T Cells Expressing Cars Directed Against HLA-0201 –Rmf WT-1 Peptide Complex Can Effectively Eradicate WT1+ A0201+ Tumor Cells in-Vitro

Abstract

Adoptive transfer of ag specific T-cells can effectively eradicate viral infections and tumors. T-cells can be targeted to specific ags expressed on tumor cells by genetic modification to express chimeric ag receptors (CARs); which are fusion proteins incorporating an ag recognition moiety (ab) and T-cell activation domains. Human T-cells genetically modified to express a CD19-targeted CAR have been demonstrated to successfully eradicate CD19+ B cell malignancies in patients with refractory disease. The CARs developed thus far have been directed against native cell-surface antigens, such as CD19, that are recognized through antibody-derived antigen-binding motifs, independent of antigen processing or MHC-restricted presentation. However, tumor associated intracellular self ags such as WT-1 require processing following which antigenic epitopes are presented on the tumor cell surface in complex with HLA class-I or class-II. A strategy for targeting T-cells to specific peptide epitopes of such self ags expressed on tumor cells involves genetically introducing TCR mimic CARs containing a scFv ab directed against the specific peptide-MHC complexes. CARs containing a Fab directed against an HLA-A0201-NY-ESO-1 epitope have been generated, but functional data is lacking. We generated a TCR-like CAR containing a scFv specific for an HLA/peptide complex; the WT-1 RMFPNAPYL peptide presented by HLA-A0201 (A2-RMF CAR). The original A2-RMF ScFv, isolated using phage display technology, had a low binding affinity (Kd= 2.3 x 10-7 M). T-cells were transduced to express the A2-RMF CAR using MSCV retroviral vector. However, CAR transduced T-cells bearing the low affinity A2-RMF ScFv did not demonstrate any binding to the A2-RMF tetramers, and did not demonstrate any in-vitro cytotoxic activity against A2+/WT-1+ tumor cell targets. We subsequently developed an affinity matured ScFv (Kd= 3.08 x 10-9 M) to determine if ScFv binding affinity correlated with functional activity. T-cells transduced to express CARs bearing the affinity matured A2-RMF ScFv demonstrated strong binding to the A2-RMF tetramer in all transduced cells. The affinity matured A2-RMF CAR modified T-cells were able to kill A2+/WT-1+ solid tumor or B cell leukemia cell lines (29% -40% specific lysis) at E:T ratios of 50:1 and 20:1, in comparison to 1-4% specific lysis for HLA A2-/WT-1+ or A2+/WT-1- control tumor cell lines. These data demonstrate that a TCR-mimic CAR bearing scFv against an A2-RMF complex, can bind to A2-RMF tetramers and effectively lyse tumor cell targets co-expressing HLA A2 and WT1 in an HLA restricted and antigen specific manner. The results also suggest that the binding affinity of the antibody is important for effective functional activity of CAR modified T-cells directed against epitopes of self tumor associated antigens.