B-Cell Maturation Antigen (BCMA)-Specific, Centyrin^TM-Based, PiggyBac^TM-Transposed CAR-T Memory Stem Cells Are Effective Against p53^-/- and Patient-Derived Multiple Myeloma Tumors

Introduction: Recent studies with chimeric antigen receptor (CAR)-T cells targeting B-cell maturation antigen (BCMA) have shown promise in treatment of patients with relapsed/refractory multiple myeloma (MM). However, current strategies are impeded by an excess of short-lived effector cells that contribute to poor durability and acute toxicity, host-immune responses against murine scFv CARs and low transduction efficiencies. We developed a novel CAR-T cell product, P-BCMA-101, which utilizes a non-immunoglobulin, fully-human alternative scaffold Centyrin™ molecule fused to a standard second-generation CAR molecule (a "CARTyrin") and manufactured with the non-viral piggyBac™ (PB) DNA transposon system. The PB transgene encodes three genes, an iCasp9-based safety switch for rapid elimination of the product if needed, an anti-BCMA CARTyrin, and a selection gene for manufacture of a nearly 100% pure CAR⁺ product. In contrast to CAR-T cells generated with conventional methods, including viral modification that results in product largely comprised of more differentiated T cell subsets, P-BCMA-101 is predominantly long-lived T-stem cell memory cells (60-80% T_SCM). Here, we report in vitro and in vivo results of P-BCMA-101 against MM cell lines (MM.1S, either WT or p53^-/- ("p53KO"), which models the p53 abnormality occurring in 30-50% of relapsed/refractory MM patients) and primary MM tumor cells, including plasma cell leukemias (PCL).

Methods: P-BCMA-101 functional activity was assessed in vitro against the MM.1S cell line and primary MM tumor cells obtained from MM patients using flow cytometry-based cytotoxicity, CFSE proliferation, and multiplex cytokine induction assays. The efficacy of P-BCMA-101 was also tested in vivo against WT MM.1S cells, p53KO MM.1S cells, and primary patient MM xenografts administered into human fetal bone and implanted in NSG mice.

Results: P-BCMA-101 exhibited potent anti-tumor activity in vitro as measured by specific lysis of MM.1S cells and primary MM tumor cells, as well as significant proliferation of both CD4⁺ and CD8⁺ P-BCMA-101 cells. Further, multiplex cytokine analysis of co-culture supernatants revealed a predominant release of IFN-γ and GM-CSF.

To assess efficacy in vivo, NSG mice were injected IV with luciferase⁺ WT or p53KO MM.1S cells. After 21 days, animals received a single IV dose of P-BCMA-101. All untreated animals exhibited rapid tumor growth accompanied by marked increase in serum M-protein levels and bioluminescent imaging (BLI) and quickly succumbed to disease. In contrast, 100% of WT MM.1S-challenged mice that received P-BCMA-101 rapidly eliminated tumors within 3-7 days as measured by BLI and all mice survived until study end at 60 days post-treatment. In 40% of mice, a relapse of tumor was observed by BLI, but was subsequently controlled without re-administration of P-BCMA-101. Similarly, while p53KO MM.1S tumors grew more aggressively than tumors derived from the parental WT MM.1S line, they were also controlled to low levels by P-BCMA-101 treatment in 4 of 5 of the mice. Interestingly, one animal maintained tumor at a stable and moderate level, similar to stable disease sometimes observed in human patients.

Lastly, to assess efficacy against primary MM tumor cells, CD138-purified cells from MM patient bone marrow or peripheral blood (PCL) were injected into the PDX model. Mice with measurable serum M-protein levels were treated with a single dose of P-BCMA-101. M-protein levels rapidly decreased to undetectable levels within 7 days in 100% of treated mice demonstrating rapid elimination of primary MM tumor cells residing within the human bone chip xenograft. Furthermore, these mice survived beyond 60 days post-treatment, showing P-BCMA-101 treatment results in durable remissions.

Conclusions: Results demonstrate that this first-in-class Centyrin-based, PB-transposed CAR-T memory stem cell therapeutic, P-BCMA-101, exhibited specific, potent, and durable anti-tumor activity against BCMA⁺ myeloma cells both in vitro and in vivo . P-BCMA-101 was also effective against p53KO MM.1S cells, a high-risk MM subtype occurring in a large percentage of patients who are resistant to all standards of care. These results support clinical investigation of P-BCMA-101 for the treatment of patients with relapsed/refractory MM, including the significantly large subset with tumors that have perturbations in the p53 pathway.