Chimeric antigen receptor (CAR)-T cells directed against B-cell maturation antigen (BCMA), have yielded impressive results in clinical trials for multiply relapsed/refractory multiple myeloma (MM). However, progression-free survival is short, demonstrating a need for improvements. While numerous advances have been made to improve this treatment modality, these have not always translated to superior clinical outcomes. A major limitation is that novel therapeutics are often not tested in a relevant in vivo model. These novel strategies have been largely tested only in immunodeficient models of MM. To better recapitulate the host-tumour-effector cell interactions, particularly in the TME, we sought to develop a system to study CAR-T cell activity in the Vk*MYC model of MM. Here, we report on the first known successful model of CAR-T cell activity in an immunocompetent model of MM.

We sought to identify a BCMA CAR construct that is capable of binding to both human and mouse BCMA. A novel nanobody recognizing BCMA was developed by immunizing llamas with human BCMA and the resulting single-domain antibodies (sdAbs) were subsequently isolated and cloned. These sdAbs were then screened for binding to mouse BCMA. A binder capable of recognizing both human and mouse BCMA was identified and cloned into our murine CAR construct, with CD28 and CD3z signaling domains, herein referred to as sdBCMA2.CD28.CD3z. Murine CAR-T cells were then generated by transducing activated healthy murine splenocytes with a retroviral vector carrying the sdBCMA2.CD28.CD3z construct. These CAR-T cells were then characterized in vitro using standard cytotoxicity, proliferation, and cytokine release assays. To assess activity in an immunocompetent model, female C57BL/6J mice were injected intravenously with Vk12598 Vk*MYC cells. CAR-T cells were later administered intravenously. Mice were bled weekly to quantify M-protein disease burden by serum protein electrophoresis and for phenotypic characterization of circulating cells.

When co-cultured with BCMA+ human MM cell lines and with Vk*MYC cell lines, murine sdBCMA2.CD28.CD3z CAR-T cells effectively lysed target cells, proliferated, and released inflammatory cytokines. To assess the in vivo activity of these CAR-T cells, we injected intravenously female C57BL/6J mice with Vk12598 cells, and then administered sdBCMA2.CD28.CD3z CAR-T cells intravenously. While there was some evidence of survival improvement, the median overall survival (mOS) of 35 days compared to 28 days, for CAR-T vs. Mock T-cells, respectively, the difference was non-significant, and the survival benefit was not durable. To address this, we incorporated lymphodepletion by total body irradiation (TBI) followed by CAR-T cell administration the next day. By incorporating lymphodepletion prior to CAR-T cell administration we were able to significantly reduce M-protein surrogates of disease burden, as well as achieve durable remissions in the TBI + CAR-T arm with an mOS not yet reached after experimental day 91, compared to an mOS of 42 days in the TBI + Mock T-cell arm. The addition of TBI also significantly improved the engraftment of CAR-T cells, as assessed by flow cytometry. The TBI + CAR-T arm also had significantly increased IL-6, IL-1b, IL-2, and TNF-a in the serum, compared to the TBI + Mock arm, at 7 days post administration of CAR-T cells.

The durable remission achieved using the combination of our novel sdBCMA2.CD28.CD3z CAR-T cells with lymphodepletion is a pivotal advancement in modelling MM CAR-T cell activity in this clinically relevant model system. Our approach represents the first time that CAR-T cells were successfully used to treat the Vk12598 model of MM that faithfully recapitulates relapsed MM. The incorporation of lymphodepletion significantly improves CAR-T cell engraftment, in line with clinical CAR-T cell therapy paradigms. The cytokine profile observed is consistent with the cytokine elevations typically observed after CAR-T cell administration in patients. As our construct is capable of binding to both human and mouse BCMA, we are therefore able to fully characterize our lead assets in a relevant model system. Our strategy will enable further research to dissect host-tumour-effector cell interactions, to better inform the design and characterization of novel CAR-T cell therapies in MM.

Bergsagel:Pfizer: Research Funding; Omeros: Consultancy; Radmetrix: Consultancy; Novartis: Patents & Royalties: Royalty for hCRBN mice; Janssen: Consultancy; CellCentric: Consultancy; Salarius: Consultancy; Mayo Clinic: Patents & Royalties: Royalty for hCRBN and Vk*MYC mice; Aptitude Health: Honoraria. Chesi:Novartis: Patents & Royalties: Licensing hCRBN mice; Pfizer: Patents & Royalties: licensing Vk*MYC lines, Research Funding. Hay:BMS: Honoraria; Janssen: Research Funding; Kite/Gilead: Honoraria; Novartis: Honoraria.

2023
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