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3438 Trogocytosis-Resistant CAR T Cells Exhibit Increased Persistence and Prevent Antigen Escape

Program: Oral and Poster Abstracts
Session: 703. Cellular Immunotherapies: Basic and Translational: Poster II
Hematology Disease Topics & Pathways:
Research, Biological therapies, Translational Research, Chimeric Antigen Receptor (CAR)-T Cell Therapies, Therapies
Sunday, December 10, 2023, 6:00 PM-8:00 PM

Kenneth A. Dietze, BS1*, Michael L. Olson, PhD2*, Etse Gebru, BS3*, Djordje Atanackovic, MD3, Aaron P. Rapoport, MD3 and Tim Luetkens, MD1,3*

1Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD
2Division of Hematologic Neoplasia, Dana Farber Cancer Institute, Boston, MA
3University of Maryland Greenebaum Comprehensive Cancer Center, Baltimore, MD

Chimeric Antigen Receptor (CAR) T cell therapy is a highly effective treatment for cancer patients in which T cells are genetically modified to express a CAR targeting an antigen expressed on the surface of cancer cells. While CAR T cells have shown remarkable efficacy, many patients receiving CAR T cell therapy relapse within five years of treatment. It has recently been shown that tumor cells can escape killing by CAR T cells using a mechanism called trogocytosis. CAR-mediated trogocytosis (CMT) involves the transfer of cell surface proteins from tumor cells to CAR T cells. Antigen transfer by CMT results in antigen-negative tumor cells and reduced CAR T cell persistence due to increased CAR T cell exhaustion and fratricide. To date, the molecular processes causing CMT remain poorly understood and there are currently no approaches to therapeutically target CMT.

To track CMT in real-time, we first developed a custom luciferase complementation assay with a detection limit of approximately 100 CAR T cells displaying the target antigen (CompLuc). Using this assay, we found that CMT is effector-target ratio and time-dependent and can be modulated by inhibiting actin polymerization, a process essential for key T cell functions including vesicle formation, protein transport, and target cell killing.

To develop a universal approach to inhibit CMT in CAR T cells without altering tumor cell killing, we investigated the effect of inhibiting adhesion, endocytosis, vesicle formation, and proteolytic degradation using various small-molecule inhibitors. We found that inhibition of the cysteine protease Cathepsin B with the small molecule inhibitor Ca-074-Me significantly reduced CMT as determined by CompLuc assay and flow cytometry. Performing a luciferase-based cytotoxicity assay, we found that Cathepsin B inhibition does not affect tumor cell killing. Based on these data, we hypothesized that Cathepsin B inhibition could be a potential therapeutic approach to limit CMT.

Cathepsin B activity in human cells is regulated by the human proteins Cystatin A and Cystatin B. Structural data of Cathepsin B in complex with Ca-074-Me or Cystatin A show that both molecules appear to sterically block access of substrates to the active site cysteine of Cathepsin B. We therefore explored if overexpression of cystatins in CAR T cells could be an efficient approach to inhibit CMT. We found that overexpression of Cystatin A significantly reduced Cathepsin B activity in CAR T cells and limited transfer of tumor antigen to CAR T cells. We also observed that cystatin overexpression reduced loss of antigen from the tumor cell surface, indicating that Cathepsin B inhibition blocks CMT at an early stage of the trogocytic process. Importantly, Cystatin A overexpression did not alter CAR T cell expansion, tumor cell killing, or CAR T cell phenotype, indicating that this approach selectively inhibits CMT and that key CAR T cell functions remain intact. Cystatin overexpression also led to significantly increased tumor-specific CAR T cell numbers (78% of non-targeting CAR T cells) compared to wild-type CAR T cells (61% of non-targeting CAR T cells, p = 0.0298) after 24 h exposure to tumor cells, indicating that selective inhibition of CMT prevents CAR T cell fratricide and/or exhaustion.

To further reduce presence of antigen on CAR T cells following CMT, we next developed a system to actively degrade the trogocytosed protein in CAR T cells. Specifically, we developed a fusion protein combining the SH2 domain of the Src kinase Lyn, which binds with high affinity to the intracellular domain of CD19, with the von Hippel-Lindau E3 ubiquitin ligase. We show that expression of this fusion protein within CAR T cells rapidly targets trogocytosed CD19 for degradation and eliminates its presence on the surface of CAR T cells. Trogocytosed antigen degradation also increased CAR T cell survival at the end of in vitro co-culture, indicating reduced CAR T cell fratricide and/or exhaustion.

Taken together, we provide the first preclinical proof-of-concept that, using a fully human genetic engineering approach, CMT can be targeted without compromising essential CAR T cell functions. This represents a promising approach to improve CAR T cell efficacy and limit the occurrence of relapse in patients receiving CAR T cell therapy.

Disclosures: No relevant conflicts of interest to declare.

*signifies non-member of ASH