Abstract 580: MM3 CAR T: A novel tumor targeting and T cell redirection platform for multiple myeloma
Authors: McCachren Samuel S. , Manalo Julia , Pendleton Katherine , Ballestas Mary E. , Dhodapkar Kavita M. , Kemp Melissa L. , Cooper Max D. , Dhodapkar Madhav V. 
Published: June 2022

Abstract
Novel strategies to target tumors are needed to enhance the efficacy of immune redirection therapies. The detection of tumors based on mammalian immunoglobulin scFvs has been a significant innovation in immunotherapy but is impacted by limits of immune tolerance and immune system evolution. The discovery of unique variable lymphocyte receptors (VLRs) - analogues to antibodies in evolutionarily distant jawless vertebrates - in the Cooper lab (Pancer, Nature) provides an opportunity to target antigens beyond those recognized by conventional antibodies. A particular VLR, termed MM3, was found to identify a distinct epitope specific to human plasma cells (PCs) and plasmablasts (PBs), created by CD38 dimerization with activity as an NAD+ glycohydrolase (Yu, JCI Insight). CD38 is an extensively explored target for treating multiple myeloma (MM); however, specific scFv-based targeting of CD38 on malignant plasma cells is difficult, as the protein is expressed on a variety of other immune cell lineages. Therefore, MM3 provides a unique, novel mechanism to target CD38 in a tumor-specific manner in MM and other plasma cell dyscrasias. Here, we present a CAR T cell with an MM3 binding domain replacing the standard scFv. Our CAR construct consists of the MM3 binding domain, a CD8 hinge and transmembrane domain, and 4-1BB and CD3zeta intracellular domains. Staining of MM patient bone marrow specimens revealed that the strong specificity for PCs and PBs previously observed in healthy donors was maintained in MM bone marrow, illustrating the ability of MM3 to target the malignant PC population. To assess in vitro function of the MM3 CAR T cells, flow cytometry analysis of co-cultures using healthy donor-derived MM3 CAR T cells and target cell lines revealed specific increased degranulation (CD107a expression), activation (CD69 expression), and killing (target cell Annexin V binding) over untransduced control T cells only in cell lines sensitive to MM3 binding, with no differences in negative control cell lines, demonstrating specific recognition and killing of target cells. Preliminary in vivo studies in the MISTRG6 myeloma xenograft mouse model were performed using intrafemorally injected INA-6, an IL-6-dependent MM cell line, which MM3 binds after growth in vivo. After treatment with MM3 CAR T cells via intravenous injection, we observed that the MM3 CAR T cells specifically homed to and were retained in the injected femur along with the tumor cells, suggesting tumor recognition and potential for in vivo efficacy. In summary, these data demonstrate that VLR-based targeting may allow design of novel CAR constructs. Specifically, the VLR MM3 is a promising novel, non-scFv mechanism for targeting CD38 in a malignant PC-specific manner, and the MM3 CAR T cells have shown promising preliminary activity. Therefore, the VLR-based MM3 CAR T cell platform may provide a novel strategy to improve T cell redirection to target tumors in MM.
Citation Format: Samuel S. McCachren, Julia Manalo, Katherine Pendleton, Mary E. Ballestas, Kavita M. Dhodapkar, Melissa L. Kemp, Max D. Cooper, Madhav V. Dhodapkar. MM3 CAR T: A novel tumor targeting and T cell redirection platform for multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 580.