Title:  Method of inhibiting HIV infection with CD44 and anti-CD44 antibodies

United States Patent:  6,432,405

Issued:  August 13, 2002

Inventors:  Weinberg; J. Brice (Durham, NC); Haynes; Barton F. (Durham, NC)

Assignee:  Duke University (Durham, NC)

Appl. No.:  753851

Filed:  December 2, 1996

The present invention relates to a method of inhibiting HIV infection of cells susceptible to HIV infection. The method comprises contacting such cells with an agent (such as an anti-CD44 antibody) that inhibits CD44-facilitated HIV infection of the cells.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method of treating inflammation and immune-mediated tissue damage, such as occurs, for example, in the course of autoimmune diseases.

In one embodiment, the present invention relates to a method of suppressing T cell activation in an human comprising administering to the human the CD44 protein, or derivative or peptide portion thereof, in an amount sufficient to effect suppression. Examples of CD44 peptides suitable for use in the present method include those sets forth in Table 1.

 

Administration can be by injection or topical application (for example topically applied to the eye). Injection can be made directly into a skin lesion.

An additional form of the CD44 molecule that may be used as an immunosuppressive agent is a recombinantly produced CD44 molecule or a portion of the CD44 molecule produced by recombinant DNA technology. An example of such a form of CD44 has been reported by Aruffo, A et al Cell 61:1303-1313, 1990. This form of CD44 has been recombinately engineered to contain portions of the immunoglobulin protein constant domains. The addition of immunoglobulin domains to the extracellular domain of CD44 yielded in molecule called CD44-Rg-2 that has the properties of being secluded by COS cells when a plasmid containing this CD44-Rg2 gene was transfected into COS cells (Aruffo et al. Cell 61:1303-1313, 1990). The presence of immunoglobulin on the extracellular domain of CD44 would also have the potential advantage of increasing the circulating half-life of the CD44 molecule when administered to humans or animals.

Production of CD44-Rg-2 fusion construct: CD44-Rg-2 plasmid can be transfected into COS cells using DEAE dextran as described in Seed and PNAS 84: 3365-3369, 1987 and Aruffo, A et al Cell 61: 1303-1313, 1990. Semi-confluent COS cells plated on 100 mm plates will be transfected. Twelve hours after transfection, cells are trypsinized, seeded onto fresh 100 mm dishes and allowed to grow for 7-10 days. On the fourth day 5 ml fresh media, 10% calf serum are added per dish. Supernatants are harvested and stored at 4^oC.

Purification of CD44-Rg protein: Twelve hours following transfection, a fraction of the COS cells transfected are seeded into flasks. Thirty-six hours post-transfection, the cells are washed with PBS and overlayed with cysteine-methionine media for 30 min. [³⁵ Methionine and [³⁵ S]Methionine and [35S] Cysteine will be added to a final concentration of 150 .mu.Cl/ml, and the cells will be allowed to incorporate the label overnight. The supernatants will be harvested and incubated with 200 .mu.l of protein A-Trisacryl at 4^oC. for 12 hours. The beads will be collected by centrifugation and washed in 10 ml of PBS, 1 Nonidet P-40. The beads will then be eluted 200 .mu.l of 1% SDS.

In another embodiment, the present invention relates to a method of inhibiting various types of cellular interactions including macrophage T cell interactions and lymphocyte and macrophage interactions with endothelial cells. The invention further relates to a method of inhibiting CD44-monocyte IL1 release. These methods also involve the administration of an effective amount of the CD44 protein or derivative or portion thereof to an animal in need of such treatment.

CD44 protein suitable for use in the present method can be isolated from synovial tissue (preferably, human synovial tissue) or the protein can be produced recombinantly. Synthetic peptides reflective of discrete regions of the CD44 molecule can be made by standard techniques.

One skilled in the art will appreciate that the amounts to be administered for any particular treatment protocol can readily be determined. The CD44 protein, peptide or derivative can be administered together with a pharmaceutically acceptable carrier.

In yet another embodiment, the present invention relates to a method of transporting a drug or cytotoxic agent to a site of inflammation in an animal comprising administering to the animal the CD44 protein, or peptide or derivative thereof, linked, preferably covalently, to the drug or cytotoxic agent.

Examples of drugs to be targeted to organ- specific sites of inflammation are non-steroidal anti-inflammatory agents, forms of glucocorticosteroids, and cytoxic agents such as cyclophosphamide. By either incorporating these agents in liposomes bearing CD44 molecules, or by physically linking CD44 molecules to these drugs, one could achieve selective targeting or homing of the drug-CD44 complexes to sites of upregulated CD44 expression, that is sites of inflammation.

In a further embodiment, the present invention relates to a method of transporting a drug or cytotoxic agent to a site of inflammation in an animal comprising administering to the animal CD44 protein, or peptide or derivative thereof, and a drug or cytotoxic agent wherein both are incorporated into a liposome.

Claim 1 of 12 Claims

What is claimed is:

1. A method of inhibiting CD44-facilitated HIV infection of a mononuclear phagocyte susceptible to infection with a strain of HIV comprising contacting said mononuclear phagocyte with an anti-CD44 antibody in an amount such that said antibody binds to CD44 molecules present on the surface of said mononuclear phagocyte and thereby inhibits said CD44-facilitated infection of said mononuclear phagocyte by said strain of a HIV.

 

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