Abstract Background: CD38 is a transmembrane protein that is often overexpressed in multiple myeloma (MM) and other hematologic cancers. The success of anti-CD38 monoclonal antibodies, like daratumumab and isatuximab, in the treatment of MM supports the potential of CD38-targeting therapy candidates in development, including chimeric antigen receptor (CAR) engineered natural killer (NK) cells. Because CD38 is expressed on normal hematopoietic cells and other tissues, it is important to assess the affinity and epitope-specificity of the CD38-targeting moiety to limit potential on-target/off-tumor toxicities. To determine how different biophysical characteristics impact CAR activity, we identified a diverse panel of CD38-specific antibodies, which were shown to display a wide range of affinities and epitopes towards CD38 and converted these clones into CARs to evaluate their expression and target-specific and non-specific activation in a high-throughput (HTP) CD38-knockout (CD38 KO) Jurkat screen. Methods: Antibodies against CD38 were identified through hybridoma screening. Humanized mice were immunized with the extracellular domain of CD38, and lymphocytes from lymph nodes were fused with SP2/0 cells to generate hybridoma cells. Supernatants from single cell sorted hybridoma clones were screened against CD38+ and CD38 KO Jurkat cells and were assessed for affinity and epitope via surface plasmon resonance detection with the Carterra LSA. 96 unique clones were converted into CARs and transduced into CD38 KO Jurkat cells. CAR-expressing Jurkat cells were assayed for CAR expression and activation (via CD69 expression) when cultured overnight in the presence or absence of MM.1R target cells. Seven CARs were selected for additional characterization in primary NK cells. NK cells from healthy donors were expanded on our proprietary NKSTIM cell line, knocked out for CD38 using a CRISPR-Cas system, and transduced with CD38 CAR constructs. CD38 CAR NK-mediated cytotoxicity against MM.1S target cells was assessed by IncuCyte® S3 live cell analysis system. Results: Hybridoma screening identified many unique CD38-specific antibodies, displaying a wide range of affinities and epitopes towards CD38. Despite these biophysical differences, many of these antibodies, when converted to CARs, confer target-specific activity. Multiple epitopes on CD38 may be targeted and even low affinity antibodies were sufficient for target-specific CAR activation. Furthermore, the seven selected CAR candidates that were expressed in CD38 KO donor NK cells displayed potent in vitro target cell killing against MM.1S target cells. Conclusions: In summary, we have identified and characterized a range of CD38-specific antibodies. When converted into CARs, these targeting moieties mediate target-specific activation, despite differences in CD38 epitope and in affinity, demonstrating the utility of HTP CAR screening. Moreover, expression of the top seven CAR candidates in CD38 KO NK cells resulted in potent target cell killing, supporting further pre-clinical evaluation in relevant models. Citation Format: Emily N Kang, Jacinda T Chen, Bing Li, Kate Jamboretz, Nitin Patel, Daniel Bedinger, Kyle T Pratt, Jessica Hsieh, Luxuan TE Buren, Chao Guo, Ivan Chan, James B Trager, Sasha Lazetic. Screening and characterization of CD38 chimeric antigen receptors for the development of natural killer cell-based therapies [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A044.
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Dec 1, 2023
Fan Dai + 6
Open Access
