AU706927B2 - Articles of manufacture for removing HIV-1 from a sample, and methods of using same - Google Patents

Description

WO 96/02575 PCTfUS95/08805 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 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 100 units of blood contaminated with HIV-I enter the U.S.blood supply each year. As primary HIV-l isolates are WO 96/02575 PCT1US95/08805 2 fairly robust, the infectious potential of contaminated blood survives storage of blood components Thus, administration of contaminated components to transfusion recipients often, perhaps invariably, leads to HIV-1 transmission. The subsequent infection is usually fatal, and can be transmitted to others. Consequently, public confidence in the safety of donated blood has been reduced.

A practical method for the efficient and safe elimination of HIV-1 from donated blood would therefore be an important contribution to public health.

Contamination of donated blood can arise as a result of several scenarios. Perhaps the most common source of contamination results from the donation of blood by individuals who have been infected with HIV-1, but who have not yet developed antibodies detectable by standard screening assays. These pre-seroconversion cases can be highly viremic. During the acute phase of HIV-1 infection, virus titres in excess of 104 TCID 50 /ml can be found in plasma in the absence of detectable HIV-1 antibodies (6,7) Studies have shown that it is possible to detect antibodies by ELISA 4 days before they register in a conventional antibody-detection ELISA, and after plasma virus titres decline by more than 100-fold from their peak values. The infectious potential of such individuals is obvious. As the HIV epidemic spreads into the low-risk population, the efficacy of pre-donation screening may diminish, allowing more such contaminated units into the blood supply. Another potential source of contamination may arise from random error during screening procedures, resulting in the use of contaminated samples from antibodypositive, asymptomatic individuals. It is also possible that some antibody-positive samples may not be detectable by current screening tests, either because the antibody concentration in the plasma is extremely low, or because an WO96/02575 PCT/US95/08805 3 immune response is not raised against those epitopes best represented in ELISA-based screening assays. Such cases have been documented and as HIV-I diversifies in sequence and more exotic strains are introduced into the 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 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-1 found during the acute, antibodynegative phase of infection and with the lower titres of HIV-1 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-1 but which are not suitable for treating whole blood or blood components containing cells or platelets.

CD4-based molecules include recombinant soluble CD4 (sCD4; V1-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 WO 96/02575 PCT1US95/08805 4 three important antiviral properties (12-15): 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 inhibition of the intercellular spread of virus from infected cells to uninfected cells by inhibiting HIV-1 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-1 with the greatest overall potency when compared to monoclonal antibodies or HIV immune globulin This is not surprising since CD4 is the major receptor for HIV-1 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-1 exhibit greater sensitivity to neutralization by CD4-based molecules as compared to primary isolates in vitro, all clinical isolates of HIV-1 tested are inhibited by CD4-based molecules at concentrations achievable in humans.

Little or no toxicity or immunogenicity has been observed in clinical trials with CD4-based molecules In addition, administration of CD4-Ig to chimpanzees prior to challenge with HIV-1,I 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-1), most neutralizing antibodies in sera from naturally infected humans are directed against the outer envelope glycoprotein, WO 96/02575 PCT/US95/08805 (20-30). The V3 loop of HIV-1 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-l-infected chimpanzee model 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-1-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 crossreactive as CD4-based molecules, and none is as potent at neutralizing laboratory-adapted or primary HIV-1 isolates in vitro Several studies have demonstrated that some combinations of CD4-based molecules and Mabs directed against gpl20 or gp41 are synergistic in neutralizing HIV-1 (44-48).

As noted above, primary HIV-1 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 dissociation or shedding (15, 49).

