WO2001043779A2

WO2001043779A2 - Anti-hiv-1 conjugates for treatment of hiv disease

Info

Publication number: WO2001043779A2
Application number: PCT/US2000/034032
Authority: WO
Inventors: Michael Fung; David Thomas
Original assignee: Tanox, Inc.
Priority date: 1999-12-16
Filing date: 2000-12-15
Publication date: 2001-06-21
Also published as: AU2265701A; WO2001043779A3

Abstract

The present invention provides a conjugate of an anti-CD4 molecule and an antichemokine receptor molecule. These molecules may be antibodies. The anti-CD4 molecule may be an antibody that inhibits HIV-1 infection of CD4+ cells and also inhibits syncytia formation between CD4+ cells, but does not cause immunosuppression in humans. The anti-chemokine receptor antibody may be an anti-CCR5 molecule. The invention also provides for conjugate of an anti-CD4 molecule and an HIV-1 fusion inhibiting peptide. Both types of conjugates may be formed by chemical methods and may or may not include a linker. Numerous types of linkers may be used. Both types of conjugates may also be expressed as recombinant proteins with or without linkers. The invention also provides a method of treating HIV-1 disease by providing a conjugate to a subject. The conjugate may be provided by providing the subject with a recombinant nucleic acid in a viral vector.

Description

ANTI-HIV-1 CONJUGATES FOR TREATMENT OF HIV DISEASE

SPECIFICATION

Field of the Invention The invention relates to conjugates of anti-CD4 molecules with anti-chemokine co-receptor molecules or anti-HIV-1 fusion peptides, a method of treating HIV-1 infection with such conjugates, a method of preventing HIV-1 entry into CD4+ cells with such conjugates, a method of preventing syncytia formation between CD4+ cells with such conjugates, and a method of delivering the conjugates to cells.

Background of the Invention The CD4 surface molecule on T cells and monocytes/macrophages is known as the primary receptor for HIV-1 binding. Upon binding to CD4, the virus can enter and infect these cells. The virus replicates inside the infected cells to produce more viral particles which are then released to infect other cells. The result is the decline of CD4+ T cell numbers. Since CD4+ T cells are pivotal in mediating immune response, the depletion of CD4+ T cells results in generalized immunosuppression, which then leads to secondary infections or cancer, the clinical hallmarks of AIDS.

Along with CD4, several co-receptors on T cells and/or monocytes/macrophages for the virus have been identified and implicated in entry of the virus into cells. These chemokine co-receptors include CCR5 and others.

Anti-CD4 antibodies have long been suggested as a potential prevention or treatment for HIV-1 infection. These antibodies prevent viral entry into the cell. A problem with most of these antibodies is that they themselves can cause immunosuppression by their binding to CD4. However, one unique anti-CD4 monoclonal antibody has been developed which does not cause immunosuppression, but nevertheless inhibits infection of CD4+T cells and monocytes/macrophages by HIV-1 virions and blocks fusion between infected cells and CD4+ target cells. This monoclonal antibody is designated 5A8, and is described and claimed in U.S. Patent No. 5,871,732, incorporated herein by reference. Similar monoclonal antibodies might be developed using conventional techniques and detected using the methods of that disclosure.

A number of anti-chemokine receptor monoclonal antibodies have also been developed. These include LeukoSite, Inc.'s (now Millennium Pharmaceuticals, Inc.) 2D7 anti-CCR5 antibody, which is commercially available from Pharmingen, Inc. Such antibodies have also been implicated in interfering with HIV-1 infection of target cells

It is also possible to block HIV-1 infection of cells with a fusion inhibiting peptide, for example, the peptide discussed in Canadian Patent Application 2,208,420 (PCT US95/16733, WO96/19495) of Trimeris, Inc, incorporated by reference herein. These fusion inhibiting peptides inhibit the fusion of two discontinuous regions of HIV-1 gp41 and thus prevent the fusion between the virus and the target cells. These fusion inhibiting peptides derived from HIV-1 gp41 include the amino acid sequences YTSLIHSLIEESQNQQEKJ ffiQELLELDKWASLWNWF (SEQ. ID. NO. 1), designated YT36WF (amino acid residue numbers 642 to 677 of gpl60 of LAI strain) and WMEWDREINNYTSLIHSLIEESQNQQEKNEQELL (SEQ. ID. NO. 2), designated WM34LL (amino acid residue numbers 632 to 665 of gpl60 LAI strain), as well as all variant sequences in the corresponding regions in other strains and clades.

These peptides and others that may be involved in or capable or preventing fusion of HIV-1 to a target cell are described in R. Furuta et al., Nature Structural Biology, 1998; 5: 276-279; M. Kilby et al., Nature Medicine, 1998; 4: 1302-1307; C. Wild et al, Proc. Natl. Acad. Sci. USA, 1994; 91 : 9770-9774; D. Eckert et al, Cell, 1999; 99:103- 115; and J. Sodroski, Cell, 1999; 99: 243-246.

