Abstract 1868: Novel immune checkpoint inhibitor: In vivo and in vitro efficacy of anti-CNTN4 antibody, GENA-104A16 in the models with increased CNTN4 expression
Authors: Cha Mi Young , Yu Hyunkyung , Jeon Bu-Nam , Ha Youngeun , Kim Hyunuk , Kim Yunyeon , Chung Joo-Yeon , Kim Gihyeon , Park Changho , Park Kyung Mi , Park Hansoo 
Published: June 2023

Abstract
We previously suggested that contactin 4 (CNTN4) negatively regulates T cell activity through binding with amyloid precursor protein on T cells. In addition, we confirmed that CNTN4 is highly expressed in the various types of tumors such as gallbladder, pancreas, stomach, endometrium, liver, prostate, and bladder cancer and melanoma with positive rates over 70% through immunohistochemistry analysis. However, murine cancer cells expressed CNTN4 at a very low level, differently from human tumors or cancer cells. Therefore, it was limited in assessing the anti-tumor efficacy by targeting CNTN4 in syngeneic mice models. Following this background, we have prepared CT26 murine cancer cells overexpressing CNTN4 (CT26/CNTN4) and evaluated anti-tumor efficacy by treating GENA-104A16, the anti-CNTN4 humanized monoclonal antibody in the CT26/CNTN4 syngeneic mice model. The treatment of GENA-104A16 induced fairly improved in vivo efficacy in the model compared to the CT26 WT model. Moreover, to identify the in vivo immune modulatory function of GENA-104A16, we analyzed the proportion of tumor-infiltrated immune cells in CT26/CNTN4 tumors through flow cytometry and single-cell analysis. As a result, the population of Treg cells decreased in the tumors of GENA-104A16-treated mice. In addition, the total amount of tumor-infiltrated immune cells increased by 2-fold, CD4+ T cells by 4-fold, and cytotoxic CD8+ T cells by 1.5-fold in tumors of mice administrated with GENA-104A16. We also performed transcriptomic analysis on samples collected from IgG-treated CT26 WT tumors and CT26/CNTN4 tumors treated with IgG or GENA-104A16. Comparative analysis of gene expression using the Gene Set Enrichment Analysis Hallmark showed that the gene sets of TNFα signaling, IFNγ response, inflammatory response, and apoptosis were decreased in CT26/CNTN4 tumors than in CT26 WT tumors. And the majority of the two gene sets, TNFα signaling and IFNγ response in CT26/CNTN4 tumors, increased by the administration of GENA-104A16. These results indicate that the overexpression of CNTN4 reduces the activity of tumor-infiltrating lymphocytes, resulting in decreased cytokine secretion, which is restored by administering GENA-104A16. Furthermore, we investigated in vitro T cell-mediated cytotoxicity of GENA-104A16 on U2OS (human osteosarcoma cell) endogenously expressing CNTN4 together with CNTN4-knocked out U2OS. GENA-104A16 increased T cell-mediated cytotoxicity when CNTN4-expressing U2OS cancer cells were co-cultured with T cells. Collectively, the function of CNTN4 in negative immune regulation was confirmed repeatedly through the present studies. And the expression level of CNTN4 on tumors was essential for the anti-tumor activity of GENA-104A16. Therefore, the expression of CNTN4 could be a potential biomarker for monitoring clinical responses to anti-CNTN4 therapy.
Citation Format: Mi Young Cha, Hyunkyung Yu, Bu-Nam Jeon, Youngeun Ha, Hyunuk Kim, Yunyeon Kim, Joo-Yeon Chung, Gihyeon Kim, Changho Park, Kyung Mi Park, Hansoo Park. Novel immune checkpoint inhibitor: In vivo and in vitro efficacy of anti-CNTN4 antibody, GENA-104A16 in the models with increased CNTN4 expression [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 1868.