Hypoimmune, Allogeneic CD22-Directed CAR T Cells That Evade Innate and Adaptive Immune Rejection for the Treatment of Large B Cell Lymphoma Patients That Are Relapsed/Refractory to CD19-Directed CAR T Cell Therapy

Abstract

Despite the success of CD19CAR therapy against large B-cell lymphoma, up to 60% of CD19-directed CAR T cell treated patients relapse after, or are refractory to, treatment. Of the relapsed/refractory population a significant subset of patients, around 30%, have CD19-negative tumor and a short median survival prognosis - necessitating the implementation of rapid intervention strategies (Atilla, Transl Oncol 2022; Spiegel, Blood 2021). Sana is developing “off-the shelf” allogeneic CD22-directed CAR T cells that may enable rapid intervention and induce clinically meaningful activity for these CD19 CAR T cell relapsed/refractory patients. Here we describe methods to create hypoimmune (HIP), allogeneic CD22-directed CAR T cells using a clinically validated CD22CAR that: 1) support adaptive immune cell evasion via B2M and CIITA gene disruption, 2) promote persistence and innate immune cell evasion by overexpression of CD47, and 3) mitigate GvHD via TRAC gene disruption, (Hu, Nat Commun 2023). HIP, allogeneic CD22-directed CAR T cells (CD22 HIP CAR T cells) were derived from healthy donor CD8 + and CD4 + T cells that were gene-edited to disrupt the B2M, CIITA, and TRAC (TCR) genes and transduced to overexpress CD47 and express a second-generation, clinically validated CD22-directed CAR. Gene-edited Mock T cells and unedited, allogeneic CD47-CD22CAR T cells were generated as donor-matched controls. The CD22 HIP CAR T cells were evaluated for immune evasive activity using a previously described in vitro xCelligence cytotoxicity assay (Hu, Nat Commun 2023). The T cells were further evaluated in vitro for dose-dependent short-term and long-term cytolytic activity analysis via luciferase or IncuCyte assay, respectively. Correlate cytokine responses were performed by meso-scale discovery analysis and T cell expansion by IncuCyte classification. Systemic NALM and RAJI parental or CD19 antigen knockout tumor models (IV injection) were executed in a NSG or NSG-SGM3 mouse background. xCelligence analysis confirmed that CD22 HIP CAR T cells were protected from NK and macrophage recognition; whereas Mock control T cells that lack HLA expression succumbed to NK cell and macrophage killing within 5-hours of the 50-hour assay due to the absence of CD47 protection. Furthermore, CD22 HIP CAR T cells elicited cytotoxicity and robust cytokine responses (e.g. IFNγ, IL-2) in a dose-dependent manner comparable to allogeneic CD47-CD22CAR T cells when cultured with CD22 +/CD19 + or CD22 +/CD19 - NALM, RAJI, or K562 targets. The CD22 HIP CAR T cell and allogeneic CD47-CD22CAR T cell control produced low level IFNγ (<400pg/mL) and Granzyme B (<100pg/mL) when cultured alone or with CD22 - tumor targets. These basal levels of IFNγ and Granzyme B did not have long-term cytolytic effects on CD22 - tumor cells, nor was any significant level of IL-2 produced by CD22 HIP CAR T cells cultured alone, or in the presence of CD22-negative tumor targets. Indeed, CD22 HIP CAR T cell proliferation was dependent upon CD22 + tumor co-culture. NALM and RAJI CD19 knockout in vivo tumor models demonstrated that CD22 HIP CAR T cells significantly reduced flux (Day 21 AUC vs Mock: NALM and RAJI (p=0.0001)) and prolonged survival (Median vs Mock: p<0.004). Furthermore, CD22 HIP CAR T cells elicited comparable, dose-dependent antitumor activity relative to allogeneic CD47-CD22CAR T cell controls in a systemic NALM tumor model. Our data provide evidence that CD22 HIP CAR T cells elicit robust antitumor activity in an antigen and dose-dependent manner. Furthermore, these studies demonstrate that CD22 HIP CAR T cells are agnostic to CD19 expression and selectively elicit pharmacologic activity against, or in response to, CD22 + tumor. Results also demonstrate that the HIP approach to generate allogeneic CAR T cells that evade innate and adaptive immune rejection can be expanded as a platform to generate CAR T cells against target antigens alternative to CD19. Collectively, the data suggest that CD22 HIP CAR T cells display a combination of antigen-specific pharmacologic activity and immune evasion that support their progression into human clinical studies for the treatment of CD19 CAR T cell-refractory patients.