Summary of the Invention The present invention relates to a conjugate comprising an anti-CD4 molecule and an anti-chemokine receptor molecule. In a preferred embodiment the molecules are antibodies. Here and throughout the specification and claims, antibodies refers to whole antibodies and antibody fragments or molecules including antibody fragments, including, but not limited to, single chain antibodies, humanized antibodies, Delmmunised™ antibodies, and Fab, F(ab')₂, V_H, V_L, Fd, and single or double chain Fv fragments. In a more preferred embodiment, the anti-CD4 molecule does not result in immunosuppression when administered to a human, but does block HIV-1 infection of CD4+ cells and does block syncytia formation between CD4+ cells. In a preferred embodiment, the anti-chemokine receptor molecule is an anti-CCR5 antibody.

In a preferred embodiment, the molecules are conjugated by a chemical method. In a more preferred embodiment, the molecules are conjugated by a linker. In another more preferred embodiment, the molecules are antibodies and the anti-CD4 molecule is a single chain antibody which is conjugated to the C-terminal end of the anti-chemokine receptor molecule. In another preferred embodiment, the molecules are expressed in a single recombinant protein. In another preferred embodiment, an anti-CD4 molecule is conjugated to an HIV-

1 fusion inhibiting peptide. In a more preferred embodiment, the anti-CD4 molecule is an antibody In a more preferred embodiment, the anti-CD4 molecule does not result in immunosuppression when administered to a human, but does block HIV-1 infection of CD4+ cells and does block syncytia formation between CD4+ cells. In another more preferred embodiment, the fusion inhibiting peptide has the sequence of SEQ. ID. NO. 1 or SEQ. ID. NO. 2.

In a preferred embodiment, the molecules are conjugated by a chemical method. In a more preferred embodiment, the molecules are conjugated by a linker. In another more preferred embodiment, the anti-CD4 molecule is an antibody and the fusion inhibiting peptide is conjugated to the C-terminal end of the anti-CD4 antibody. In another preferred embodiment, the molecules are expressed in a single recombinant protein.

The present invention also comprises a method of preventing HIV-1 entry into target cells as well as prevention or treatment of HIV-1 infection, or syncytia formation between target cells. The method comprises administering to the subject an anti-CD4 antibody/anti-chemokine receptor antibody conjugate or an anti-CD4 antibody/HIV- 1 fusion inhibiting peptide conjugate in an amount effective to prevent HIV-1 entry into cells, or prevent syncytia formation between target cells, or in an amount effective to prevent or treat an HIV-1 infection. In a preferred embodiment, an effective amount of the conjugate is provided to a subject by administering to the subject a viral vector comprising a recombinant nucleic acid encoding the conjugate, under conditions whereby the recombinant sequence is expressed as a recombinant protein that may be secreted and may enter the circulation and is targeted to cells to prevent HIV-1 entry into target cells or to prevent syncytia formation between cells.

Brief Description of the Drawings

Figure 1 : Figure la: Conjugate of an anti-CD4 antibody and an anti-chemokine receptor antibody. Figure lb: Conjugate of an anti-CD4 antibody and a fusion inhibiting peptide.

Figure 2: Conjugate of an and anti-CD4 antibody to an anti-CCR5 antibody in the absence of a linker.

Figure 3 : Conjugate of an anti-CD4 antibody to a fusion inhibiting peptide in the absence of a linker.

Figure 4: Conjugate of an anti-CD4 antibody to an anti-CCR5 antibody with a linker. Figure 5 : Recombinant protein conjugate of an anti-CD4 antibody and an anti-

CCR5 antibody.

Detailed Description of the Invention The invention provides conjugates of anti-CD4 molecules and anti-chemokine receptor molecules which may be used to prevent HIV-1 entry into target cells or syncytia formation between cells and may be helpful in HIV-1 disease treatment. By blocking both CD4 and a chemokine receptor, HIV-1 entry into or infection of target cells should be more effectively inhibited. Furthermore, conjugated antibodies may display a synergistic improvement in blocking HIV-1 infection relative to simultaneously administered, unconjugated antibodies. This synergistic effect may be expected because HIV-1 must have access to CD4 and a chemokine receptor in close proximity to one another in order to infect a target cell. The conjugated antibodies, like unconjugated antibodies, may bind to chemokine receptors or CD4 in relative isolation, but if the second antigen is near the first, both should be blocked because both of the conjugated antibodies will bind. In fact, the increased stability of double binding may target the conjugate to proximate targets. In contrast, unconjugated antibodies will not block CD4 and chemokine receptors in close proximity with such a degree of selectivity and certainty.

