Abstract 3200: Non-engineered T cells with specific anticancer effects in KRAS-mutated colorectal cancer

Abstract

Abstract Cytokine-induced killer (CIK) cell therapy is known as a useful treatment for cancer immunotherapy due to its anticancer activity and safety. However, its clinical efficacy is limited due to its lack of antigen specificity and low in vivo persistence. KRAS mutations play an important role in promoting tumorigenesis in cancer. But, its clinical application as a biomarker for cancer treatment is still difficult. Non-engineered T cell therapy approaches could be a promising strategy for treating KRAS-mutated cancer. Herein we developed non-engineered T cells empowered with specific anticancer effects in KRAS-mutated colorectal cancer (CRC), and investigated its properties and anticancer effects compared to CIK cells. A recombinant overlapping peptide was designed to overlap the KRAS protein by 15 amino acids linked by a cathepsin S cleavage site (LRMK). It contained KRASG12D, KRASG12V, KRASG13D, and KRASWT epitope peptides and other KRAS protein regions. Peripheral blood mononuclear cells (PBMCs) from CRC patients with KRAS mutations were stimulated with the recombinant overlapping peptides in vitro for two days in the presence of interleukin (IL)-4, granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor (TNF)-α, IL-1β, and prostaglandin 2, and expanded using the 12-day CIK cell expansion method in the presence of IL-2 (200 IU/mL). To verify the enrichment of KRAS mutant-specific T cells, the expanded T cells were restimulated by pulsed monocyte-derived dendritic cells with KRAS mutant antigen for two days and the frequency of KRAS mutation-specific T cells was determined using the interferon (INF)-γ capture assay. Red fluorescent protein (RFP)-expressing HLA-matched cell lines and cell line-derived spheroids T3M10 (KRASG12D) and MCF-7 (KRASWT) were used for the Incucyte immune cell killing assay. T-cell phenotypes were determined by flow cytometry. The expanded non-engineered T cells stimulated with KRAS mutant antigen showed skewed CD4 T cell differentiation and increased central memory T cells compared to CIK cells, suggesting that they may have more long-term immunity and functionality than CIK cells. The KRASG12D-specific T cells were enriched 12-13% (9.4-fold) by in vitro stimulation compared to 1.3% in CIK cells. Anticancer activity in a 2D-culture model was significantly higher in KRASG12D-specific enriched T cells (82.5%) than in CIK cells (64.9%) co-cultured with T3M10 cells for 48 h. In a 3D spheroid-culture model, increased cytotoxicity was also detectable in the KRASG12D-specific enriched T cells (13.2% at 72 h and 14.1% at 142 h) compared to CIK cells. These results demonstrated that non-engineered T cells with KRAS mutated-specific anticancer activity are an ideal candidate for KRAS mutant cancer-targeted immunotherapy. Further studies are necessary to verify the anticancer effects in vivo. Citation Format: Sunki Lim, Yujeong Gho, Seoyoung Kim, Min Ki Kim, Jong Seob Park, Wang Jun Lee, Young-Kwan Lee, Hyoun Jong Moon, Excelsisbio.Inc, Goyang, Korea. Non-engineered T cells with specific anticancer effects in KRAS-mutated colorectal cancer [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 3200.