In the past ten years, various strategies that use bispecific antibodies to redirect the activity to T cells against tumor cells have been developed. Despite the advance of several of these agents into clinical trials, there remain significant problems with the bispecific antibody approach. Chief among these problems has been the inability to sustain an active T cell infiltrate at the site of solid tumors. The overall goal of the proposed experiments is to sustain T cell activity at the site of the tumor and redirect the efficient lysis of tumor cells. Bispecific agents that target T cells to the high affinity folate receptor (FR), found on most ovarian carcinomas and some brain tumors, can be produced easily by attaching folate to any anti-T cell antibody of interest. This method will allow evaluation of the tumor-dependent T cell activating potential of various conjugates, including folate conjugates of antibodies to the T cell receptor/CD3 complex, CD28, and LFA-1. Multivalent ligation of these molecules on the surface of T cells stimulates full activation of T cells under normal physiological conditions. In contrast, ligation of the T cell molecule CTLA4 with its ligand B7 leads to T cell inactivation. Inhibition of the CTLA4:B7 interaction can sustain the activity of T cells. Based on these rationales the various folate/anti-T cell antibody conjugates will be tested alone and in combination with monovalent forms (scFv and Fab) of an anti-CTLA4 antibody.
In Specific Aim 1, in vitro assays will measure T cell proliferation and cytotoxicity after incubation of T cells with folate/anti-TCR conjugates, anti-CTLA4 antibody fragments and FR tumor cells.
In Specific Aim 2, mice treated in vivo with various antibody agents will be examined ex vivo for cytotoxicity by splenic T cells or peritoneal exudate cells and by immunohistochemical analysis of T cell infiltrates within tumors. The goal of this aim is to identify agents that optimally maintain activated T cells within the tumor.
In Specific Aim 3, antibodies and/or folate-conjugates will be tested for their ability to eliminate FR+ tumors in three murine model systems: (a) immunologically defined 2C TCR/RAG mice transplanted with a human FR+ tumor; (b) immunocompetent mice transplanted with a syngeneic FR+ tumor; and (c) immunocompetent transgenic mice that develop endogenous FR+ brain tumors. Endpoints will include survival, tumor progression, and testing for tumor-specific memory by re-transplantation of the appropriate tumor.
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| Orr, Brent A; Carr, Lori M; Wittrup, K Dane et al. (2003) Rapid method for measuring ScFv thermal stability by yeast surface display. Biotechnol Prog 19:631-8 |
| Roy, E J; Gawlick, U; Orr, B A et al. (2000) IL-12 treatment of endogenously arising murine brain tumors. J Immunol 165:7293-9 |