Numerous studies have demonstrated that the envelope glycoprotein of HIV-1, gpl20, may have a direct role in the pathogenesis of HIV-1 infection. Some potential mechanisms for gpl20 in immunopathogenesis of HIV-1 infection are described by Fauci, A. and Rosenberg, Z. (In: Textbook of WO 96/02575 PCTIUS95/08805 6 AIDS Medicine, Broder et al. eds., 1994, Williams and Wilkins, Baltimore, MD, pp 55-75). These mechanisms include binding of gpl20 to CD4 on CD4' cells resulting in immunosuppression or apoptosis. The removal of gpl20 from the blood or other bodily fluids of HIV-l-infected individuals would reduce these pathogenic effects and improve clinical outcome. Removal might be accomplished, for example, by attaching CD4-based proteins or antibodies to a filter and circulating blood through this filter (plasmapheresis). This procedure could also be used to remove HIV-1 virions from the blood which would also be beneficial.

WO 96/02575 PCT/US95/08805 7 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-I 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 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-I 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-i 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-i as a result of contact with the sample which comprises contacting the sample with a suitable amount of an WO 96/02575 PCTIUS95/08805 8 aqueous-soluble agent capable of specifically forming a complex with a domain present on an HIV-1 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-1 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-I as a result of contact with the sample which comprises the steps of 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-1 envelope glycoprotein, thereby forming a complex between the agent and HIV-l if present in the sample; and removing any complex so formed from the resulting sample, so as to thereby reduce the likelihood of a subject's becoming infected with HIV-I 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-I or HIV 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-I or HIV-I 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 WO 96/02575 PCT/US95/08805 9 steps of contacting the sample with a suitable amount of an aqueous-soluble agent which 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 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-I 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-1 as a result of contact with the sample which comprises the steps of 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-I 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-I envelope glycoprotein, so as to form a complex between the agent and HIV-I if present in the sample, with the proviso that step may either precede or follow step 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-1 as a result of contact with the sample which comprises the WO 96/02575 PCT/US95/08805 steps of 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-i envelope glycoprotein; and (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-I envelope glycoprotein, 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, with the proviso that step may either precede or follow step 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-i as a result of contact with the sample which comprises the steps of 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-I envelope glycoprotein; and (I) contacting the sample with a suitable amount of an aqueous-soluble agent which is capable of specifically forming a complex with a domain present on an HIV-I envelope glycoprotein, and comprises a moiety capable of specifically forming a complex with a known ligand, thereby forming a complex between the agent and HIV-I 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 may either precede or follow step WO 96/02575 PCT/US9508805 11 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-I as a result of contact with the sample which comprises, in separate compartments: 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-1 envelope glycoprotein; and an aqueous-soluble agent capable of specifically forming a complex with a domain present on an HIV-1 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-1 as a result of contact with the sample which comprises, in separate compartments: 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-I envelope glycoprotein; an aqueoussoluble agent which is capable of specifically forming a complex with a domain present on an HIV-l 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; and 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 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-1 as a result of contact with the sample which comprises, in WO 96/02575 PCT/US95/08805 12 separate compartments: an aqueous-soluble agent which is capable of specifically forming a complex with a domain present on an HIV-1 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 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-1 or HIV-1 gpl20 envelope glycoprotein present in a bodily fluid sample which comprises the above-described article of manufacture.

WO 96/02575 PCTIUS95/08805 13 Brief Description of the Fiqrure Figure 1 CD4-IgG2 effectively neutralizes HIV-i in the plasma of HIV-1 infected individuals.

p:\OPER\MRO\31954-95.SPE- 26/2/97 -14- Detailed Description of the Invention The plasmids pT4B, CD4IgG2-pcDNA1, CD4-kLC-pRcCMV, CD4-IgG 2 HC-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. ATCC Accession No. 40952 was deposited on 31 January, 1991. ATCC Accession Nos. 75193 and 75194 were deposited on 30 January, 1992.

ATCC Accession Nos: 40952, 75193 and 75194 were deposited for the purpose of Australian I Patent No. 660662, published on 7 September, 1992. ATCC Accession No. 68389 was deposited for the purpose of United States Patent No. 5,126,433, issued on 30 June, 1992.