The preferred anti-CD4 antibody of this invention is SA8, or other similar antibodies as described in the background that do not result in immunosuppression. The chemokine receptors include CCR5, CXCR4, CCR2b, CCR3, CCR8, STRL33, GPRl , V28, ChemR23, GPRl 5 and AP5. The anti-CD4 antibody should be non- immunosuppressive and can include, for example, the binding portion of the antibody 5A8. The preferred chemokine receptor target is CCR5. The conjugates are designed to bind to both CD4 and the chemokine receptor simultaneously, to block the sequential steps of HIV-1 entry into the target cell.

HIV-1 infection may also be inhibited by conjugating a fusion inhibiting peptide as described above to an anti-CD4 molecule. This conjugation should also exhibit synergistic effects when compared to administration of unconjugated anit-CD4 molecules and fusion inhibiting peptides. First, because it is conjugated to an anti-CD4 molecule, the fusion inhibiting peptide should localize better than unconjugated protein to the vicinity of the T cell membrane where it is to exert its effects. Additionally, even if HIV- 1 manages to bind to nearby CD4 or cytokine receptors, its ability to infect will still be hampered by the peptide.

Similar effects may be obtained by conjugating a fusion inhibiting peptide to an anti-cytokine receptor antibody such as one of the antibodies described above. The fusion inhibiting peptide of the invention may be YT36WF or WM34LL. It may also include other peptides described in the art and now or later known to inhibit the fusion of HIV-1 to the target cell. Such peptides may be isolated from HIV-1 proteins or may be discovered through techniques such as biophage panning. The fusion inhibiting peptides may be conjugated to the C-terminal or N-terminal end of the anti-CD4 molecule, which could be a single chain, Fv fragment, Fab, another fragment, or a whole monoclonal antibody.

One preferred construct for the conjugate is a non-immunosuppresive anti-CD4 monoclonal antibody (preferably 5A8) with a single chain Fv fragment of a monoclonal antibody targeting a chemokine receptor, preferably CCR5, conjugated to the C-terminal end of each heavy chain. This non- flexible construct is well-suited for binding to both CD4 and the chemokine receptor where these targets are in proximity, but not directly associated or joined together on the cell surface. Alternatively, the antibody portion of the conjugate could be an anti-chemokine receptor antibody, (e.g. anti-CCR5) with the single chain Fv fragment of anti-CD4 (preferably 5 A8) linked to the C-terminal end of the heavy chain of the anti-CCR5 antibody.

Another preferred construct for the conjugate is non-immunosuppresive anti-CD4 monoclonal antibody (preferably 5 A8) with a single chain Fv fragment of a monoclonal antibody targeting a chemokine receptor, preferably CCR5, conjugated to the C-terminal end of each heavy chain (or the alternative construct described above), and with a flexible linker joining the antibody and the fragment. The flexible linker allows this construct to bend, so that it can bind to both CD4 and the chemokine receptor where these targets are directly associated or joined together on the cell surface. Such a linker may also be included in an anti-CD4/fusion inhibitor construct.

Any of the constructs (or the alternative constructs) described above can also be in a univalent form, with one heavy chain and one light chain of either the CD4-binding molecule or the chemokine receptor-binding molecule conjugated, at the C-terminal end of the heavy chain, with one single chain fragment targeting the appropriate receptor. An analogous construct could be used to make the conjugates of anti-CD4 binding molecule with a fusion inhibiting peptide and anti-CCR5 binding molecule with a fusion inhibiting peptide. These constructs can either include or not include a flexible linker, as desired in view of the considerations described above.

The invention can also include a number of variations of the conjugates described above, including small molecules or peptides capable of binding CD4, a chemokine receptor or HIV-1 gp41 conjugated together, both with and without a flexible linker. Although only preferred embodiments of the invention are specifically described above, it will be appreciated that modifications and variations of the invention are possible without departing from the spirit and intended scope of the invention.

The following non-limiting examples are provided to more clearly illustrate the aspects of the invention and are not intended to limit the scope of the invention. Examples Example : Antibodies

The various embodiments of the invention described herein may include a monoclonal antibody against CD4 or CCR5 or other chemokines. In a preferred embodiment, the anti-CD4 antibody is an antibody as described in U.S. Pat. No. 5,871,732 to Burkly, et al. Such an antibody binds to human CD4, does not block binding of HIV gpl20 to human CD4 and does not result in immunosuppression when administered to a human, but does block HIV-induced syncytia formation between CD4+ cells. Further, in a preferred embodiment, the anti-chemokine antibody is anti-CCR5 antibody is 2D7, an HIV-infection-blocking antibody commercially available from Pharmingen. Other HIV infection-blocking anti-chemokine antibodies may also be used.

Any monoclonal antibody of this invention may be a chimeric or humanized antibody, a human antibody, or a Delmmunised™ antibody. It might also consist of a whole antibody, a Fab, F(ab')₂, or single or double chain Fv fragment , a single chain antibody, or other form of antibody fragment. The antibody may be produced by any recombinant method known to the art and may be produced in vivo or in vitro.

