Title:  Targeted cytolysis of HIV-infected cells by chimeric CD4 receptor-bearing cells

United States Patent:  6,753,162

Issued:  June 22, 2004

Inventors:  Seed; Brian (Boston, MA); Banapour; Babak (Boston, MA); Romeo; Charles (Belmont, MA); Kolanus; Waldemar (Watertown, MA)

Assignee:  The General Hospital Corporation (Boston, MA)

Appl. No.:  394388

Filed:  February 24, 1995

Abstract

Disclosed is a method of directing a cellular immune response against an HIV-infected cell in a mammal involving administering to the mammal an effective amount of therapeutic cells which express a membrane-bound, proteinaceous chimeric receptor comprising (a) an extracellular portion which includes a fragment of CD4 which is capable of specifically recognizing and binding the HIV-infected cell but which does not mediate HIV infection and (b) an intracellular portion which is capable of signalling the therapeutic cell to destroy the receptor-bound HIV-infected cell. Also disclosed is a second method of treating HIV in a mammal involving administering to the mammal an effective amount of therapeutic cells expressing a membrane-bound, proteinaceous chimeric receptor comprising an extracellular portion which includes a fragment of CD4 which is capable of specifically recognizing and binding the HIV-infected cell but which does not mediate HIV infection. Also disclosed are cells which express the chimeric receptors and DNA and vectors encoding the chimeric receptors.

SUMMARY OF HE INVENTION

In general, the invention features a method of directing a cellular immune response against an HIV-infected cell in a mammal. The method involves administering to the mammal an effective amount of therapeutic cells, the therapeutic cells expressing a membrane-bound, proteinaceous chimeric receptor comprising (a) an extracellular portion which includes a fragment of CD4 which is capable of specifically recognizing and binding the HIV-infected cell but which does not mediate HIV infection and (b) an intracellular portion which is capable of signalling the therapeutic cell to destroy the receptor-bound HIV-infected cell.

In a related aspect, the invention features a cell which expresses a proteinaceous membrane-bound chimeric receptor which comprises (a) an extracellular portion which includes a fragment of CD4 which is capable of specifically recognizing and binding the HIV-infected cell but which does not mediate HIV infection and (b) an intracellular portion which is capable of signalling the therapeutic cell to destroy the receptor-bound HIV-infected cell.

In a second aspect, the invention features a method of treating HIV in a mammal involving administering to the mammal an effective amount of therapeutic cells, the therapeutic cells expressing a membrane-bound, proteinaceous chimeric receptor comprising an extracellular portion which includes a fragment of CD4 which is capable of specifically recognizing and binding the HIV-infected cell but which does not mediate HIV infection.

In a related aspect, the invention features a cell which expresses a membrane-bound, proteinaceous chimeric receptor comprising an extracellular portion which includes a fragment of CD4 which is capable of specifically recognizing and binding the HIV-infected cell but which does not mediate HIV infection.

In preferred embodiments of both the first and second aspects, the CD4 fragment is amino acids 1-394 of CD4 or is amino acids 1-200 of CD4; the CD4 fragment is separated from the intracellular portion by the CD7 transmembrane domain shown in FIG. 26 or by the hinge, CH2, and CH3 domains of the human IgG1 molecule shown in FIG. 25; and the CD4 fragment is separated from the therapeutic cell by at least 48 angstroms (and preferably, by at least 72 angstroms). In preferred embodiments of the first aspect, the intracellular portion is the signal-transducing portion of a T cell receptor protein (for example, .zeta.), a B cell receptor protein, or an Fc receptor protein; and the therapeutic cells are selected from the group consisting of: (a) T lymphocytes; (b) cytotoxic T lymphocytes; (c) natural killer cells; (d) neutrophils; (e) granulocytes; (f) macrophages; (g) mast cells; (h) HeLa cells; and (i) embryonic stem cells (ES).

In other related aspects, the invention features DNA encoding a chimeric receptor of the invention; and a vector including that chimeric receptor DNA.

Although the specific embodiment of the present invention is a chimera between CD4 and zeta, any receptor chain having a similar function to these molecules, e.g., in granulocytes or B lymphocytes, could be used for the purposes disclosed here. The distinguishing features of a desirable immune cell trigger molecule comprises the ability to be expressed autonomously (i.e., as a single chain), the ability to be fused to an extracellular CD4 domain such that the resultant chimera is present on the surface of a therapeutic cell, and the ability to initiate cellular effector programs upon aggregation secondary to encounter with a target ligand.

At present the most convenient method for delivery of the chimeras to immune system cells is through some form of genetic therapy. However reconstituting immune system cells with chimeric receptors by mixture of the cells with suitably solubilized purified chimeric protein would also result in the formation of an engineered cell population capable of responding to HIV-infected targets. Similar approaches have been used, for example, to introduce the CD4 molecule into erythrocytes for therapeutic purposes. In this case the engineered cell population would not be capable of self renewal.

The present invention relates to functional and simplified chimeras between CD4 fragments and T cell receptor, B cell receptor, and Fc receptor subunits which are capable of directing immune cells to recognize and lyse HIV-infected cells. The method for directing the cellular response in a mammal comprises administering an effective amount of therapeutic cells (for example, cytotoxic T lymphocytes) to the mammal, the cells being capable of recognizing and destroying the HIV-infected cell.

The invention also includes the chimeric receptor proteins which direct the cytotoxic T lymphocytes to recognize and lyse HIV-infected cells, the host cells transformed with a vector comprising the chimeric receptors, and antibodies directed against the chimeric receptors.

These and other non-limiting embodiments of the present invention will be apparent to those of skill from the following detailed description of the invention.

In the following detailed description, reference will be made to various methodologies known to those of skill in the art of molecular biology and immunology. Publications and other materials setting forth such known methodologies to which reference is made are incorporated herein by reference in their entireties as though set forth in full.

Standard reference works setting forth the general principles of recombinant DNA technology include Watson et al., Molecular Biology of the Gene, volumes I and II, the Benjamin/Cummings Publishing Company, Inc., publisher, Menlo Park, Calif. (1987); Darnell et al., Molecular Cell Biology, Scientific American Books, Inc., publisher, New York, N.Y. (1986); Lewin, Genes II, John Wiley & Sons, publishers, New York, N.Y. (1985); Old et al., Principles of Gene Manipulation: An Introduction to Genetic Engineering, 2nd ed., University of California Press, publisher, Berkeley, Calif. (1981); Maniatis et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, publisher, Cold Spring Harbor, N.Y. (1989); and Ausubel et al., Current Protocols in Molecular Biology, Wiley Press, New York, N.Y. (1989).

Claim 1 of 16 Claims

What is claimed is:

1. A cell which expresses a proteinaceous cell membrane-bound chimeric receptor, said receptor comprising (i) an extracellular CD4 portion which specifically recognizes and binds HIV or an HIV-infected cell and (ii) a transmembrane domain, wherein said chimeric receptor does not mediate HIV infection of said cell expressing said chimeric receptor.

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