Abstract 4790: Immunostimulatory effects of GENA-104A16, a monoclonal anti-CNTN4 antibody, on tumor-infiltrating lymphocytes in a colon cancer liver metastasis model

Abstract Introduction: Immune checkpoint inhibitors (ICIs) have significantly improved response rates and survival, becoming a cornerstone in the treatments for various cancers. ICIs are effective when their target immune checkpoints, most notably PD-L1, are expressed in tumor cells, and are less effective when they are not. CNTN4 is a novel immune checkpoint expressed independently to previous targets of ICIs. GENA-104A16, a humanized anti-CNTN4 IgG4 (S228P) antibody, binds to CNTN4 on tumor cells and blocks its interaction with amyloid precursor protein (APP) on T cells. This disruption neutralizes immune-suppressive activity and enhances anti-tumor immune responses. This study aimed to further investigate the immunostimulatory effects of GENA-104A16 in a colon cancer liver metastasis model, which is known to recapitulate the resistance of ICIs. Method: CT26 mouse colon cancer cells overexpressing mCNTN4 (CT26/Cntn4) were inoculated into the spleen. One day later, mice were treated with either isotype control (n=10), GENA-104A16 (n=10), or atezolizumab (n=9) via intraperitoneal injection (3 mg/kg) on a q3d schedule. On Day 11 post-inoculation, animals were sacrificed, and livers were harvested for formalin fixation and paraffin embedding preparation. Tumor-infiltrating lymphocytes (TILs) within hepatic tumor nodules were analyzed using an artificial intelligence (AI)-powered immunohistochemistry (IHC) analyzer applied to CD4 or CD8 stained liver sections containing tumor nodules. CD4+ or CD8+ TILs within the hepatic tumor area were quantified on whole slide images both as a percentage (%) of total cells and by cell density. Results: GENA-104A16 treatment significantly increased CD4+ T cells as well as CD8+ T cells in both metrics (CD4+ %: 17.5, CD4+ density: 1470; CD8+ %: 12.6, CD8+ density: 1124) compared to isotype control (CD4+ %: 10.0, CD4+ density: 760; CD8+ %: 7.0, CD8+ density: 615). Meanwhile, atezolizumab treatment did not significantly increase TILs (CD4+ %: 14.0, CD4+ density: 1056; CD8+ %: 6.7, CD8+ density: 587) in the current model, which showed similar expression levels of CNTN4 and PD-L1. Notably, an inverse correlation trend was observed between TILs quantification and tumor nodule count across all samples, regardless of the treatment group. Conclusions: Our findings demonstrate a significant increase in CD4+ and CD8+ TILs following treatment with GENA-104A16 in a clinically relevant model, suggesting a potential for enhanced anti-tumor immunity. These results support the clinical evaluation of GENA-104A16 and underscore the potential of TILs quantified by an AI-powered IHC analyzer as a predictive biomarker for its therapeutic efficacy. Citation Format: Hyunkyung Yu, Taebum Lee, Gyeongyeon Kim, Sanghoon Song, Youngeun Ha, Soo Ick Cho, Kitae Park, Hyunuk Kim, Mi Young Cha. Immunostimulatory effects of GENA-104A16, a monoclonal anti-CNTN4 antibody, on tumor-infiltrating lymphocytes in a colon cancer liver metastasis model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4790.