Example 1A: Production of Fab or Ffab'^ antibody Fragments Generally, a preferred method for generating Fab antibody fragments comprises digestion of the selected antibody with papain and cysteine to generate IgG, Fab and Fc. A solution of the components is then loaded on a column containing Protein A. IgG and Fc to bind the column and Fab is present in the effluent. Similarly, a divalent F(ab' )₂ fragment may be produced by digestion of the antibody with pepsin, followed by chromatographic separation.

Example IB: Production of Single Chain Antibodies

Single chain antibodies ("ScFv") are preferred for making the conjugates. The method of their determination and construction is described, for example, in U.S. Patent No. 4,946,778 to Ladner, et al., incorporated herein by reference. Generally, such antibodies are produced by introducing into host cells a nucleic acid that encodes a single chain polypeptide with binding affinity for an antigen comprising the binding portion of the light chain variable region of an antibody, the binding portion of the heavy chain variable region of an antibody, and a polypeptide linker linking the light and heavy chain variable region binding portions into a single chain polypeptide having binding affinity for an antigen. In the present invention, the polypeptide, or ScFv, may have binding affinity for CD4, CCR5 or another chemokine.

Example lC:Chimeric and Humanized antibodies

Chimeric antibodies are produced by recombinant processes well known in the art, and, generally, have an animal (e.g. murine) variable region and a human constant region. Humanized antibodies correspond more closely to the sequence of human antibodies than do chimeric antibodies. In a humanized antibody, only the complementarity determining regions (CDRs) which are responsible for antigen binding and specificity are animal-derived and have an amino acid sequence corresponding to the animal antibody, and substantially all of the remaining portions of the molecule (except, in some cases, small portions of the framework regions within the variable region) are human-derived and have a corresponding amino acid sequence to a human antibody. See L. Riechmann et al., Nature, 1988; 332: 323-327; United States Patent No. 5,225,539 (Medical Research Council); U.S. Patent Nos. 5,585,089; 5,693,761; 5,693,762; and 5,530,101 (Protein Design Labs, Inc.), incorporated herein by reference.

Example ID: Delmπninised™ antibodies

Delmmunised™ antibodies are antibodies in which the potential T cell epitopes have been eliminated, as described in International Patent Application PCT/GB98/01473, incorporated herein by reference. Therefore, immunogenicity in humans is expected to be eliminated or substantially reduced when they are applied in vivo.

Example IE: Human Antibodies

Human antibodies can be made by several different methods, including by use of human immunoglobulin expression libraries (Stratagene Corp., La Jolla, California;

Cambridge Antibody Technology Ltd., London, England) to produce fragments of human antibodies (V_H, V_L, FV, Fd, Fab, or (Fab') ₂), and using these fragments to construct whole human antibodies by fusion of the appropriate portion thereto, using techniques similar to those for producing chimeric antibodies. Human antibodies can also be produced in transgenic mice with a human immunoglobulin genome. Such mice are available from Abgenix, Inc., Fremont, California, and Medarex, Inc., Annandale, New Jersey. In addition to connecting the heavy and light chain Fv regions to form a single chain peptide, Fab can be constructed and expressed by similar means (M.J. Evans et al., J. Immunol. Meth., 1995; 184: 123-138), incorporated by reference herein.

All of the wholly and partially human antibodies are less immunogenic to humans than wholly murine or animal-derived antibodies, as are the single chain antibodies. All these molecules are therefore less likely to evoke an immune or allergic response. Consequently, they are better suited for in vivo administration in humans than wholly animal antibodies, especially when repeated or long-term administration is necessary.

Example 2: HIV-1 Fusion Inhibiting Peptides

HIV-1 fusion inhibiting peptides may be detected and derived through any methods known to the art or later discovered. Such peptides are characterized by their ability to inhibit or prevent the fusion of the HIV-1 virus to a target cell, such as a CD4+ T cell. Two peptides, YT36WF and WM34LL (SEQ. ID. NOS. 1 and 2, respectively) are known to prevent such fusion. These peptides are derived from HIV-1 proteins involved in the fusion process. Other peptides might also be derived from HIV-1 proteins implicated in fusion.

The invention also encompasses non-HIV-1 -derived peptides or HIV-1 peptides derived from proteins not involved in fusion. Such peptides may be discovered by any technique that tests their ability to inhibit HIV-1 fusion to a target cell. For instance, phage biopanning can be used to identify such peptides. See G. Ehrlich and W. Bailon, Methods Mol Biol. 2000;147:195-208; and J. Devlin et al, Science 1990; 249: 404-406, incoφorated by reference herein, for phage biopanning techniques. Example 3: Producing Conjugated Antibodies Without Linkers

Antibodies may be conjugated by linking the Fc region of one antibody to the Fc region of the second antibody. In a preferred embodiment, one antibody or antibody fragment is linked to the C-terminal end of the heavy chain of a second antibody or antibody fragment. Conjugation may be accomplished by conventional means of attaching other proteins to the C-terminal end of the heavy chain.

