV-l as a result of contact with the sample which comprises the steps of

(a) contacting the sample under suitable conditions with the article of manufacture of claim 1 ; and

(b) (i) contacting the sample with a suitable amount of an aqueous-soluble agent capable of specifically forming a complex with a domain present on an HIV-l envelope glycoprotein, thereby forming a complex between the agent and HIV-l if present in the sample, and

(ii) removing any complex so formed from the resulting sample, with the proviso that step (a) may either precede or follow step (b) .

25. A method of treating a bodily fluid sample so as to substantially reduce the likelihood of a subject's becoming infected with HIV-l as a result of contact with the sample which comprises the steps of

(b) (i) contacting the sample with a suitable amount of an aqueous-soluble agent which (1) is capable of specifically forming a complex with a domain present on an HIV-l envelope glycoprotein, and (2) comprises a moiety capable of specifically forming a complex with a known ligand, thereby forming a complex between the agent and HIV 1 if present in the sample, and

(ii) removing any complex so formed from the resulting sample by contacting the resulting sample with an immobilized form of the known ligand, with the proviso that step (a) may either precede or follow step (b) .

26. The method of any one of claims 12, 13, 14, and 21-25, further comprising the step of removing CD4+ cells from the bodily fluid sample.

27. The method of any one of claims 12, 13, 14, and 21-25, wherein the bodily fluid sample is selected from the group consisting of whole blood, a red blood cell preparation, a platelet preparation, and semen.

28. A kit for treating a bodily fluid sample so as to substantially reduce the likelihood of a subject's becoming infected with HIV-l as a result of contact with the sample which comprises the article of manufacture of claim 1.

29. A kit for treating a bodily fluid sample so as to substantially reduce the likelihood of a subject's becoming infected with HIV-l as a result of contact with the sample which comprises, in separate compartments:

(a) the article of manufacture of claim 1; and

(b) an aqueous-soluble agent capable of specifically forming a complex with a domain present on an HIV-l envelope glycoprotein.

30. A kit for treating a bodily fluid sample so as to substantially reduce the likelihood of a subject's becoming infected with HIV-l as a result of contact with the sample which comprises, in separate compartments :

(a) the article of manufacture of claim 1;

(b) an aqueous-soluble agent which (1) is capable of specifically forming a complex with a domain present on an HIV-l envelope glycoprotein, and (2) comprises a moiety capable of specifically forming a complex with a known ligand, which moiety permits the removal of the agent from a sample via contact with an immobilized form of the known ligand; and

(c) an article of manufacture comprising a solid support having operably affixed thereto the known ligand capable of specifically forming a complex with the moiety of the aqueous- soluble agent of step (b) .

31. A kit for treating a bodily fluid sample so as to substantially reduce the likelihood of a subject's becoming infected with HIV-l as a result of contact with the sample which comprises, in separate compartments :

(a) the aqueous-soluble agent of claim 11; and

(b) an article of manufacture comprising a solid support having operably affixed thereto the known ligand capable of specifically forming a complex with the moiety of the aqueous-soluble agent of claim 11.

32. A kit for reducing the amount of HIV-l or HIV-l gpl20 envelope glycoprotein present in a bodily fluid sample which comprises the article of manufacture of claim 1.

33. A kit of claim 32, wherein the bodily fluid is blood.