P-03.05 scFv-dependent anti-tumor efficacy of CAR-T cells targeting novel antigens in acute myeloid leukemia

<h3>Background</h3> Acute myeloid leukemia (AML) is an aggressive malignancy associated with poor prognosis, high relapse rates, and resistance to standard therapies. While chimeric antigen receptor (CAR) T-cell therapies targeting cell surface proteins offer potential, their efficacy in AML is limited due to the overlap of target protein expression between leukemic and healthy hematopoietic cells. To address these challenges, we utilized proteomics and surface glycoproteomics screening approaches to identify novel cell surface proteins specifically upregulated in AML patients. Leveraging these discoveries, we generated antigen specific antibodies (scFvs) and engineered third-generation CAR-T cells to target the identified AML-specific proteins, thereby enhancing and redirecting CAR-T cell efficacy against AML. <h3>Materials and Methods</h3> To identify AML-specific surface proteins, we performed LC/LC-MS on total and isolated surface glycoproteomes from AML patient samples. Antigen-specific antibodies (scFvs) for the discovered targets were then generated using a phage display library. The scFvs were incorporated into a GMP-compliant third-generation CAR backbone and expressed in primary human T cells by gammaretroviral transduction. The functionality and specificity of the engineered CAR-T cells were assessed through co-culture assays at 1:1 and 1:4 effector-to-target ratios. Anti-tumor efficacy was evaluated against antigen positive tumor cell lines <i>in vitro.</i> <h3>Results</h3> Detailed analysis of proteomics and glycoproteomics datasets identified 9 upregulated surface proteins in leukemia stem cells and AML, including 5 novel targets reported here for the first time. We produced 19 unique scFvs targeting extracellular domain epitopes that in CAR format, showed robust 20–80% CAR expression in engineered primary human T cells. CARs evaluated against CD206, ITGA4 and LILRB1 antigens targeted and eliminated antigen-positive tumor cells after 24 h at 1:1 and 1:4 effector-to-target ratios. Notably, anti-CD206 CAR-T cells eradicated nearly all K562 antigen-positive cells within 72 hours of co-culture. We observed a 10–15% increase in production of effector cytokines TNF-α and IFN-γ from both CD4⁺ and CD8⁺ CAR-T cells compared to non-transduced T cells. Functional differences between CARs incorporating different scFvs were found allowing their further evaluation and selection. <h3>Conclusions</h3> We present an approach to enrich surface glycoproteins in AML patients and identify AML-specific antigens through detailed proteomics analysis. The utility of these identified targets was evaluated by engineering CAR-T cells with precise targeting capabilities. These CAR-T cells, equipped with unique scFvs, demonstrated anti-tumor efficacy against antigen-positive AML cell lines <i>in vitro</i>. This approach paves the way for the discovery of new AML targets while effectively leveraging current immunotherapeutic tools. J.J. Brysting: None. S.M. Abdin: None. F. Schuler: None. Y. Liu: None. E. Cetin: None. B. Ince: None. J. Frenz: None. A.K. Jayavelu: None. J.J. Schwarz: None. T. Sauer: None. J.B. Zaugg: None. C. Müller-Tidow: None.