Tandem CAR-T cells targeting CLDN18.2 and NKG2DL for treatment of gastric cancer.

Abstract

4030 Background: Gastric cancer (GC) is one of most common malignant tumor which is fifth for melanoma incidence and second for mortality rates worldwide. Novel therapeutic strategies are urgently needed. Recently people are exploring the potential of chimeric antigen receptor T (CAR-T) cell therapy in GC, yet the clinical outcome is limited. Reportedly, the bispecific CAR-T cell with tandem scFvs exhibited superior response rates or synergistic response compared with CAR-T cell with single scFv. It's reported that Claudin18.2 (CLDN18.2) is the most potential target of gastric cancer. Here, we construct a novel bispecific CAR-T cells (KD-496), which simultaneously recognize NKG2D ligands and CLDN18.2, to show superior antitumor efficacy and safety in vitro and in vivo. Methods: Gastric cancer cell lines as well as GC patient tissue samples were evaluated for NKG2DL and CLDN18.2 expression. The KD-496 CAR-T cells showed antigen-specific stimulation by cytokine secretion and tumor cell cytotoxicity assay. The patient tumor tissue fragments (8 mm3) were implanted subcutaneously into B-NDG mice to establish PDX models. When the tumor volume reached 50-100mm3, the mice received a single treatment of 5 million KD-496 CAR-T cells intravenously. The effect of KD-496 CAR-T in vivo was detected by measuring tumor volume twice a week. Results: CLDN18.2 and NKG2DL were detected on NUGC4, AGS-18.2 and MKN-28-18.2 cells and most of screened BM patient samples. The bispecific CAR-T cell KD-496 was generated with CD8 hinge region and transmembrane region, 4-1BB costimulatory region and CD3 zeta region. The KD-496 expression was > 50% on the surface of T cells confirmed by flow cytometry. Co-incubation of KD-496 CAR-T cells with NUGC4 and MKN-28-18.2 cell specifically upregulates IFN-γ cytokines and strongly lysis tumor cells even at low E:T ratio (50-60% at 4:1). Strikingly, KD-496 CAR more efficiently eliminated xenograft tumors in vivo and did not exhibit significant treatment-related toxicity in the treated mice. No obvious pathological changes were found in the tested organs. Conclusions: Our work construct a tandem CAR molecule targeting two tumor-associated antigens, NKG2DL and CLDN18.2, and found that Tan-CAR-T cells（KD-496）distinctly recognize the antigens and exhibited superior antitumor effect. KD-496 CAR-T cells potently respond to GC and more efficient tumor elimination than single CAR such as KD-025 and KD-182 CAR-T cells in PDX model with no obvious safety issue. The results support future clinical trial of KD-496 CAR in patients with GC, where the need for effective treatment is great.