Abstract 912: Single cell analysis reveals tumor evolution and CAR T cell dysfunction associated with tumor resistance
Authors: Yang Yanping , Louie Raymond , Puc Janusz , Vedvyas Yogindra , Alcaina Yago , Min Irene M. , Britz Matt , Luciani Fabio , Jin Moonsoo M. 
Published: April 2023

Purpose: While there have been extensive studies about defining T cell states associated with response to checkpoint immunotherapy, relatively little is known about the crosstalk between tumor and tumor-infiltrating chimeric antigen receptor (CAR) T cells and how it leads to tumor resistance and CAR T cell dysfunction. Here we employ a two-pronged approach using CAR T cell imaging and single cell multi-omics techniques to elucidate the complex tumor-CAR T cell interactions in a gastric cancer xenograft model.

Methods: Human CAR-T cells targeting epithelial cell adhesion molecule were engineered to co-express somatostatin receptor 2 (SSTR2) for positron emission tomography (PET) imaging using 18F-NOTA-Octreotide radiotracer. NSG mice were inoculated subcutaneously with firefly luciferase-expressing MKN-45 cells (1 × 106 per mouse) and treated 5 days later with 10 × 106 SSTR2-expressing CAR-T or non-transduced T cells. Tumor growth and CAR T cell expansion were monitored near synchronously by bioluminescence and PET-CT imaging, respectively. Resistant or relapsing tumors were harvested and MACS sorted into human CD45+ tumor-infiltrating T cells (TILs) and CD45-depleted tumor cells. CAR T cell products and MKN-45 tumor cell line prior to injection were included in single cell analysis for comparison.

Results: TCR clonal analysis supports that CAR T cells in resistant tumors are proliferative and clonally expanded, which finding is corroborated by whole-body CAR T cell imaging. Highly expanded clones were observed within the CD4+ CAR T cell compartment, especially within the regulatory T (Treg) cell cluster. CD8+ CAR T cells underwent less clonal expansion with most clones found within the exhausted cells (LAG3, PDCD1, and TIGIT, LAYN and SOX4), whereas non-transduced control CD8+ T cells did not express high levels of exhaustion associated genes. Differential gene expression (DGE) analysis between TILs and pre-infusion CAR T cells showed increased expression of LAYN, CXCL13, LMNA, and DUSP4 in TILs. The most upregulated DUSP4 was previously found to be overexpressed in Treg and aged T cells, and the DUSP4 induction is linked to a defective adaptive immune response. In addition, we observed the loss of cell-surface CAR in TILs, likely from chronic exposure to antigens in resistant tumors. Furthermore, DGE analysis reveals that under CAR T cell treatment MKN-45 tumors upregulated interferon alpha inducible protein 27 (IFI27), TNFRSF14 and a group of pathways related to metabolic activity and antigen processing and presentation, implying the evolution of tumor cells to resist T cell killing.

Conclusions: Our findings highlight the complex tumor-T cell interplay as tumor evades CAR T cell killing. Tumor resistance is associated with CAR T cell transition from cytotoxic to dysfunctional phenotypes, the suppression of expanded Treg cells, and the acquisition of resistant features by tumors.

Citation Format: Yanping Yang, Raymond Louie, Janusz Puc, Yogindra Vedvyas, Yago Alcaina, Irene M. Min, Matt Britz, Fabio Luciani, Moonsoo M. Jin. Single cell analysis reveals tumor evolution and CAR T cell dysfunction associated with tumor resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 912.