193. Efficacy and Safety of Immunotherapy with Chimeric Antigen Receptor Targeting WT1 and HLA-A24:02 pMHC Complex

Abstract

Recent success in the treatment of patients with B cell malignancy by CD19-CAR encourages the development of successive CAR therapy targeting broad range of tumor-associated antigens. However, the search for the appropriate target molecule for CAR, other than B cell markers, has not met the needs. The molecules recognized by CAR is generally limited to the cellular surface molecules, making difficult the search for the tumor-specific targets. Inspired by the physiological recognition of epitope peptide and MHC molecule (pMHC) by T cells, we have generated a series of antibodies that recognize the pMHC complexes with peptides derived from tumor antigens expressed intracellularly. Screening a phage display library of human antibody scFv, we isolated an scFv antibody clone “WT#213” that can specifically recognize WT1 p235-243 peptide (CMTWNQMNL) complexed with HLA-A24:02 molecule. We have constructed a retroviral vector that encodes the CAR consists of WT#213 and intracellular signal transduction domains of CD3ζ and GITR (WT#213 CAR). We confirmed the specific recognition of endogenous WT1-expressing cells by the CAR-T cells with the intracellular cytokine staining and the 51Cr release cytotoxic assay. Moreover, we demonstrated the effectiveness of adoptive cell therapy with WT#213 CAR against the WT1 expressing HLA-A24:02 positive human leukemia cells, utilizing NOG immunodeficient mice. To evaluate the safety of the WT#213 CAR, we predicted the potential property of WT#213 CAR to cross-react to normal tissues in humans. We conducted alanine scan analysis of WT1p235-243 peptide that was recognized by WT#213 CAR as well as the TCR derived from CTL clone TAK-1 which recognizes same epitope peptide in association with HLA-A24:02 to define the amino acids that were critically important in the recognition by the WT#213 CAR or TAK-1-derived TCR. After BLAST search, we synthesized the normal protein-derived peptides with potential risk of cross-reactivity, and tested the recognition of these peptides by WT#213 CAR or TAK-1-derived TCR. Although the critical peptides, and therefore the peptides with potential risk, were quite different between the WT#213 CAR and TAK-1-derived TCR, none of these peptides showed the stimulation of WT#213 CAR or TAK-1-derived TCR. The results here suggest that the immunotherapy with WT#213 CAR will be effective for the treatment of the leukemia patients without the predicted risk in the evaluation we performed.View Large Image | Download PowerPoint Slide