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-1 envelope glycoprotein.

The solid support may be any solid support known in the art to which the agent can be 20 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.

i In the preferred embodiment, the receptacle is a bag specifically intended for the WO 96/02575 PCT/US95/08805 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 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-1 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-1 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-1 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.

\1 WO 96/02575 PCT/US95/08805 16 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-1 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-IgG1 heterotetramer.

In the preferred embodiment, the CD4-immunoconjugate is a heterotetramer comprising two heavy chains and two light chains, both heavy chains being either IgG2 heavy chains or chimeric CD4-IgG2 heavy chains, and both light chains being either 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

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WO 96/02575 PCT/US95/08805 17 manner permitting the formation of a complex between the affixed agent and the domain present on an HIV-1 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-1 envelope glycoprotein" means capable of forming a complex with a domain present on an HIV-1 envelope glycoprotein but not capable of forming a complex with any other domain.

As used herein, "HIV-1" is synonymous with the terms "HIV-1 particle," "HIV-1 virion" or "HIV-1 virus." In one embodiment, the HIV-1 envelope glycoprotein is the HIV-1 gpl20 envelope glycoprotein. In another embodiment, the HIV-1 envelope glycoprotein is the HIV-1 gp41 envelope glycoprotein.

In one embodiment, the domain present on the HIV-1 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-1. In the preferred embodiment, the conserved domain present on the HIV-1 envelope glycoprotein is the CD4-binding domain of the HIV-1 gpl20 envelope glycoprotein.

In another embodiment, the domain present on the HIV-1 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-1 gpl20 envelope glycoprotein.

This invention further provides an article of manufacture comprising a solid support having operably affixed thereto

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WO 96/02575 PCT/US95/08805 18 a plurality of agents each capable of specifically forming a complex with a domain present on an HIV-1 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.

This invention further provides an aqueous-soluble agent which is capable of specifically forming a complex with a domain present on an HIV-1 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.

As used herein, "aqueous-soluble" means capable of existing in soluble form in water at 4 0 C at a concentration of at least IpM.

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, oligohistidine/nickel ion, glutathione S-transferase/glutathione, and (c) biotin/streptavidin.

This invention further provides a method of treating a WO 96/02575 PCTIUS95/0805 19 bodily fluid sample so as to remove therefrom HIV-1 or HIV 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-1 envelope glycoprotein, thereby removing therefrom HIV-1 or HIV-1 gpl20 envelope glycoprotein if present in the sample.

As used herein, "treating a bodily fluid sample so as to remove therefrom HIV-1" means either rendering the HIV-1 in the bodily fluid sample unable to invade CD4' cells, physically separating HIV-1 from the bodily fluid sample, or a combination of and with the proviso that the HIV-1 present in the resulting sample and capable of invading CD4 cells does not exceed 50% of the amount of such HIV-1 present in the sample prior to removing HIV-1. 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-1 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-1. Such conditions are known to those skilled in the art.

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-1 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 WO 96/02575 PCT/US95/08805 complex with a domain present on an HIV-1 envelope glycoprotein, so as to form a complex between the agent and HIV-1 if present in the sample and thereby reduce the likelihood of a subject's becoming infected with HIV-1 as a result of contact with the sample.

This invention provides a method of substantially reducing the amount of HIV-1 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-1 envelope glycoprotein, so as to form a complex between the agent and HIV-1 if present in the sample and thereby reduce the amount of HIV-1 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-1 virions and/or HIV-1 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-1 means reducing the likelihood of the subject's becoming infected with HIV-1 by at least two-fold. For example, if a subject has a 1% chance of becoming infected with HIV-1, a two-fold reduction in the likelihood of the subject's becoming infected with HIV-1 would result in the subject's having a chance of becoming infected with HIV-1. In one embodiment, substantially reducing the likelihood of the WO96/02575 PCT/US95/08805 21 subject's becoming infected with HIV-1 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-1 means reducing the likelihood by at least 100-fold.

