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1979 Novel Protease-Mediated Double Antigen Recognizing Chimeric Antigen Receptor (CAR) Enhances Directionality of CAR-T Cell Activity and Improves Target Cell Specificity

Program: Oral and Poster Abstracts
Session: 703. Cellular Immunotherapies: Basic and Translational: Poster I
Hematology Disease Topics & Pathways:
Research, Fundamental Science, Biological therapies, Translational Research, Chimeric Antigen Receptor (CAR)-T Cell Therapies, Therapies, immunology, Biological Processes
Saturday, December 10, 2022, 5:30 PM-7:30 PM

Satoru Aoyama, M.D., Ph.D.1,2*, Ayako Nogami, M.D., Ph.D.1,3, Sadakatsu Ikeda, M.D., Ph.D.2* and Takehiko Mori, M.D., Ph.D.1*

1Department of Hematology, Tokyo Medical and Dental University (TMDU), Tokyo, Japan
2Department of Precision Cancer Medicine, Tokyo Medical and Dental University (TMDU), Tokyo, Japan
3Department of Laboratory Medicine, Tokyo Medical and Dental University (TMDU), Tokyo, Japan

Recently, Chimeric Antigen Receptor (CAR)-T cell therapies show excellent therapeutic effect against various hematologic malignancies, including B-cell leukemia, B-cell lymphoma, and multiple myeloma. Expansion of target diseases of this attractive immunotherapy is one of the most important issues. To identify the novel specific tumor antigen as CAR-T cell target is the standard way to accomplish this goal. However, recent CAR-T cells recognize single tumor antigen, so normal tissues expressing the target antigen are possible to be attacked by CAR-T cells. For example, anti-CD19 CAR-T cells against B-cell malignancies kill not only tumor cells but also normal B-cells, so patients receiving the CAR-T cells showed severe immune deficiency. Improvement of target cell recognition system of CAR-T cells minimizes this adverse effect and contributes to expand target diseases.

To improve target-cell-specificity of CAR-T cell, we established a protease-mediated regulatory CAR, named “Scissors-CAR” (panel A). This regulatory CAR is constituted with extracellular antigen recognition portion and cytoplasmic Human Immunodeficiency Virus protease (HIVPR) catalytic domain. When CAR-T cells expressing both anti-A Scissors-CAR and anti-B Counterpart-CAR that has HIVPR recognition polypeptide contact with target tumor expressing both antigen-A and B, these two CARs interact on cell membrane. This interaction induces Scissors-CAR-mediated cleavage of target polypeptide and structural change of Counterpart-CAR. Previously, we reported double antigen recognition system using Scissors-CAR. Previous Counterpart-CAR named “Signal-CAR” had HIVPR recognition polypeptide upstream of CD3-zeta, so Scissors-CAR-mediated cleavage broke the CAR protein structure and suppressed CAR-T cell activity (panel B, named “Negative system”). This system allowed CAR-T cells to directional attack against target cells expressing only antigen-A and protect the cells expressing both antigen-A and B.

Here, we established another double-antigen recognition system using Scissors-CAR. In many cases including both hematologic malignancies and solid tumors, additional ectopic antigens are expressed on malignant cells. If CAR-T cells empowered to directional attack against target cells expressing both antigen-A and B, much more tumors can be treated with this tumor immunotherapy (panel C, named “Positive system”). To establish this directional CAR-T cells, we constructed novel Counterpart-CAR named as “Activating-CAR” constituted with target recognition portion, co-stimulatory domain, CD3-zeta, and protein destabilization motif known as degron. Between CD3-zeta and degron motif, HIVPR recognition polypeptide was inserted. In vitro analysis revealed that this degron motif destabilized Activating-CAR and HIVPR-mediated cleavage and that release of degron motif increased Activating-CAR stability. Furthermore, cell proliferation of Jurkat cells expressing both anti-CD19 Activating-CAR and anti-HER2 Scissors-CAR was enhanced co-cultivated with target cells expressing both CD19 and HER2.

From these results, combined use of Activating-CAR and Scissors-CAR allowed CAR-T cells to directionally activate against target cells expressing two antigens recognized by each CAR. By choosing the Counterpart-CAR from “Signal-CAR” or “Activating-CAR”, this protease-mediated double antigen recognition system could optimize target-cell-specificity of CAR-T cells for the individual tumor and contribute to expand the target tumors of CAR-T cell therapy.

Disclosures: No relevant conflicts of interest to declare.

*signifies non-member of ASH