Abstract 1264: Mutated CD80 may facilitate T-cell activation by inhibiting PDL1-PD1 suppression and by costimulating.

Abstract

Abstract CD80 functions as a costimulatory signal to activate T cells when it binds to CD28 or as an apoptotic signal when it binds to CTLA4. We recently identified another function for CD80 and showed that co-expression of CD80 by Program Death Ligand-1+ (PDL1+) human tumor cells prevents PDL1 binding to its receptor PD1 on activated T cells, and enhances activation of tumor-reactive T cells. To distinguish if CD80 is facilitating T cell activation by preventing PDL1-PD1 interactions and/or by functioning as a costimulatory molecule, we have generated a recombinant CD80 molecule that does not bind to CD28. Since we hypothesize that CD80 may be a therapeutic for inhibiting tumor-mediated immune suppression via PDL1-PD1 interactions, the recombinant CD80 is also designed to not interact with CTLA4. Using site directed mutagenesis we generated CD80 with the following mutations: H96A, L97A, and E99A (CD8096,97,99). CD28 and CTLA4 bind to 35.3% and 37.4%, respectively, of tumor cells expressing wild type CD80. In contrast, only 1.2% and 1.4% of tumor cells expressing CD8096,97,99 bind CD28 and CTLA4, respectively, demonstrating that the mutant CD80 does not bind to CD28 or CTLA4. Previous studies demonstrated that CD80 prevents PDL1-PD1 interactions by binding to PDL1 and obscuring its detection. Only 7.05% of CD8096,97,99 mutants have detectable PDL1 (vs. 98% of untransfected parental human tumor cells), demonstrating that the CD8096,97,99 mutant does not bind CD28 or CTLA4, but retains the ability to inhibit PDL1-PD1 interactions. We are also generating a soluble form of CD8096,97,99 (sCD8096,97,99) that could be administered to cancer patients to prevent tumor cell-mediated PDL1 immune suppression. The construct for the soluble form was generated by ligating the two extracellular domains of CD80 containing the 96,97,99 mutations to the Fc region of human IgG1, followed by incorporation into the Pet21a+ plasmid containing the T7 promoter. Recombinant protein will be isolated from transformed E. coli using a protein G affinity column, and tested in functional experiments with PDL1+ tumor cells to determine if CD80 increases T-cell activation by inhibiting PDL1-PD-1 immune suppression and/or by facilitating costimulation. This work was funded, in part, by grants from the HHMI Precollege and Undergraduate Science Education Program (52006949), CBI (T32 GM066706), and NIH (R01CA84232 and RO1CA115880). Citation Format: Sonia P. Dalal, Samuel Haile, Preethi Somasundaram, Suzanne Ostrand-Rosenberg. Mutated CD80 may facilitate T-cell activation by inhibiting PDL1-PD1 suppression and by costimulating. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1264. doi:10.1158/1538-7445.AM2013-1264