Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing
Reexamination Certificate
1994-04-29
2003-10-14
Wehbé, Anne M. (Department: 1632)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
C514S453000, C424S130100, C424S278100
Reexamination Certificate
active
06632789
ABSTRACT:
BACKGROUND OF THE INVENTION
The induction of antigen-specific T cell responses involves multiple interactions between cell surface receptors on T cells and ligands on antigen presenting cells (APCs). The primary interaction is between the T cell receptor (TCR)/CD3 complex on a T cell and a major histocompatibility complex (MHC) molecule/antigenic peptide complex on an antigen presenting cell. This interaction triggers a primary, antigen-specific, activation signal in the T cell. In addition to the primary activation signal, induction of T cell responses requires a second, costimulatory signal. In the absence of proper costimulation, TCR signaling can induce a state of anergy in the T cell. Subsequent appropriate presentation of antigen to an anergic T cell fails to elicit a proper response (see Schwartz, R. H. (1990)
Science
248:1349).
A costimulatory signal can be triggered in a T cell through a T cell surface receptor, such as CD28. For example, it has been demonstrated that suboptimal polyclonal stimulation of T cells (e.g. by anti-CD3 antibodies or phorbol ester, either of which can provide a primary activation signal) can be potentiated by crosslinking of CD28 with anti-CD28 antibodies (Linsley, P. S. et al. (1991)
J. Exp. Med.
173:721; Gimmi, C. D. et al. (1991)
Proc. Natl. Acad. Sci. USA
88:6575). Moreover, stimulation of CD28 can prevent the induction of anergy in T cell clones (Harding, F. A. (1992)
Nature
356:607-609). Natural ligands for CD28 have been identified on APCs. CD28 ligands include members of the B7 family of proteins, such as B7-1(CD80) and B7-2 (B70) (Freedman, A. S. et al. (1987)
J. Immunol.
137:3260-3267; Freeman, G. J. et al. (1989)
J. Immunol.
143:2714-2722; Freeman, G. J. et al. (1991)
J. Exp. Med.
174:625-631; Freeman, G. J. et al. (1993)
Science
26:909-911; Azuma, M. et al. (1993)
Nature
366:76-79; Freeman, G. J. et al. (1993)
J. Exp. Med.
178:2185-2192). In addition to CD28, proteins of the B7 family have been shown to bind another surface receptor on T cells related to CD28, termed CTLA4, which may also play a role in T cell costimulation (Linsley, P. S. (1991)
J. Exp. Med.
174:561-569; Freeman, G. J. et al. (1993)
Science
262:909-911).
The elucidation of the receptor:ligand relationship of CD28/CTLA4 and the B7 family of proteins, and the role of this interaction in costimulation, has led to therapeutic approaches involving manipulation of the extracellular interactions of surface receptors on T cells which bind costimulatory molecules. For example, a CTLA4Ig fusion protein, which binds to both B7-1 and B7-2 and blocks their interaction with CD28/CTLA4, has been used to inhibit rejection of allogeneic and xenogeneic grafts (see e.g., Turka, L. A. et al. (1992)
Proc. Natl. Acad. Sci. USA
89:11102-11105; Lenschow, D. J. et al. (1992)
Science
257:789-792). Similarly, antibodies reactive with B7-1 and/or B7-2 have been used to inhibit T cell proliferation and IL-2 production in vitro and inhibit primary immune responses to antigen in vivo (Hathcock K. S. et al. (1993)
Science
262:905-907; Azuma, M. et al. (1993)
Nature
366:76-79; Powers, G. D. et al. (1994)
Cell. Immunol.
153:298-311; Chen C. et al. (1994)
J. Immunol.
152:2105-2114). Together, these studies indicate that T cell surface receptors which bind costimulatory molecules such as B7-1 and B7-2 are desirable targets for manipulating immune responses.