As used herein, "the subject's becoming infected with HIV-1" means the invasion of the subject's own cells by HIV-1.

As used herein, contact with a bodily fluid sample is any contact sufficient to cause HIV-1 in the sample to be transmitted to the subject's body, and thereby infect the subject with HIV-1.

The amount of aqueous-soluble agent suitable to substantially reduce the likelihood of a subject's becoming infected with HIV-1 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 IpM and about 10mM. In the preferred embodiment, the suitable amount of aqueous-soluble agent is an amount between about IpM and about 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 IgG2 heavy chains or chimeric CD4-IgG2 heavy chains, and both light chains being either 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 WO 96/02575 PCT/US95/08805 22 chains are CD4 chimeras.

The chimeric CD4-IgG2 heavy chains may be the heavy chains encoded by the expression vector designated CD4-IgG2HCpRcCMV (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).

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

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 when the agent would cause undesirable effects in a subject to whom it is administered).

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-1 as WO 96/02575 PCT/US95/08805 23 a result of contact with the sample which comprises the steps of contacting the sample with a suitable amount of an aqueous-soluble agent which is capable of specifically forming a complex with a domain present on an HIV-1 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 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-I as a result of contact with the sample.

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-I as a result of contact with the sample which comprises the steps of 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-1 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-1 envelope glycoprotein, so as to form a complex between the agent and WO 96/02575 PCTIUS95/08805 24 HIV-I if present in the sample, with the proviso that step may either precede or follow step 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-1 as a result of contact with the sample which comprises the steps of 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 (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 may either precede or follow step 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-I as a result of contact with the sample which comprises the steps of 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-I envelope glycoprotein; and (I) contacting the sample with a suitable amount of an aqueous-soluble agent which is capable of specifically forming a complex with a domain present on an HIV-l envelope glycoprotein, and comprises a moiety capable of specifically forming a complex with a known ligand, WO 96/02575 PCT/US95/08805 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 may either precede or follow step 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-I in an HIV-l-infected subject. Bodily fluids include, but are not limited to, whole blood or derivatives thereof 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 citrate) and preservatives dextrose) In one embodiment, the contacting steps of the methods of the subject invention are performed at about 4 0 C. In WO 96/02575 PCTITS95/08805 26 another embodiment, the contacting steps of the methods of the subject invention are performed at about 20 0 C. In still another embodiment, the contacting steps of the methods of the subject invention are performed at about 37 0

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-I as a result of contact with the sample which comprises, in separate compartments: 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-1 envelope glycoprotein; and an aqueous-soluble agent capable of specifically forming a complex with a domain present on an HIV-I 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-I as a result of contact with the sample which comprises, in separate compartments: 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-1 envelope glycoprotein; an aqueoussoluble agent which is capable of specifically forming a complex with a domain present on an HIV-i envelope glycoprotein, and comprises a moiety capable of specifically forming a complex with a known ligand, which WO 96/02575 PCTUS95/08805 27 moiety permits the removal of the agent from a sample via contact with an immobilized form of the known ligand; and 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) 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-I as a result of contact with the sample which comprises, in separate compartments: an aqueous-soluble agent which is capable of specifically forming a complex with a domain present on an HIV-1 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 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-1 or HIV-1 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) WO 96/02575 PCT/US95/08805 28 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.

