Type: Oral
Session: 702. CAR-T Cell Therapies: Basic and Translational: Novel Targets and Therapeutic Approaches for CAR-T Cells
Hematology Disease Topics & Pathways:
Research, Fundamental Science, Translational Research, Lymphomas, Non-Hodgkin lymphoma, B Cell lymphoma, Plasma Cell Disorders, Diseases, Lymphoid Malignancies
Methods: We obtained a hybridoma cell line (clone B7/21) producing anti-HLA-pan-DP monoclonal antibody (mAb) and identified the CDR sequences of the mAb's VH and VL chains. We inserted these into an IgG4 (EQ) backbone plasmid to create an expression vector (Mandal et al., Nature Cancer 2023). Lentiviral transduction of primary human T cells from a healthy donor created CAR-T cells. In vitro cytotoxic assays used various lymphoma, multiple myeloma (MM), and blastic plasmacytoid dendritic cell neoplasm (BPDCN) cell lines, as well as primary lymphoma cells. An in vivo mouse experiment was performed using NSG mice.
Results: Analyzing CoMMpass mRNA data (release IA19), we evaluated HLA-DP expression of myeloma cells derived from patients. Surprisingly, HLA-DP was significantly higher at relapse compared to diagnosis (P < 0.0001), as well as in t(4;14) positive cases (P=0.007), but not in del-17p and 1q-gain. HLA-DP CARs were efficacious in in vitro cytotoxicity assays targeting lymphoma cell lines such as JeKo-1, Namalwa, and Toledo, the MM cell line JJN-3, and the BPDCN cell line PMDCN05, depending on the level of HLA-DP expression. The in vitro cytotoxicity of HLA-DP CARs was comparable to CD19 CARs against JeKo-1 cells, similar to BCMA CARs against JJN-3 cells, and akin to CD123 CARs against PMDCN05 cells. HLA-DP and CD19 CARs killed 75% and 100% of JeKo-1 at 0.5:1 and 1:1 effector:tumor (E:T) ratio, respectively. Similarly, HLA-DP and BCMA CARs killed 90% and 100% of JJN-3 at 0.5:1 and 1:1 E:T ratio, respectively. We did not detect cytotoxicity of HLA-DP CARs against HLA-DP KO JeKo-1 and JJN-3, demonstrating specificity. Additionally, cytotoxicity against 8 primary B-cell lymphomas derived from lymph nodes (2 follicular lymphomas (FL), 3 diffuse large B-cell lymphomas (DLBCL), 2 mantle cell lymphomas (MCL), and 1 Burkitt lymphoma (BL)) that expressed HLA-DP was assessed. We observed complete cytotoxicity versus FL and DLBCL, but not MCL and BL. Finally, we assessed the cytotoxicity against JeKo-1 cells using an in vivo mouse model. HLA-pan-DP CARs showed a significantly longer survival benefit compared to empty CARs and no treatment (P < 0.05) and no difference to CD19 CARs.
Since HLA-DP is reported to be expressed on normal hematological cells such as B-cells, monocytes, macrophages, dendritic cells, as well as Schwann cells and endothelial cells, we assessed the cytotoxicity of HLA-pan-DP CARs against these cells. HLA-DP CARs killed B-cells, monocytes, macrophages, and dendritic cells, but not T-cells, NK-cells, and neutrophils. Using an xCelligence-based assay for alterations in target cell impedance upon loss of viability, no cytotoxic effects were found against Schwann cells (HE193, ipn02.3) and endothelial cells (HUVECs). We also assessed the influence of HLA-pan-DP CARs on hematopoiesis by colony-forming assays using methylcellulose-based medium. HLA-pan-DP CARs significantly reduced the number of colonies in CFU-M compared to empty CARs (P<0.001) but not in BFU-E, indicating a negative impact on B-cell and monocytic lineage hematopoiesis.
Conclusion: Anti-HLA-pan-DP CAR-T is a promising candidate for further preclinical development for the treatment of various HLA-DP positive hematological malignancies such as B-cell lymphomas, MM, and particularly BPDCN, for which there are no effective treatments except for CD123 targeted immunotherapy (SL-401). Due to expected suppression of normal hematopoiesis by HLA-pan-DP CAR-T, this approach would likely be best-implemented as a bridge to hematopoietic stem cell transplantation.
Disclosures: Takamatsu: Adaptive Biotechnologies: Consultancy; Sanofi: Honoraria; Bristol-Myers Squibb: Honoraria, Research Funding; SRL: Consultancy; Janssen: Honoraria; Ono: Honoraria. Inagaki: GenVivo, Inc.: Current Employment. Nagato: Wakunaga Pharmaceutical: Current Employment. Tatsuno: Wakunaga Pharmaceutical: Current Employment. Wiita: Indapta Therapeutics, LLC: Current equity holder in private company; Sanofi: Honoraria; Protocol Intelligence, LLC: Current equity holder in private company.
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