Example 1
Anti-MICA CAR was constructed by fusing the B2 scFv to human CD28 hinge-transmembrane-cytoplasmic domains (residues 135-220) and CD3 ζ cytoplasmic domain (residues 52-164). The anti-MICA construct was then cloned into a retroviral vector pFB-neo (Stratagene, Palo Alto, CA), and expressed in T cells.
The cells were analyzed for expression of the MICA CAR through a B3Z Assay. B3Z assays were performed according to known methods (Shastri & Gonzalez (1993) J. Immunol. 150:2724-36). B3Z (B3xZ.8) is a CD8+ T cell hybridoma that expresses LacZ in response to activation of T cell receptors specific for the SIINFEKL peptide (SEQ ID NO:70) (OVA-immunodominant peptide) in the context of H-2Kb MHC class I molecules. CAR signaling via CD3ζ (CD3-zeta) will induce LacZ expression in a similar manner. Briefly, 105 B3Z or MICA-specific CAR-transduced B3Z cells at ratios of 10:1, 5:1 and 1:1 E:T (effector (B3Z cell):target (tumor cells) were co-cultured in flat-bottom 96-well plates with ID8-GFP, ID8-GFP-MICA, P815 or P815-MICA tumor cells for 24 hours (FIGS. 2A and 2B). P815 is a murine mastocytoma cell line, H-2d haplotype. ID8 is a mouse ovarian carcinoma cell line. ID8-GFP is a ID8 cell line transduced with the reporter green fluorescent protein (GFP)-expressing lentiviral particles. Both cells were engineered to express human MICA. The plates were spun, and the cell pellets were lysed and incubated with CPRG for detection of LacZ activity. Absorbance at 595 nm was measured by using an enzyme-linked immunosorbent assay plate reader after 6 hours.
The results of this analysis, presented in FIGS. 2A-2B, indicated that the anti-MICA CAR (“B2”) was functional as it induced CAR-mediated activation in the presence of MICA on the target cells (“ID8-GFP-MICA” and “P815-MICA”). The B3Z cells alone, which were included in each assay and do not express a MICA binding CAR, did not respond tumor cells when MICA was not present. That is, incubation of the B3Z T cells and tumor cells (either ID8 or P815) alone yielded no signaling activation, as evidenced by there being little or no detectable signal from the LacZ reporter, even in the presence of MICA-expressing tumor cells (see “B3Z+ID8-GFP”, “B3Z+ID8-GFP-MICA” samples in FIG. 2A, and “B3Z+P815”, and “B3Z+P815-MICA” samples in FIG. 2B). While the sample “B3Z+ID8-GFP-MICA” produced some TCR activation, this was at the highest ratio of E:T (1:1). Likewise, incubation of B3Z T cells with MICA CAR cells in the presence of tumor cells alone did not yield an appreciable TCR activation signal (“B2+ID8-GFP” in FIG. 2A, and “B2+P815” in FIG. 2B). In contrast, the presence of MICA CAR cells (“B2”) clearly amplified the TCR activation at all E:T ratios (“B2+ID8-GFP-MICA” in FIG. 2A and “B2+P815-MICA” in FIG. 2B).