WO 96/02575 PCTIUS95/08805 29 Experimental Details A. Introduction Primary viruses appear to become more sensitive to sCD4 if incubated therewith for prolonged periods at 4 0 C rather than at 37 0 C. Preliminary results indicate that neutralization by anti-V3-loop HuMAbs is also more sensitive at 4°C than at 370C, 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 In the subject invention, particular advantages are found in using a tetrameric CD4-immunoglobulin protein (CD4-IgG2) formed by the fusion of the Vl 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-1, including primary isolates of the virus. Of additional importance in the subject invention is the observation that CD4-IgG2 effectively neutralizes HIV-1 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 General sCD4, CD4-gammal chimeric heavy chain homodimers, CD4-gamma2 chimeric heavy chain homodimers and CD4-IgG2 chimeric heterotetramers may be obtained from Progenics WO 96/02575 PCT/US95/08805 Pharmaceuticals, Inc. (Tarrytown, New York). Mouse antibody [9205] to the V3 loop of gpl20, derived from the HIV-ITLvIIB isolate, may be obtained from DuPont NEN Research Products (Wilmington, Delaware). Human antibody 2F5 to a conserved epitope of HIV-1 gp41 may be obtained from Viral Testing Systems Corporation (Houston, Texas) and from Waldheim Pharmazeutika (Vienna, Austria).

sCD4 Soluble CD4 (a genetically-engineered, water-soluble extracellular fragment of human CD4) is disclosed, for example, in Patent Cooperation Treaty International Publication No. WO 88/01304. Soluble CD4 is also commercially available.

Soluble CD4, also designated sCD4, may be produced by truncating pT4B (ATCC No. 68389) after the V4J4 domain.

Such DNA fragments terminate before the transmembrane segment, which begins at approximately nucleotide position 1264.

Purification and characterization of soluble CD4 fragments is greatly enhanced by constructing a cell line (preferably mammalian) which overexpresses the secreted protein fragment. Strategies which allow the overexpression of proteins may be employed in bacteria, yeast, insect and mammalian systems. Inducible expression systems may also be employed in bacteria and yeast to overproduce proteins which may be toxic if constitutively expressed.

Overexpression of soluble CD4 fragments may be accomplished by amplifying a soluble CD4 expression vector, resulting in constitutive overexpression. The amplification of dihydrofolate reductase (dhfr) genes by growth in progressively increased concentrations of the drug methotrexate, an antagonist of dhfr, is widely employed.

WO 96/02575 PCTIUS95/08805 31 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 BclI sites of the mammalian expression vector DSP modified to contain the SV-40 early promoter and the bovine growth hormone polyadenylation sequence. Through WO 96/02575 PCT/US95/08805 32 the use of synthetic linkers, the HaeII (bp 124) HpaII (bp 1252) fragment of pT4B is inserted between the KpnI and XbaI sites of the plasmid pUC18. A soluble CD4 expression vector 5 is created by ligating: 1. a 0.95 kb BglII SacI fragment of modified DSP which contains the 1.8 kb EcoRI-BamHI fragment of pT4B (this segment contains the SV40 early promoter, the CD4 leader sequence, and the amino terminal portion of the extracellular CD4 sequence); 2. the 0.66 kb SacI XbaI fragment of the pUC18 plasmid containing the HaeII-HpaII fragment of pT4B (this segment contains the carboxy terminal portion of the extracellular CD4 sequence followed by a TAA termination codon inserted after valine 371); and 3. the 2.48 kb BglII XbaI fragment of modified DSP which contains the bovine growth hormone polyadenylation sequence.

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

DXB-11, a clone of Chinese hamster ovary cells deficient in dhfr, is transfected with the soluble CD4 expression plasmid. The DXB-11 transformants are then grown in F12 medium, without hypoxanthine or thymidine, containing dialyzed fetal bovine serum. Clones are selected and WO 96/02575 D"Tr'/UfS T noo 33 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.

CD4-IqG2 Chimeras 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 Asepharose 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 WO 96/02575 PCT/US95/08805 34 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 IgG-free fetal calf serum. Conditioned media was collected, clarified by centrifugation, and diluted 1:1 with PBS either with or without detergent 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 100mM 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 4 column volumes of 50mM BES pH 7.0, 100mM NaCl, 6 column volumes of 50mM BES pH 7.0 225mM NaC1, 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 NaC1.

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 Img/ml.