In a preferred embodiment, incubation of the two antibodies to be joined in a glutaraldehyde solution with Tris buffer should induce conjugate formation. See Current Protocols in Molecular Biology, John Wiley and Sons, Inc., 2000, pp. 11.1.4-11.1.5 and Short Protocols in Molecular Biology, 3rd ed., John Wiley and Sons, Inc., 1995, p. 1 1-4, incoφorated by reference herein.

In another preferred embodiment, the antibodies or antibody fragments may be conjugated by first chemically or recombinantly modifying one antibody or antibody fragment to contain a carbohydrate side chain such as that present on and used for conjugation of horseradish peroxidase to antibodies. The antibody could then be conjugated to another antibody or antibody fragment using a protocol similar to that employed for horseradish peroxidase conjugation. See Current Protocols in Molecular Biology, John Wiley and Sons, Inc., 2000, pp. 11.1.1-11.1.3 and Short Protocols in Molecular Biology, 3rd ed., John Wiley and Sons, Inc., 1995, pp. 11-3 - 11-4, incoφorated by reference herein.

Conjugated antibodies may also be produced by any recombinant methods known to the art. In such cases, the recombinant nucleic acid should encode at least the heavy and light chain variable region sequences responsible for binding to the antigen of both of the antibodies to be joined. The nucleic acid should additionally encode a product that is capable of binding to both of the antigens.

Example 4: Antibody-Fusion Inhibiting Peptide Conjugates Without Linkers

Fusion inhibiting peptides may be conjugated to the C-terminal or N-terminal end of an anti-CD4 or anti-chemokine antibody or antibody fragment. Such fusion inhibiting peptides preferably prevent fusion between the HIV-1 vims and its target cell. In a more preferred embodiment, the peptides are YT36WF (SEQ. ID. NO. 1) or WM34LL (SEQ. ID. NO. 2).

The peptide may be conjugated to the antibody by chemical means, including those used to conjugate other proteins such as horseradish peroxidase or alkaline phosphatase to antibodies. Addition of a carbohydrate chain to the peptide may be necessary for conjugation following a horseradish peroxidase protocol. See Current Protocols in Molecular Biology, John Wiley and Sons, Inc., 2000, pp. 11.1.1-11.1.7 and Short Protocols in Molecular Biology, 3rd ed., John Wiley and Sons, Inc., 1995, p. 11-3 — 11-4, incoφorated herein by reference. Conjugated antibodies and fusion inhibiting peptides might also be produced by recombinant methods. In such an instance, the recombinant nucleic acid should encode at least the heavy and light chain variable region sequences responsible for binding to the antigen of the antibody and the fusion inhibiting peptide sequence. The nucleic acid should additionally encode a product that is capable of binding to the antigens and inhibiting fusion of the HIV-1 vims to a target cell.

The recombinant proteins of this Example and Example 3 may be produced in vivo or in vitro. For in vitro production, in a preferred embodiment cultured cells are transformed or transfected with the recombinant nucleic acid. Such cells or conjugates produced thereby may then be administered to an individual.

Example 5: Linkers

The flexible peptide linker in the preferred embodiment can be any of a number of linkers, including Ser-Cys, (Gly)₄-Cys, (His)₆-(Gly)₄-Cys, chelating agents, and chemical or disulfide couplings. The linker may be attached by covalent or ionic bonds. Examples of covalent linker include, but are not limited to, sulfhydryl and maleimide linkages. Examples of ionic bond linkages include, but are not limited to, cationic molecules such as poly-L-lysine (PLL) and polyethylene glycol-PLL (PEG- PLL). Additional linkers include biocompatible polymers having an average weight of 200 to 20,000 daltons which may be chemically modified to be used as linkers. More specifically, any chemical linker may be used, provided that it does not affect the reactivity of any conjugated antibody or antibody fragment or the fusion inhibiting function of any conjugated peptide.

One example of a linker comprises a covalent bond through a maleimide- sulfhydryl linkage. In a preferred embodiment, a sulfhydryl reactive group is introduced to one antibody or antibody fragment or peptide and a sulfhydryl reactive group is introduced to the other antibody or antibody fragment or peptide.

More specifically, in this embodiment, N-succinimidyl S-acelythioacetate (SATA) may be used to introduce a sulfhydryl group onto one antibody, antibody fragment, or peptide. Sulfosuccinimidyl 4-(N-maleimidomethyl) cyclo hexane-1- carboxylate (sulfo-SMCC) may then used to introduce a maleimide group onto the second antibody, antibody fragment or peptide. The two molecules comprising the sulfhydryl and maleimide groups may then be incubated together to product a conjugate with a stable thioether linkage. Conjugates may be similarly produced using other disulfide bonds.

