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Adapter-CAR-T-cells versus Acute Myeloid Leukemia and Leukemia Stem Cells

Applicant Dr. Christian Seitz, since 2/2021
Subject Area Hematology, Oncology
Pediatric and Adolescent Medicine
Term from 2019 to 2023
Project identifier Deutsche Forschungsgemeinschaft (DFG) - Project number 411791562
 
Final Report Year 2023

Final Report Abstract

Clinical translation of chimeric antigen receptor (CAR) expressing T cells (CAR-T) in acute myeloid leukemia (AML) so far has not resulted in the impressive clinical responses seen in B-phenotypic malignancies. Main reasons are the significant inter- and intratumoral heterogeneity in antigen expression, leading to resistance to therapy due to antigen evasion, as well as the promiscuous expression of potential target antigen on vitally essential tissues, particularly cells in myelopoiesis, resulting in unacceptable toxicities. To address these limitations, we developed the AdapterCAR-T (AdCAR-T) platform. AdCAR technology is based on a split recognition/activation design, in which effector cells are redirected to target antigens via biotin-conjugated adapter molecules (AMs), allowing qualitative and temporal control of CAR-T function for improved safety (on/off switch) as well as combinatorial targeting to avoid antigen evasion. The Overarching goal of this study was to utilize the AdCAR-T platform to improve immunotherapy against AML. To this end we analyzed target antigen expression on primary pediatric AML patient samples as well as cell lines, demonstrating that CD33, CD38 and CD371 (CLL1) are most prominently expressed. Co-expression analyses demonstrated significant inter- and intratumoral heterogeneity in target antigen expression. Next, AMs against 20 target antigens were generated and functionally tested, demonstrating antigen-specific activity in vitro and in vivo. Intratumoral heterogeneity was experimentally addressed by multiple antigen knockouts, demonstrating that combinatorial targeting (2 to 5 AMs) is highly efficient to treat heterogenic disease, both in vitro and in vivo. Evaluating AdCAR-T in patient derived xenograft (PDX) models, we found significant therapy associated plasticity in antigen expression requiring rational combinatorial targeting to achieve complete remission. As a novel strategy to address promiscuous antigen expression, we were able to demonstrate that AdCAR-T are capable to differential target and selectively eliminate cancer cells by integration of multiplex antigen expression profiles. To identify additional AML-associated mechanisms of resistance to CAR-T, we analyzed the expression of immune checkpoint ligands on primary pediatric AML patient samples and cell lines, demonstrating expression of The TIGIT-ligand CD112 and CD155 as well as HLA-DR on naïve blasts as wells as induction of PD-L1 expression after AdCAR-T treatment. CRISPR/Cas9 mediated knock out of TIGIT in primary AdCAR-T resulted in significant improvement of anti-leukemic activity of AdCAR-T. In conclusion, our results underscore the suitability of the AdCAR-T platform to address the specific needs for CAR-T therapies in AML, improving safety (on/off switch) and efficacy, enabling individual combinatorial targeting to cope with intratumoral heterogeneity and plasticity. Clinical trials are in preparation to be initiated in 2024.

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