WO 96/02575 PCTIUS95/08805 Co-expression of CD4-IqG2HC-pRcCMV and CD4-kLC-pRcCMV in mammalian cells to produce CD4-IqG2 chimeric heterotetramer 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 (p 4 10 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 Tween) in WO96/02575 PCTIUS95/08805 36 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 After extensive washing, the bound material is eluted with 100mM glycine/HCl, pH 3.5, directly into an aliquot of 1M Tris.HCl pH 8.0 to immediately neutralize the eluted fractions. Fractions are then analyzed by SDS-PAGE under reducing and non-reducing conditions followed by silver staining and pooled.

Production of monoclonal anti-qpl20 and anti-qp41 antibodies The anti-gpl20 and anti-gp41 monoclonal antibodies used in the subject invention are commercially available. It is also possible for one skilled in the art to make human, murine, or humanized murine anti-gpl20 or anti-gp41 antibodies by a variety of techniques.

For example it is possible to make human monoclonal antigp41 antibodies as described infra. Briefly, peripheral blood mononuclear cells (PBMCs) are isolated from the blood of HIV-1-infected individuals who exhibit anti-gp41 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 in 96-well tissue culture plates at limiting dilution.

Colonies of EBV-immortalized B lymphocytes grow out and 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 to immunoprecipitate gp41 from metabolically radiolabelled cells expressing gpl20/gp41. Also, colonies producing antigp41 antibodies may be identified by western blotting.

Colonies producing monoclonal antibodies specific for the WO96/02575 PCTIUS95/08805 37 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-1 envelope glycoprotein, or its ability to neutralize HIV-1. These parameters can be tested following immobilization by testing the ability of the immobilized agent to bind gpl20 and neutralize HIV-1 in plasma.

WO 96/02575 PCTUS95/08805 38 Second, the bound agent should be stable when stored or exposed to blood or blood products under the appropriate conditions for normal use storage for several days at 4 0 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.

WO 96/02575 PCT/US95/08805 39 Second Series of Experiments 1. Binding and neutralization of HIV-1 by CD4-based molecules and antibodies Applicants have demonstrated that CD4-based proteins and antibodies to HIV-1 bind gpl20 from different strains of the virus and neutralize many strains of HIV-1, including strains from different genetic clades. In these studies, the CD4-based proteins were more broadly reactive with gpl20 from different HIV-1 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-1 neutralizing properties. Moreover, CD4-IgG2 was capable of neutralizing a primary HIV-1 isolate obtained from an individual who presented in New York with symptoms of primary HIV-1 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-1 in contaminated blood.

The above data support the concept that CD4-based molecules and antibodies to HIV-1 could be used as soluble or immobilized reagents to reduce the infectivity of HIV-1 contaminated blood either by binding to and removing HIV-1 virions, or by neutralizing the virus. The CD4-based molecules and antibodies to HIV-1 could also be used to bind and remove HIV-1 and/or HIV-1 gpl20 from the blood of HIV-1-infected individuals by plasmapheresis. Moreover, these agents are reactive against many different strains of HIV-1 obtained from around the world.

2. CD4-based proteins effectively neutralize HIV-1 in the WO 96/02575 PCT/US95/08805 plasma of HIV-1 infected individuals.

