GITR domain inside CAR co-stimulates activity of CAR-T cells against cancer

Abstract

T cells expressing Chimeric antigen receptors or CAR-T cells are used as a novel treatment against hematological and solid cancers. In this report, we designed CAR with glucocorticoid-induced TNFR-related protein (GITR) co-stimulatory domain to study its ability to co-activate CAR-T cells. EGFR-GITR-CD3 CAR-T cells were cytotoxic against EGFR-positive: pancreatic and ovarian cancer cells but not against EGFR-negative cancer cells. The cytotoxic activity of EGFR-GITR-CD3 CAR-T cells was comparable or better than EGFR-28-CD3 or EGFR-41BB-CD3 CAR-T cells. We designed also EGFR-CD3-GITR-CAR and EGFR-ΔGITR-CD3 with deleted 184-192 amino-acids of co-stimulatory GITR domain, and showed that EGFR-GITR-CD3 had significantly higher cytotoxic activity against EGFR-positive cells. The EGFR-GITR-CD3 cells secreted significantly higher levels of IFN-gamma than EGFR-CD3-GITR and EGFR-ΔGITR-CD3 cells. In addition, Mesothelin-GITR-CD3 CAR-T cells also killed mesothelin-positive ovarian cancer cell lines, and pancreatic cancer cells. Moreover, CD19-GITR-CD3 CAR-T cells had significant cytotoxic activity against CD19-positive cancer cells and in Raji xenograft tumors. Thus, our results clearly show that GITR co-stimulatory domain can be used as a novel co-stimulatory domain in CAR-T cells.

A Real-time Potency Assay for Chimeric Antigen Receptor T Cells Targeting Solid and Hematological Cancer Cells

“…In contrast to end point data which is a mere "snap shot", the continuous impedance profile of the xCELLigence instrument clearly illuminates subtle differences in the killing kinetics of the different treatments ( Figure 5C). The results obtained here are consistent with those of an in vivo study 30 . Step 1: Seed the target cells (i.e., tumor cells) into the well of an E-Plate.…”

“…Over the past 15 years the xCELLigence assay has been validated for assessing the potency of natural killer (NK) cells, T cells, CAR T cells, checkpoint inhibitors, bispecific antibodies, oncolytic viruses, and some combination therapies 17,29,30,31,32,33,34 . Recently, the xCELLigence potency assay was evaluated for manufacturing T-cell receptor (TCR)-engineered T-cells 35 .…”

DCLK1 Monoclonal Antibody-Based CAR-T Cells as a Novel Treatment Strategy against Human Colorectal Cancers

“…These data support our hypothesis that the TSC population can be targeted using a DCLK1-specific CAR-T. Here, we report that, in collaboration with ProMab Inc., we have developed a novel CAR-T based on the DCLK1 ScFv containing a CD28 transmembrane and co-stimulatory domain and CD3ζ activation domain [39][40][41][42][43][44][45][46]. CAR-T cells generated using DCLK1 ScFv (CBT-511) demonstrated~20% CAR expression and significantly induced CRC cell cytotoxicity.…”

“…This personalized approach to cancer therapy enables a patient's own T cells to be programmed to attack and eliminate their specific cancer. Typically, the tumor antigen specific targeting region is a single-chain antibody variable fragment (ScFv) fused to a hinge, transmembrane domain, and co-stimulatory domains (CD28, 4-1BB, CD27 or others) to stimulate the immune response, as well as a CD3ζ activation domain [38][39][40][41][42][43].…”

Mechanisms underlying CD19-positive ALL relapse after anti-CD19 CAR T cell therapy and associated strategies

“…There are many other costimulatory domains, such as Dap10 costimulatory domains, 2B4 costimulatory domains, GITR domains, CD150 signaling lymphocytic activation molecule (SLAM), a 150-kDa protein termed as M150. Different costimulatory domain induces differential effects in CAR-expressing T cells [57][58][59][60][61].…”