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Background Immune rejection mediated by Natural Killer (NK cells) is a significant barrier to allogeneic cell therapy. CD38 is highly expressed on NK cells and patients treated with anti-CD38 monoclonal antibodies become NK cell deficient. 1 2 These observations suggest that anti-CD38 antibodies could be used as a preconditioning regimen to protect allogeneic CAR-T cells from immune rejection. However, expression of CD38 on T cells poses a potential obstacle to this approach.Methods To assess CD38 expression following antigen engagement, CAR-T cells were co-cultured with antigen-expressing target cells and analyzed by flow cytometry. In vivo co-administration of anti-CD38 monoclonal antibodies with CD38+ CAR-T cells resulted in their depletion, highlighting a key challenge in therapeutic settings. To overcome this, we engineered CAR-T cells to evade antibody mediated clearance using two complementary approaches: CD38 gene knock-out and epitope editing via base editing. Using base editing to perform site-directed mutagenesis, we identified a novel T116A mutation on CD38 that blocks binding to isatuximab, a CD38-directed cytolytic antibody, while preserving CD38 surface expression. Furthermore, engineered CAR-T cells were manufactured at large scale using electroporation equipment compatible with GMP protocols. The resulting CAR-T cells were adoptively transferred into immunodeficient NSG mice engrafted with CD19+ Raji cell line to evaluate in vivo CAR-T pharmacokinetics, anti-tumor efficacy, and resistance to anti-CD38 mediated depletion.Results Large scale manufacturing of base edited CAR-T cells yielded highly efficient CD38 knockout (KO) while preserving potent anti-tumor activity. Control CAR-T cells were significantly reduced in the peripheral blood of mice treated with CAR-T and anti-CD38 combination therapy, while CD38 KO CAR-T cells showed similar cell counts with or without anti-CD38 treatment. Reduced CAR-T cell abundance in the anti-CD38 treated group resulted in loss of tumor control in NSG mice. CD38 deficient CAR-T cells showed no defects in in vitro assays including cell expansion, cytotoxicity, cytokine production, or proliferation following antigen exposure. Interestingly, CD38 knockout CAR-T cells showed increased oxidative phosphorylation without diminishing glycolytic metabolism, consistent with role for CD38 in regulating cellular metabolism.Conclusions Overall, these studies demonstrate that CD38 disruption enables combining anti-CD38 antibodies with CAR-T cell therapy and show CD38 knockout CAR-T cells maintain anti-tumor efficacy in vivo. Since anti-CD38 antibodies are used in the treatment of both hematologic malignancies and autoimmune disease, this work highlights multiple engineering strategies that render CAR-T cells compatible with existing treatments and provides a new approach to combat NK cell mediated rejection of allogeneic CAR T cells.References Casneuf T, et al. Effects of daratumumab on natural killer cells and impact on clinical outcomes in relapsed or refractory multiple myeloma. Blood Advances, 2017;1(23):2105–2114. PMID: 29296857McDonnell SRP, et al. Mezagitamab in systemic lupus erythematosus: clinical and mechanistic findings of CD38 inhibition in an autoimmune disease. Lupus Sci Med. 2024;11(1):e001112.Ethics Approval Mice were maintained and used for conducting experiments in adherence to an Institutional Animal Care and Use Committee (IACUC)-approved protocol in an AAALAC-approved facility.