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    Cellular and T cell engager Immunotherapy

    (OA-09) A Syngeneic Mouse Model of CAR-T Cell Therapy Containing a Translatable Dual Human/Mouse Reactive sdAb Binder in Vk*MYC Mice Reveals Clinically-Relevant Microenvironment Changes

    Thursday, September 18, 2025
    16:10 - 16:20 East Coast USA Time
    Location: Room 718
    Introduction: Despite the therapeutic success of approved BCMA CAR-T cell therapies in RRMM, post-CAR-T relapse is near-universal and this may be due to key host-tumour-effector cell interactions of the tumour microenvironment (TME) that are not modeled in widely used immunodeficient preclinical models of CAR-T cell therapy. We therefore sought to develop a system to study CAR-T cell activity using the immunocompetent syngeneic Vk*MYC model of MM.

    Methods: Llamas were immunized with human BCMA and the resulting single-domain antibodies (sdAbs) were isolated, cloned, and screened for binding to mouse BCMA. The resulting dual species-reactive sdAb was then incorporated into a CAR construct, with CD28 and CD3z domains (sdBCMA2.28z). Murine CAR-T cells were generated by retrovirally transducing activated splenocytes, and assessed by cytotoxicity, proliferation, and cytokine release assays. For the in vivo studies, we I.V. injected C57BL/6J mice with Vk12598 Vk*MYC tumour cells. Groups were balanced based on M-protein by SPEP. The day prior to infusion with either sdBCMA2.28z or mock-transduced T cells (Mock), mice were lymphodepleted with 5Gy total body irradiation. Mice were bled weekly to assess disease burden by M-protein. In landmark studies, mice were euthanized at CAR+3d for harvest of spleens (SPL), bone marrows (BM), and peripheral blood (PB) for flow cytometry.

    Results: In vitro, sdBCMA2.28z CAR-T effectively killed BCMA+ cells, and demonstrated robust proliferation and pro-inflammatory cytokine release following exposure to BCMA+ cells, but not BCMA- cells. In vivo, TBI+CAR mice had significantly improved survival compared to TBI+Mock. We observed expansion of sdBCMA2.28z CAR-T cells that were mostly CD8+ and with an activated phenotype (CD44+) in the SPL, BM, and PB. We saw significant increases in circulating serum IL-6, IL-1b, and TNFa following CAR-T cell infusion, but not with Mock T-cells. Three days following CAR-T infusion, we saw increased CAR+ T-cells in the BM and SPL, along with decreases in CD138+B220- Vk*MYC tumour cells. We also observed an increased frequency of M1-like (CD86+) macrophages, with a slight decrease in M2-like (CD206+) macrophages in the BM of TBI+CAR-treated mice. Untreated disease-bearing mice had higher proportions of circulating myeloid-derived suppressor cells (MDSCs), and TBI+CAR-treated mice had modestly decreased polymorphonuclear MDSCs (Ly6G+ among CD11b+Gr-1+) in the spleen.

    Conclusions:

    We show improvement in survival of Vk*MYC-engrafted mice treated with sdBCMA2.28z CAR-T cells compared to Mock. The cytokine profile observed is consistent with that observed after CAR-T cell administration in patients. Our studies of the TME provide a rationale for developing immunomodulatory strategies to make the TME more permissive to CAR-T cells. Our strategy enables the study of host-tumour-effector cell interactions, which will better inform the design of novel CAR-T cell therapies in MM.