Applicants have demonstrated that CD4-IgG2 potently neutralizes clinical isolates of HIV-1 in plasma form HIV-1 infected individuals using an "ex-vivo" neutralization assay The ex vivo neutralization assay procedure was similar to that described by Daar et al., 1990, Proc Natl Acad Sci USA 87:6574-6578. Briefly, dilutions of viremic plasma from HIV-1 infected individuals were incubated with PHA-activated normal PBMC. 25ug CD4-IgG2 (or antibody) was added to each culture, with a final culture volume of approximately 1 ml. After 7 days, a p24 antigen assay was performed on the culture supernatant to assess the degree of HIV-1 replication. Several broadly neutralizing human monoclonal antibodies were tested in parallel with CD4-IgG2 in these assays (all proteins at 25ug/ml), including IgG12a broadly neutralizing MAb directed to the CD4-binding site, 19b-a cross-reactive V3 loop MAb; A-32-a broadly neutralizing MAb directed at a discontinuous epitope of (described below); 21h-aCD4 binding site MAb. The results obtained with two patients are illustrated in Figure 1. In this figure, a measure of the amount of infectious HIV-1 in the plasma samples is given by the results in control cultures (medium alone or control IgG), where virus replication could be easily detected in cultures exposed to a 125-fold or greater dilution of plasma. CD4-IgG2 potently neutralized HIV-1 in these samples, reducing virus replication to background levels even in undiluted viremic plasma. The monoclonal antibodies tested also neutralized HIV-1, but were less effective when compared to CD4-IgG2 in undiluted viremic plasma.

Similar data were obtained using viremic plasma samples from four other donors. This study demonstrates that CD4- IgG2 potently neutralizes HIV-1 present in the plasma of WO 96/02575 PCT/US95/08805 41 HIV-infected individuals, supporting the concept that CD4- IgG2 could be used to remove or inactivate HIV-1 in whole blood and blood components.

3. CD4-IqG2 covalently immobilized to aqarose beads binds p120 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 15 immobilized overnight at room temperature onto Agarose i 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 1mg CD4-IgG2/ml gel.

This CD4-IgG2 affinity gel was used to remove recombinant HIV-lAj gpl20 from the culture medium of Chinese hamster ovary cells. An enzyme-linked immunosorbent assay specific for gpl20 was used to analyze 25 the treated and untreated cell culture supernatants. It was demonstrated that immobilized CD4-IgG2.removed >90% of the at a capacity of approximately 0.6 mg gpl20/ml gel.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

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Claims (22)

1. A method of treating a bodily fluid sample so as to remove therefrom HIV-1 or HIV-1 envelope glycoprotein comprising contacting the sample under suitable conditions with an article of manufacture comprising a solid support having operably linked thereto an agent capable of specifically forming a complex with a CD4-binding domain of the HIV-1 gpl20 envelope glycoprotein.

2. The method of claim 1, wherein the solid support is selected from the group consisting of a bead, a receptacle, and a filter. i

3. The method of claims 1 or 2, wherein the agent is a CD4-based molecule.

4. The method of claims 1 or 2, wherein the CD4-based molecule is CD4- immunoconjugate. *e

5. The method of claim 4, 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 chimeric CD4-IgG2 heavy chains, and both light chains being either kappa light chains or chimeric CD4-kappa light chains, with the proviso that either both heavy chains or both light chains or all four chains are CD4 chimeras.

6. The method of claim 5, 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).

7. A method of treating bodily fluid sample so as to substantially reduce the likelihood of a subject's becoming infected with HIV-1 as a result of contact with said sample P:\OPER\MRO\31954-95.CLM 23/4/99 -47- comprising contacting said sample with a suitable amount of an aqueous-soluble agent capable of specifically forming a complex with a domain present on an HIV-1 envelope glycoprotein, so as to form a complex between the agent and HIV-1 if present in the sample.

8. A method of substantially reducing the amount of HIV-1 gpl20 envelope glycoprotein in a bodily fluid sample which comprises contacting said sample with a suitable amount of an aqueous-soluble agent capable of specifically forming a complex with a domain present on an HIV-1 envelope glycoprotein, so as to form a complex between the agent and HIV-1 if present in the sample and thereby reduce the amount of HIV-1 gpl20 envelope glycoprotein in the sample.

