Development of next generation multi-antigen targeting off-the-shelf CAR T cells for conditioning-free treatment of B-cell lymphoma

Abstract Autologous chimeric antigen receptor (CAR) T-cell therapy has had tremendous success in the treatment of hematological malignancies, yet its clinical application remains hindered by several significant limitations. Major challenges include the high cost, complex manufacturing process, the requirement for intense lymphodepleting chemotherapy prior to infusion, and the limited accessibility and scalability of the therapy. FT839 is an induced pluripotent stem cell (iPSC) derived CAR T cell designed to overcome these limitations and provide potent and versatile therapy against lymphocytic cancers. FT839 is a multiplex-engineered CAR T cell equipped with anti-CD19 and anti-CD38 dual-CAR system to target lineage specific or activated pathological cell subsets, including cells of hematologic malignancies. FT839 also incorporates Sword and ShieldTM technology, the synergistic action of a novel Alloimmune Defense Receptor (ADR), which eliminates 4-1BB+ alloreactive immune cells, and the genetic ablation of CD58 (CD58KO), which limits synapse formation with alloreactive cells to promote functional persistence and evade host alloreactive immune responses, designed to eliminate the need for intensive conditioning chemotherapy. Co-targeting of CD19 and CD38 on B cell acute lymphoblastic leukemia cell line NALM6, both in combination or individually through engineered isogenic lines (CD19+CD38+, CD19-CD38+ and CD19+CD38-) to represent cancer heterogeneity, showed enhanced and durable efficacy (91.6%, 92.8%, & 82.6% vs 52.8%, 0%, & 50%) respectively, when comparing FT839 and CD19 primary CAR T cells at low effector: target ratio. FT839 CAR T cells are also uniquely engineered to express a novel CD3-chimeric fusion receptor (CD3CFR) and a high-affinity, non-cleavable CD16 (hnCD16). These engineered attributes allow for flexible multi-antigen targeting in combination with clinically approved T cell engagers (TCEs) or monoclonal antibodies (mAbs). Indeed, in combination with the CD20-specific mAb rituximab or the CD38-specific mAb daratumumb, FT839 potently eliminated the mantle cell lymphoma cell line Jeko-1 (97.8% CD20+, 54,888 rMFI) and Burkitt's lymphoma cell line RAJI (91.6% CD38+, 101,129 rMFI), respectively, highlighting the flexible and broad multi-antigen targeting of FT839 via CAR and hnCD16 activation. FT839 also demonstrated potent control of NALM-6 xenografts in vivo, as a monotherapy, and with further deepening response in combination with mAb, compared to control. With Sword and ShieldTM engineering (ADR & CD58 KO), FT839 demonstrated resistance to potent pre-existing as well as de-novo generated host vs graft allogeneic immune responses, maintaining functional anti-tumor activity. In a 2-way MLR with HLA-mismatched donor peripheral blood mononuclear cells (PBMCs), FT839 demonstrated enhanced persistence (21x, p<0001) and reduced alloreactive expansion (4x, p<0.0005), compared to control CAR-T cells. FT839 also showed higher resistance (113% of base wells) to pre-existing allogeneic responses (primed allogeneic T cells), as compared to CD58-sufficient, ADR-negative CAR-T cells (2.4% of base wells). Finally, FT839 maintained durable anti-tumor activity against repeat challenges with NALM6 tumor cells 1.17x p=ns) even in the presence of primed allogeneic PBMCs that elicit potent reaction against mismatched cells. Compared to traditional CAR T-cells, FT839 demonstrates versatility in targeting cancer cells via multiple antigen-receptor activation pathways, enabling potent and flexible multi-antigen targeting for the successful treatment of relapsed/refractory B cell lymphomas that is otherwise challenging to treat because of its heterogenous cellular composition. Furthermore, unlike autologous and allogeneic CAR T cells, expression of ADR and deletion of CD58 ensures the functional persistence of FT839, potentially eliminating the need for intense conditioning chemotherapy regimen and ensuring broad and on-demand access for patients with relapsed/refractory B-cell leukemia/lymphoma.