Additional linkers include polymers which may be chemically modified to be used as linkers. Polymers are large non-immunogenic, biologically inert molecules comprising a chain of smaller molecules linked by covalent bonds. The polymers preferably have an average molecular weight of from about 200 to about 20,000 daltons, are biocompatible, and may be linear or branched. The polymers may be homopolymers or heteropolymers. Suitable polymers for use in the present invention include polyalkalene compounds such as polyalkalene oxides and glycols. Polyalkalene compounds include polyoxymethylene, polyethylene glycols (PEG) and oxides, and methoxypolyethyleneglycols, and derivatives thereof including for example polymethyl- ethylene glycol, polyhydroxypropylene glycol, polypropylene glycol, polymethyl propylene glycol, and polyhydroxypropylene oxide.

A preferred polymer in accordance with the present invention is PEG, which is a water-soluble polymer having the formula H(OCH₂CH₂)_nOH, wherein n is the number of repeating units and determines the average molecular weight. PEGs having average molecular weights of from 200 to 20,000 daltons are commercially available. In an especially preferred embodiment, the PEG molecule is a bifunctional PEG molecule comprising an amine reactive moiety and a sulfhydryl reactive moiety.

Other chemical compounds that can be used to produce conjugates comprising linkers comprise hetero functional molecules that have both amine reactive and sulfhydryl-reactive groups. Examples of such heterofunctional molecules include, for example, N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), succinimidyloxycarbonyl-α-methyl-(α-2-pyridyldithio) toluene (SMPT), m- maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), N-succinimidyl(4 iodoacetyl)aminobenzoate (SIAB), succinimidyl-4-(p-maleimidophenyl)butyrate (SMPB), N-(γ-maleimidobutyryloxy)succinimide ester (GMBS), succinimidyl-6- ((iodoacetyl)amino)hexonate) (SIAX), succinimidyl- 4(((iodoacetyl)amino)methyl) (SIAC), and (p-Nitrophenyl iodoacetate)(NPIA). These molecules may also contain sulfo groups, which will increase the solubility of these molecules in water. Examples include: sulfo-SPDP, sulfo-SMPT, sulfo-SIAB, sulfo-SMPB and sulfo-GMBS. When using N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), the activated

N-hydroxysuccinimidyl (NHS) ester end of SPDP reacts with amine groups in proteins or antibodies to form an amide linkage. The 2-pyridyldithiol group at the other end reacts with sulfhydryl groups on the other antibodies or peptides, to form a disulfide linkage.

When using succinimidyloxycarbonyl-a-methyl-(2pyridyldithio) toluene (SMPT), the amine reactive NHS ester at one end reacts with the first antibody or peptide. The sulfhydryl-reactive pyridyldisulfide group reacts with the second antibody or peptide.

When using m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), the amine reactive NHS ester at one end reacts with amine groups in the antibody or peptide, and at the other end is a sulfhydryl reactive group. The second antibody or peptide has a sulfhydryl group.

When using N-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB), the NHS ester of SIAB couples to primary amines on the first antibody or peptide and the iodoacetyl group at the other end can couple to a sulfhydryl residue in the second antibody or peptide. When using succinimidyl-4-(p-maleimidophenyl) butyrate (SMPB), having an amine reactive NHS ester on one end to bind to the first antibody or peptide and a sulfhydryl- reactive maleimide group on the other end, the second antibody or peptide contains a sulfhydryl group.

When using N-(γ-maleimidobutyryloxy)-succinimide ester (GMBS), a heterobifunctional crosslinker that contains an NHS ester on one end to bind to the first antibody or peptide and a sulfhydryl-reactive maleimide group on the other end, the second antibody or peptide contains a sulfhydryl group.

When using succinimidyl 6-((iodoacetyl)amino)hexonate) (SIAX), which contains an NHS ester on one end to bind to the first antibody or peptide and an iodoacetyl group which can react with sulfhydryls on the other end, the second antibody or peptide contains a sulfhydryl group

When using succinimidyl 4(((iodoacetyl)amino)methyl) (SIAC), which has a cyclohexane-1-carboxylate NHS ester on one end of the molecule which can react with amines of the first antibody or peptide, and an iodoacetyl group on the other end which can couple to sulfhydryl groups, the second antibody or peptide contains a sulfhydryl group.

When using (p-nitrophenyl iodoacetate) (NPIA), which has an activated carboxylic acid group with a p-nitrophenyl ester, the p-nitrophenyl ester couples to amine containing proteins of the first antibody or peptide. The other end can react with sulfhydryl groups to give thioether bonds. The second antibody or peptide contains a sulfhydryl group.

In the embodiments described above, the sulfhydryl group is added to either an antibody or peptide by methods known in the art, such as by using S ATA (N- Succinimidyl S-Acetythioacetyl).

