Abstract 2862: Novel immune checkpoint protein CNTN4 and the preclinical efficacy of affinity matured anti-CNTN4 antibody GENA-104A16
Authors: Cha Mi Young , yu Hnkyung , Jeon Bu-Nam , Kim Hyunuk , Ha Youngeun , Kim Yunyeon , Chung Joo-Yeon , Moon Soojung , Youn Hyunseong , Kim Han-sol , Park Kyung Mi , Park Hansoo 
Published: June 2022

Abstract
The immunotherapeutic treatment has profoundly improved the clinical outcomes in patients with advanced cancers. However, most patients still have not responded to immune checkpoint inhibitor (ICI) treatment. Thus, it is still urgent to find novel treatments for patients who have no response to ICI and acquire ICI resistance. We previously suggested that contactin 4 (CNTN4), known initially to act as an axon guidance molecule during neural development, is a novel inhibitory immune checkpoint protein. In addition, we confirmed that CNTN4 is highly expressed on the various types of human tumor tissue, and anti-CNTN4 antibodies, GENA-104A07 and GENA-104A11, show the anti-tumor efficacy by enhancing T cell activity. In the present study, we describe the role of amyloid precursor protein (APP) expressed on T cells as a binding partner of CNTN4 and the preclinical characterization of GENA-104A16 as an immune-oncology drug candidate targeting CNTN4. Firstly, we investigated the APP expression on T cells and found that APP was detected in T cells' membrane and cytosolic fraction. Its expression level was higher on activated T cells. In addition, we observed that the immune-suppressive activity of CNTN4 on APP KO T cells was significantly decreased compared with APP WT T cells, which means APP as a binding partner of CNTN4 is a core molecule for transmitting immunosuppressive signal of CNTN4 to T cells. GENA-104A16 is a humanized antibody and binds to human and mouse CNTN4 with high binding affinity, which is matured from GENA-104A07 and GENA-104A11. In the in vitro functional assays, GENA-104A16 showed the improved effects on the restoration of T cell proliferation and activation (i.e., inflammatory cytokine releases from T cells and T cell activity-related signaling cascade) suppressed by CNTN4 and on CTL (cytotoxic T lymphocytes)-mediated killing for human cancer cell lines expressing CNTN4. In line with these effects, GENA-104A16 produced higher anti-tumor effects than GENA-104A11 in the CT26 syngeneic mouse colon cancer model. To identify the in vivo immune modulatory function of GENA-104A16, we analyzed the proportion of tumor-infiltrated T cells, Treg cells, and T cell subsets expressing perforin and granzyme B in CT26 tumor tissues. As a result, the population of CD4+ T cells, CD8+ T cells, and cytotoxic CD8+ T cells expressing perforin or granzyme B tended to increase in the tumors of GENA-104A16 treated mice while decreasing the FoxP3+CD25+ Treg population. Interestingly, GENA-104A16 treatment showed better anti-tumor effects together with the elevated level of CD8+ T cells expressing perforin and granzyme B than atezolizumab treatment. Overall, these preclinical studies demonstrate that GENA-104A16 is a promising blocking antibody for CNTN4-mediated immune suppression and can serve as an effective immune-oncology drug for patients with cancer that highly express CNTN4.
Citation Format: Mi Young Cha, