Session: 703. Adoptive Immunotherapy: Mechanisms and New Approaches: Poster II
Hematology Disease Topics & Pathways:
multiple myeloma, Biological, apheresis, Adult, Diseases, Therapies, CAR-Ts, Adverse Events, Biological Processes, white blood cells, Technology and Procedures, cell expansion, immune cells, Plasma Cell Disorders, immunotherapy, Cell Lineage, Xenograft models, Lymphoid Malignancies, Study Population, Clinically relevant, immune mechanism, flow cytometry, microenvironment
We aimed to develop a fully-human BCMA CAR with long-term persistence and functional resistance to the suppressive effects of TGF-β. Initially, two fully human single chain variable fragments (scFv) specific for BCMA, derived by biopanning of a yeast display human scFv library, were characterized in a CAR format. Each of the two scFvs was cloned into a lentiviral vector CAR backbone, comprised of the CD8 hinge and transmembrane domain, 4-1BB co-stimulatory domain and CD3ζ signaling domain, and termed BCMA1 and BCMA2, respectively. BCMA2 and BCMA1 T cells, generated by lentiviral vector transduction of primary human T cells, exhibited high CAR expression at multiplicities of infection 10 to 40 (BCMA1: 64-81%; BCMA2: 84-94%), and consistently demonstrated potent cytotoxicity in an overnight co-culture with BCMA+ MM cell lines RPMI-8226 and MM1.S, but not the BCMA- 293T cells. BCMA2 CAR showed robust proliferative capacity in response to repeated, long-term exposure to MM1.S target cells, concordant with the sustained CD4 T-cell subset and high IL-2 production during the course of repeated exposure to target cells. This is in contrast to BCMA1, that showed precipitous decrease in CD4+ T-cell subset upon co-culture with MM cells, resulting in a CAR T-cell population dominated by CD8+ T-cells. Furthermore, the BCMA2 demonstrated prolonged potency in clearing tumor cells compared to BCMA1, even after continuous 20-day exposure to target cells. This was further confirmed in a mouse intradermal RPMI-8226 xenograft tumor model, in which infusion of BCMA2 T-cells resulted in the rapid and complete eradication of tumors, while BCMA1 showed a slower decline in tumor burden.
To further improve the efficacy of the BCMA2, specifically within the TGF-β-rich immunosuppressive tumor microenvironment, we developed an armored BCMA2 CAR variant, which co-expresses the dominant negative TGF-β RII bicistronically via a 2A sequence (BCMA2-TbnegCAR). In a 10 day-long co-culture assay with MM1.S targets in the presence of spiked 10 ng/ml TGF-β, BCMA2-TbnegCAR retained high proliferative capacity and potent cytotoxicity, while the unarmored BCMA2 had diminished proliferation and substantially reduced cytokine and granzyme B production.
Consistently, in the in vivo intradermal tumor model that utilizes RPMI-8226, which is a MM cell line that endogenously produces TGF-β, we observed that BCMA2-TbnegCAR treatment resulted in higher T-cell counts in the tumors and an earlier decrease of tumor burden compared to infusion with BCMA2, further demonstrating the increased efficacy and potency of the BCMA2-TbnegCAR.
In conclusion, we have designed and characterized a new fully-human BCMA2-TbnegCAR, with a novel scFv that exhibits robust proliferative capacity and persistent cytotoxicity, and remains functionally resistant to the immunosuppressive effects of TGF-β. This novel BCMA CAR can potentially improve the effectiveness and durability of the current BCMA CAR-T cell therapy.
Disclosures: Alabanza: Lentigen, a Miltenyi Biotec Company: Current Employment, Patents & Royalties: CAR-T immunotherapy. Vu: Lentigen, a Miltenyi Biotec Company: Current Employment, Patents & Royalties: CAR-T immunotherapy. Wu: Lentigen, a Miltenyi Biotec Company: Current Employment. Zhu: Lentigen, a Miltenyi Biotec Company: Current Employment, Patents & Royalties: CAR-T immunotherapy. Dropulic: Lentigen, a Miltenyi Biotec Company: Current Employment, Patents & Royalties: CAR-T immunotherapy. Schneider: Lentigen, a Miltenyi Biotec Company: Current Employment, Patents & Royalties.
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