Abstract 3201: CB-012, an allogeneic anti-CLL-1 CAR-T cell therapy engineered with next-generation CRISPR technology to resist both the immunosuppressive tumor microenvironment and immune cell-mediated rejection, for patients with relapsed or refractory acute myeloid leukemia
Authors: Garner Elizabeth , Kelly Erin , Namburi Sai , Colgan Cian , Fowler Tristan , Mutha Devin , Aviles Art , Stanaway Morena , Guo Raymond , An Zili , Degagne Emilie , Kwong George , Edwards Leslie , Jakes Emma , Shaw McKay , Schilling Benjamin , Huynh Jeremy , Luu Ricky , Sidorov Max , Mousali Rhonda , Otsmaa Mikk , Skoble Justin , Kanner Steven 
Published: April 2023

Abstract
Background: CB-012 is an allogeneic anti-CLL-1 CAR-T cell therapy in development for evaluation in relapsed or refractory acute myeloid leukemia (r/r AML). CB-012 is engineered with a next-generation CRISPR genome-editing technology to leverage both checkpoint disruption and immune cloaking to enhance persistence of CAR-T cell antitumor activity.
Methods: Caribou’s CRISPR hybrid RNA-DNA (chRDNA) guides in combination with Cas12a were used to make five edits in the manufacture of CB-012. A fully human anti-CLL-1 CAR transgene was site-specifically inserted into the TRAC gene, thereby eliminating TCR expression to prevent graft-versus-host disease. A B2M-HLA-E fusion transgene was inserted into the native B2M gene, preventing expression of all HLA class I antigens except HLA-E, to blunt both T and NK cell-mediated allograft rejection of the CAR-T cells. A knockout of the PDCD1 gene prevented PD-1 receptor expression and thus PD-L1 ligand binding, thereby prolonging antitumor activity. These multiplex genome-editing strategies were designed to enhance persistence of CB-012 antitumor activity and potentially achieve durable therapeutic responses.
Results: CB-012 CAR-T cells express a potent, fully human anti-CLL-1 scFv CAR construct and demonstrate CLL-1-dependent cytotoxicity, proliferation, and cytokine secretion in vitro using AML cell line models. In AML xenografts, robust tumor control and prolongation of survival was observed following a single dose of CB-012. In serial rechallenge assays, CB-012 cells maintained increased cytolytic potential when repeatedly challenged with CLL-1-expressing tumor cells compared to similarly engineered CAR-T cells without a PD-1 knockout, demonstrating a functional benefit of PD-1 abrogation. This advantage was modelled in vivo, conferring superior prolongation of survival with CB-012 treatment compared to similarly engineered CAR-T cells without a PD-1 knockout. Additionally, CB-012 cells expressing the B2M-HLA-E fusion demonstrated resistance to NK cell-mediated cytotoxicity in vitro.
Conclusions: CLL-1 is a compelling therapeutic target as it is highly expressed on AML tumor cells and leukemic stem cells, but not expressed on hematopoietic stem cells. It has also been established as a target in human proof-of-concept studies. CB-012 demonstrated potent and specific CLL-1-targeted cytolytic activity, and the genome-editing strategy employed to manufacture and armor CB-012 conferred a functional advantage in the context of the potentially immunosuppressive tumor microenvironment associated with r/r AML.
Citation Format: Elizabeth Garner, Erin Kelly, Sai Namburi, Cian Colgan, Tristan Fowler, Devin Mutha, Art Aviles, Morena Stanaway, Raymond Guo, Zili An, Emilie Degagne, George Kwong, Leslie Edwards, Emma Jakes, McKay Shaw, Benjamin Schilling, Jeremy Huynh, Ricky Luu, Max Sidorov, Rhonda Mousali, Mikk Otsmaa, Justin Skoble, Steven Kanner. CB-012, an allogeneic anti-CLL-1 CAR-T cell therapy engineered with next-generation CRISPR technology to resist both the immunosuppressive tumor microenvironment and immune cell-mediated rejection, for patients with relapsed or refractory acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3201.