Other heterofunctional reagents for use in chemically linking the antibodies and/or peptides to be conjugated include both carbonyl-reactive and sulfhydryl reactive groups. These reagents are especially useful for conjugating carbohydrate-containing molecules such as glycoproteins to sulfhydryl containing molecules. In intact antibodies, the Fc portion is glycosylated. A preferred carbonyl reactive functional group on these linkers is a hydrazide group that can form hydrazone bonds with aldehydes on sugars. The preferred sulfhydryl reactive functional group on the linkers comprise either a pyridal disulfate group, a disulfide group or a maleimide group. These groups react with a sulfhydryl group on the second antibody or peptide. Examples include 4 -(4-N- maleimidophenyl) butyric acid hydrazide (MPBH), 4 -(N-maleimidomethyl)cyclohexane- 1 -carboxyl-hydrazide (M₂C₂H and 3-2(2-pyridyldithio) proprionyl hydrazide (PDPH). Other useful heterofunctional reagents have an NHS ester on one end and a photoreactive aryl azide group on the other, such as N-hydroxysuccinimidyl-4- azidosalicylic acid (NHS-ASA). Other similar linkers include SANPAH and sulfo- SANPAH (N-succinimidyl-6-(4-azido-2'-nitrophenylamino)hexanoate). These linkers contain an NHS ester and a photoreactive phenylazide group. Other similar linkers include sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)- ethyl- 1 ,3'-dithioproprionate (SAND), N-Succinimidyl-4(4-Azidophenyl) 1,3'dithiopropionate (SADP) and sulfo - SADP. SADP is a photoreactive heterobi functional cross-linker that is cleavable by treatment with a disulfide reducing agent. The cross-linker contains an amine reactive NHS ester and a photoactivatable phenylazide group. SADP is first used to modify a protein (e.g., antihexon-Fab fragment) via its amine groups through the reactive NHS ester end of the cross-linker. This leads to the formation of a nucleophile-reactive dehydroazepine intermediate able to covalently couple with further amine containing compounds.

Still other useful heterobifunctional reagents comprise molecules containing sulfhydryl-reactive and photoreactive linkers, e.g. ASIB, 1 -(p Azidosalicylamido)-4- (iodoacetamido)butyrane. This linker contains a sulfhydryl-reactive pyridal disulfide group on one end and a photosensitive phenylazide group on the other. Another example is APDPN-(4-(p-Azidosalicylamido)butyl]-3'-(2'-pyridyldithio)propionamide.

Another preferred linker comprises a polymer-type of linker, e.g., a heterofunctional PEG. Preferred heterofunctional derivatives of PEG include a heterofunctional PEG with either a NHS ester or a tresyl group on one end and vinylsulfone or maleimide on the other end. Other heterofunctional derivatives of PEG include a heterofunctional PEG having a protected amine on one end and the PEG would be activated on other end, using e.g. α-hydroxy-α-amine, ω-hydroxy-α-carbonyl and ω- amino-α-carbonyl molecules. Also within the scope of the invention are branched heterofunctional PEG molecules. Preferred branched heterofunctional derivatives of PEG include a heterofunctional PEG with a tresyl group on one end and multiple maleimide groups on the other end.

Example 6: Prevention of HIV-1 Entry or Syncytia Formation The conjugates of the invention may be used to prevent either HIV-1 entry into a target cell or syncytia formation between CD4+ cells, or both. The conjugates are administered in an amount sufficient to prevent HIV-1 entry into the target cell and/or syncytia formation between infected CD4+ cells. The cells may be in culture, in a nonhuman animal, or in human. The specific activity that may be inhibited by any particular conjugate will depend on the composition of the conjugate and will be apparent to one skilled in the art. For instance, a conjugate may be designed to inhibit syncytia formation between CD4+ cells by incoφorating at least the regions of the 5A8 antibody responsible for CD4 binding. A conjugate comprising at least the CD4 binding regions of the 5A8 antibody and at least the CCR5 binding regions of the 2D7 antibody should prevent entry of HIV-1 into a CD4+ cell.

Example 7: Treatment of HIV-1 With Conjugates Produced in Vitro

The conjugates of the invention may be administered as a composition to treat or prevent HIV-1 infection, block HIV-1 entry into target cells, or block syncytia formation between cells. The dosage and mode of administration will depend on the formulation, the stage of infection and other factors which can be readily determined during routine human clinical trials or through extrapolation from animal studies.

Such compositions comprise the conjugates admixed with a suitable physiologically acceptable carrier. As used herein, the term "physiologically acceptable carrier or diluent" means any and all solvents, dispersion media, antibacterial and antifungal agents, microcapsules, liposomes, cationic lipid carriers, isotonic and absoφtion delaying agents and the like which are not incompatible with the conjugate. Fore example, the carrier may be saline, buffered saline, or phosphate buffered saline. The use of such media and agents for physiologically active substances is well known in the art. Supplementary active ingredients may also be incoφorated into the compositions. Typically, the composition may be administered by injection, either intravenously or intraperitoneally. It may also be possible to obtain compositions which may be topically or orally administered, or which may be capable of being absorbed across mucousal membranes. Specifically, particular linker molecules may facilitate absoφtion across mucousal membranes.

