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Context Target identification for CAR-T cell therapies, especially shared targets among different malignancies, remains challenging due to the limited repertoire of tumor-specific surface proteins. Intracellular proteins presented in the context of cell surface HLA provide a wide pool of potential antigens targetable through TCR-mimic antibodies. Objective and Design We hypothesized that mass spectrometry (MS)-based analysis of the presented HLA ligands of multiple cancer cell lines can be utilized to identify a shared, tumor-associated HLA ligand, which could then be targeted by TCR-mimic CAR-T cells specific for this particular HLA ligand. Results MS of HLA ligands from eight hematological and non-hematological cancer cell lines identified a shared, non-immunogenic, HLA-A*02 restricted ligand (ALNEQIARL) derived from the kinetochore-associated NDC80 gene, which was later identified in >90% (20/22) of all A*02-positive cell lines tested. A TCR-mimic scFv was prepared against this epitope, termed “NDC80-C.” CAR-T cells transduced with the NDC80-C, directed against the ALNEQIARL:HLA-A*02 complex on cancer cells, demonstrated high sensitivity and specificity for recognizing and killing multiple cancer types with a high preference for hematological malignancies (e.g., AML, ALL, DLBCL, and ALCL). In contrast, healthy leukocytes, activated B and T-cells, or hematopoietic stem cells from A*02-positive and -negative donors were not lysed. NDC80-C CAR-T cells suppressed colony formation of primary AML cells but not of healthy stem cells. Additionally, NDC80-C CAR-T cells were efficacious in mouse models against human mesothelioma and leukemia. Conclusions Our study demonstrates how MS can inform the design of tumor-agnostic TCR-mimic therapeutic platforms that target structures that are currently not druggable by small molecules, conventional CAR-T cells, T-cells, or antibodies. This strategy lays the groundwork for a potential antibody platform therapy or CAR-T cell with efficacy against highly proliferative A*02-positive cancer cells, independent of the respective cancer type.

Introduction: The Wilms' tumor 1 (WT1) protein is a tumor associated antigen that is potentially targetable by immunotherapeutic approaches. We have demonstrated the overexpression of WT1 in myeloma cells by IHC and in HLA-A*0201+ pts by staining with a high-affinity fully human IgG1 mAb (ESK1) specific to the RMFPNAPYL/HLA-A*0201 complex on malignant plasma cells. We report initial results from pts with plasma cell leukemia (PCL) or relapsed/refractory multiple myeloma (rMM) who have been treated with CD34-selected allo transplants followed by the administration of donor-derived WT1-specific T-cell infusions to induce an immunotherapeutic effect. Methods: In situ expression of WT1 was assessed by IHC analyses using a sequential double staining technique of MoAbs specific for CD138 and WT1.For staining with the RMFPNAPYL/HLA-A*0201 complex, BM samples were blocked with human FcR Blocking Reagent and then directly stained with MoAbs specific for CD38, CD56, CD45 and ESK1 or its isotype control human IgG1 and were analyzed by flow cytometry. WT1-specific T cells were generated from the original stem cell donors by sensitization of CD3+ enriched T-cell fractions with autologous APCs loaded with the pool of overlapping pentadecapeptides of WT1 (Invitrogen, Boston, MA). Cells were propagated in vitro with weekly restimulation and supplementation with IL-2 beginning at day 10-16. After 35-49 days, T-cells were harvested, counted and tested for antigen specific cytotoxicity, HLA-restriction, lack of alloreactivity and sterility. Pts received CD34-selected PBSC allografts after myeloablative cytoreduction with busulfan, melphalan and fludarabine. Pts were treated with 3 infusions of donor-derived WT1-specific T-cell infusions (5x10e6 cells/kg) starting 6 weeks post allo HSCT and at 4 weekly thereafter. Results: Marrow from all pts with immunohistochemical documented plasma cell involvement stained positive for WT1 IHC while WT1 staining remained negative in pts in CR. Only pts expressing HLA-A*0201 that stained positively for WT1 by IHC also demonstrated expression of WT1 by the RMFPNAPYL/HLA-A*0201 complex, whereas pts lacking HLA-A*0201 but with active disease stained positive for WT1 IHC but not ESK1 staining. Of 7 pts, 3 PCL and 4 rMM, treated with WT1-specific T cells, 4 pts had persistent disease post CD34-selected allotransplant. Of these 4 pts 2 pts developed a striking rise of WT1-specific T-cell frequencies and developed a complete remission post WT1 CTL infusions lasting for >2years. Conclusion: WT1 is overexpressed on malignant plasma cells and serves as a target for potential immunotherapeutic approaches in pts with multiple myeloma. Pts with persistent PCL following CD34-selected allografts treated with adoptive transfer of donor-derived WT1-specific cytotoxic T cells can achieve long lasting remission underscoring the therapeutic potential of T-cells specific for immunogenic WT1 peptides expressed on malignant plasma cells. Disclosures O'Reilly: Atara Biotherapeutics: Research Funding.