9. The method of claims 7 or 8, wherein the agent is an antibody.

10. The method of claims 7 or 8, wherein the agent is a CD4-based molecule. 00 S

11. The method of claim 10, wherein the CD4-based molecule is a CD4- immunoconjugate.

S S S12. The method of claim 11, wherein the CD 4 -immunoconjugate is a heterotetramer comprising two heavy chains and two light chains, both heavy chains being either (a) IgG2 heavy chains or chimeric CD4-IgG2 heavy chains, and both light chains being either kappa light chains or chimeric CD4-kappa light chains, with the proviso that either both heavy chains or both light chains or all four chains are CD4 chimeras.

13. The method of claim 12, 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). P:\OPER\MRO\31954-95.CLM 23/4/99 -48-

14. A method of treating a bodily fluid sample so as to substantially reduce the likelihood of a subject's becoming infected with HIV-1 as a result of contact with said sample, comprising the steps of: contacting said sample with a suitable amount of an aqueous-soluble agent capable of specifically forming a complex with a domain present on an HIV-1 envelope glycoprotein, thereby forming a complex between the agent and HIV-1 if present in the sample; and removing any complex so formed from the resulting sample, so as to thereby S* reduce the likelihood of a subject's becoming infected with HIV-1 as a result of contact with the sample. A method of treating a bodily fluid sample so as to substantially reduce the likelihood of a subject's becoming infected with HIV-1 as a result of contact with said sample which comprises the steps of: contacting said sample with a suitable amount of an aqueous-soluble agent which is capable of forming a complex with a domain present on an HIV-1 envelope glycoprotein; and (ii) comprises a moiety capable of specifically forming a complex with a known ligand, thereby forming a complex between said agent and HIV-1 if present in the sample wherein said moiety permits removal of said agent from said sample via contact with an immediate form of said known ligand; and 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-1 as a result of contact with the sample.

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16. A method of treating a bodily fluid sample so as to substantially reduce the likelihood of a subject's becoming infected with HIV-1 as a result of contact with the sample which comprises the steps of: contacting the sample under suitable conditions with an article of manufacture comprising a solid support having operably linked thereto an agent capable of specifically forming a complex with a CD4-binding domain of the HIV-1 envelope glycoprotein; and *000 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-1 S:Ii envelope glycoprotein, so as to form a complex between the agent and HIV-1 if present in the sample, with the proviso that step may either precede or follow step

17. A method of treating a bodily fluid sample so as to substantially reduce the likelihood t of a subject's becoming infected with HIV-1 as a result of contact with the sample which comprises the steps of: contacting the sample under suitable conditions with an article of manufacture comprising a solid support having operably linked thereto an agent capable of specifically forming a complex with a CD4-binding domain of the HIV-1 envelope glycoprotein; and 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-1 envelope glycoprotein, 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, with the P:\OPER\MRO\31954-95.CLM 23/4/99 proviso that step may either precede or follow step

18. A method of treating a bodily fluid sample so as to substantially reduce the likelihood of a subject's becoming infected with HIV-1 as a result of contact with the sample which comprises the steps of: contacting the sample under suitable conditions with an article of manufacture comprising a solid support having operably linked thereto an agent capable of specifically forming a complex with a CD4-binding domain of the HIV-1 i gpl20 envelope glycoprotein. contacting the sample with a suitable amount of an aqueous-soluble agent which is capable of specifically 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, 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 may either precede or follow step

19. The method according to any one of claims 1 to 18 further comprising the step of removing CD4+ cells from the bodily fluid sample. The method according to any one of claims 1 to 19 wherein the bodily fluid sample is selected from the group consisting of whole blood, a red blood cell preparation, a platelet preparation, and semen.

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21. The method according to claim 20 wherein the bodily fluid sample is blood.

22. The method according to any one of claims 1 to 21 substantially as hereinbefore described with reference to the Figures and/or Examples. DATED this TWENTY THIRD day of APRIL, 1999 Progenies Pharmaceuticals, Inc. AND Aaron Diamond AIDS Research Centre (ARARC) DAVIES COLLISON CAVE o Patent Attorneys for the Applicants a a a a.* a a a a a a a a a a