Abstract 2759: CAR-T engager proteins for the treatment of B cell cancers and acute myeloid leukemia

Abstract

Abstract Chimeric Antigen Receptor T cells targeting malignancies expressing CD19 (CAR19) have been widely successful, with products approved to treat B cell lymphomas (NHL) and B cell leukemia. A major limitation of CAR19 therapy is the steep relapse rate within 6 months of treatment, often due to the loss or diminution of tumor cell CD19 expression. ALETA-001 is a CAR-T Engager protein that contains the CD19 extracellular domain (ECD), an anti-CD20 VHH, and an anti-albumin VHH for half-life extension. When combined with CAR19 T cells, ALETA-001 triggers cytotoxicity through CD19 bound to CD20, thus increasing total target antigen density and preventing relapse due to loss of CD19 expression. ALETA-001 will enter Cancer Research UK-sponsored Phase 1/2 clinical trials in CAR19-treated NHL patients next year. We made novel variants of ALETA-001 including CAR-T Engagers with alternative anti-CD20 modules and CAR-T Engagers that target both CD20 and a second B cell cancer antigen. Further, we extended our technology to target Acute Myeloid Leukemia (AML), using novel antigen binding domains and immunomodulatory functional domains. Site-directed mutagenesis was used to make ALETA-001 variants. We mutated the complementary determining regions, CDR2 and/or CDR3, within the anti-CD20 VHH. The novel CAR-T Engager Proteins were evaluated for binding to CD19-negative/CD20-positive lymphoma cells and for cytotoxicity against those cells in the presence of CAR19 T cells. Further, CAR-T Engagers were made that contained novel CD20 binding domains and binding domains to other B cell antigens. In addition, we created a novel CAR-T engager that contains the CD19 ECD and binding domains to two AML antigens, plus an immunomodulatory domain. This novel CAR-T engager is designed to treat CD19-positive Mixed Phenotype Leukemia (MPL) and other forms of AML. We evaluated the series of CDR mutations in the ALETA-001 anti-CD20 VHH. A spectrum of activities was observed, with mutations that retained full CD20 binding and cytotoxic activity to those with partial or no activity. A series of constructs with distinct anti-CD20 binding domains were also evaluated, with some found to be equipotent with ALETA-001. Constructs binding both CD20 and a second B cell tumor antigen were prepared and assayed and found to have potent activity against either or both antigens. Finally, CAR-T Engagers directed to AML antigens were able to bind each antigen individually, and mediated potent toxicity with the addition of CAR19 T cells. We conclude that the CAR-T Engager platform offers robust and modular anti-tumor functionality, featuring potent multi-antigen targeting for diverse indications. Finally, we can incorporate other useful immunomodulatory activities into Engager proteins. Citation Format: Paul D. Rennert, Lihe Su, Lan Wu, Roy R. Lobb, Christine Ambrose. CAR-T engager proteins for the treatment of B cell cancers and acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2759.