HLA-Pan-DP CAR-T Demonstrates Cytotoxicity Against Multiple Hematological Malignancies
Authors: Takamatsu Hiroyuki , Ramos Emilio , Patino-Escobar Bonell , Kasap Corynn , Izgutdina Adila , Werner Juwita , Inagaki Akihito , Yoroidaka Takeshi , Salangsang Fernando , Phojanakong Paul , Camara Serrano Juan Antonio , Steri Veronica , Stieglitz Elliot , Nagato Masaki , Tatsuno Satoshi , Wiita Arun P 
Published: December 2024

Background: Relapse of hematological malignancies often originates from residual cancer and/or its initiating cells. Target antigens on cells, such as CD19 and BCMA, are often downregulated after immunotherapy (Orlando et al., Nature Medicine 2018; Dhodapkar et al., Blood Cancer Discovery 2022). In this study, we explored anti-HLA-pan-DP, which is expressed across a wide range of B-cell lineages from immature to mature cells, as a target of immunotherapy. HLA-DPB1 allele-mismatched allogeneic hematopoietic stem cell transplantations have shown longer survival, suggesting a favorable graft-versus-leukemia effect and a low risk of GvHD. Therefore, anti-HLA-DP immunotherapy might be tolerable from the viewpoint of adverse effects derived from cytotoxicity against normal cells.

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 &lt; 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 &lt; 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&lt;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.