While the extracellular interactions between CD28/CTLA4 with their ligands have been characterized in some detail, little is known regarding the intracellular events that occur in a T cell following ligation of these molecules. T cell costimulation is thought to involve an intracellular signal transduction pathway distinct from signalling through the TCR since the costimulatory pathway is resistant to the inhibitory effects of cyclosporin A (see June, C. H. et al. (1990)
Immunology Today
11:211-216) Protein tyrosine phosphorylation has been shown to occur in T cells upon CD28 ligation and it has been demonstrated that a protein tyrosine kinase inhibitor, herbimycin A, can inhibit CD28-induced IL-2 production (Vandenberghe, P. et al. (1992)
J. Exp. Med.
175:951-960; Lu, Y. et al. (1992)
J. Immunol.
149:24-29).
SUMMARY OF THE INVENTION
This invention relates to the regulation of T cell responses by manipulation of intracellular signal transduction. In particular, intracellulal signalling events which occur upon costimulation of a T cell are manipulated. The invention encompasses methods for inhibiting or stimulating T cell responses by inhibiting or stimulating one or more intracellular signals which result from ligation of a surface receptor on a T cell which binds a costimulatory molecule. It has now been discovered that CD28 receptor stimulation leads to the production of D3-phosphoinositides within a T cell. Moreover, it has been discovered that inhibition of the activity of phosphatidylinositol 3-kinase in a T cell can inhibit T cell responses, such as lymphokine production and cellular proliferation. These discoveries indicate a functional role for D3-phosphoinositides in a costimulatory signal transduction pathway and provide phosphatidylinositol 3-kinase as an intracellular target for modulation of T cell responses. Accordingly, intracellular signalling events involving D3-phosphoinositides can be modulated either to inhibit a costimulatory signal and thereby induce T cell unresponsiveness, or to trigger a costimulatory signal and thereby generate a T cell response. In addition, novel screening assays for identifying inhibitors or activators of phosphatidylinositol 3-kinase, which can be used to inhibit or stimulate a T cell response, are within the scope of the invention.
One aspect of the invention pertains to methods for inhibiting a response by a T cell which expresses a surface receptor that binds a costimulatory molecule. These methods involve contacting the T cell with an agent which inhibits production of D3-phosphoinositides in the T cell. In one embodiment, the agent is an inhibitor of a phosphatidylinositol 3-kinase, such as the fungal metabolite wortmannin or the bioflavenoid quercetin, or derivatives or analogues thereof (e.g. LY294002). In another embodiment of the method of the invention, the T cell is contacted with at least one additional agent which inhibits a different intracellular signal associated with costimulation, such as protein tyrosine phosphorylation. For example, the T cell can be contacted both with an inhibitor of phosphatidylinositol 3-kinase and with an inhibitor of a protein tyrosine kinase. A preferred inhibitor of a protein tyrosine kinase is herbimycin A. Alternatively, protein tyrosine phosphorylation can be inhibited in a T cell by a tyrosine phosphatase or an activator of a tyrosine phosphatase. In this embodiment, the T cell can be contacted with an inhibitor of phosphatidylinositol 3-kinase and with a molecule, e.g., an antibody, which binds to and activates a cellular tyrosine phosphatase, such as CD45 or Hcph.
The invention also provides methods for inducing unresponsiveness to an antigen in a T cell by triggering a primary, antigen-specific signal in a T cell while interfering with an intracellular signal associated with costimulation in the T cell. As a result of interfering with costimulatory signal transduction, the T cell fails to receive a proper costimulatory signal in the presence of the antigen and antigen-specific unresponsiveness is induced in the T cell. To induce T cell unresponsiveness, an antigen-specific T cell is contacted with the antigen in a form suitable for stimulation of a primary activation signal in the T cell, together with an agent which inhibits production of D-3 phosphoinositides in the T cell. For example, a T cell can be contacted with an antigen presented by an APC together with an inhibitor of phosphatidylinositol 3-kinase, such as wortmannin or quercetin or derivatives or analogues thereof (e.g. LY294002). Additionally, other intracellular signals associated with costimulation, such as protein tyrosine phosphorylation
Lahive & Cockfield LLP
Mandragouras Esq. Amy E.
Smith, Esq. DeAnn F.
The United States of America as represented by the Secretary of
Wehbe Anne M.
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