Before administration to patients, formulants may preferably be added to the composition for in vivo use. For example, these formulants may include oils, polymers, vitamins, carbohydrates, amino acids, salts, buffers, albumin, surfactants, or bulking agents. Carbohydrates may include sugar or sugar alcohols such as mono-, di-, or polysaccharides, or water-soluble glucans.

Example 8: Treatment of HIV-1 With Conjugates Produced in Vivo

Patients may also be treated by enabling the patient to internally produce a recombinant conjugate as described in Examples 3 and 4. For production of a conjugate in an individual, the recombinant nucleic acid is introduced to a production cell capable of expressing the sequence as a recombinant protein. The nucleic acid may be introduced via any viral vector known to the art and capable of accommodating a nucleic acid of the desired length. The viral vector should also be capable of transferring the recombinant nucleic acid to the production cell such that the recombinant protein may be produced within the cell. The production cell should be chosen so that the recombinant protein may be secreted, enter the circulation and be targeted to cells to prevent HIV-1 entry into target cells or syncytia formation between cells. The viral vector may be administered to the production cells within or outside of the patient. The viral vector should be chosen so that less immunogenic vectors are employed if repeated treatments will be necessary. In a preferred embodiment, a viral vector capable of permanent introduction of the recombinant nucleic acid into the host cell, such as a RNA-virus with reverse transcriptase, may be employed for long-term treatment. In a more preferred embodiment, the production cell for the RNA viral vector is a stem cell.

Claims

CLAIMS What Is Claimed Is:

I . A conjugate comprising an anti-CD4 molecule and an anti-chemokine receptor molecule. 2. The conjugate of claim 1, wherein the anti-CD4 molecule is an antibody.

3. The conjugate of claim 1, wherein the anti-chemokine receptor molecule is an antibody.

4. The conjugate of claim 2, wherein the anti-CD4 antibody is not immunosuppressive when administered to a human, and is capable of inhibiting HIV-1 entry into and/or infection of CD4+ cells and blocking syncytia formation between CD4+ cells.

5. The conjugate of claim 3, wherein the anti-chemokine receptor antibody is an anti-CCR5 antibody.

6. The conjugate of claim 1, wherein the anti-CD4 molecule is conjugated to the anti-chemokine receptor molecule by a chemical method.

7. The conjugate of claim 6, wherein the molecules are conjugated through a linker.

8. The conjugate of claim 6, wherein the anti-CD4 molecule is a single chain antibody fragment and is conjugated to the C-terminal end of the anti-chemokine receptor molecule, which is also an antibody. 9. The conjugate of claim 1, wherein the anti-CD4 molecule and the anti-cytokine molecule comprisea single recombinant protein. 10. A conjugate comprising an anti-CD4 molecule and an HIV-1 fusion inhibiting peptide.

I I . The conjugate of claim 10, wherein the anti-CD4 molecule is an antibody. 12. The conjugate of claim 10, wherein the fusion inhibiting peptide has the sequence of SEQ. ID. NO. 1 or SEQ. ID. NO. 2. 13. The conjugate of claim 11 , wherein the anti-CD4 antibody is not immunosuppressive when administered to a human, and is capable of inhibiting HIV-1 entry into and/or infection of CD4+ cells and blocking syncytia formation between CD4+ cells.

14. The conjugate of claim 10, wherein the anti-CD4 molecule is conjugated to the fusion inhibiting peptide by a chemical method.

15. The conjugate of claim 14, wherein the molecule are conjugated through a linker.

16. The conjugate of claim 14, wherein the fusion inhibiting peptide is conjugated to the C-terminal end of the anti-CD4 molecule, which is an antibody.

17. The conjugate of claim 10, wherein the anti-CD4 molecule and fusion inhibiting peptide comprise a single recombinant protein.

18. A method of preventing HIV-1 entry into target cells, as well as prevention or treatment of HIV-1 infection, comprising administering to the subject an anti- CD4 antibody/anti-chemokine receptor antibody conjugate or an anti-CD4 antibody/HIV- 1 fusion inhibiting peptide conjugate in an amount effective to prevent HIV-1 entry into target cells and/or syncytia formation between target cells, and or an amount effective to prevent or treat HIV-1 infection.

19. The method of claim 18, wherein an effective amount of the conjugate is provided to a subject by administering to a subject a viral vector comprising a recombinant nucleic acid encoding the conjugate, under conditions whereby said recombinant sequence is expressed as a recombinant protein that may be secreted and may enter the circulation and is targeted to cells to prevent HIV-1 entry into target cells and/or syncytia formation between target cells.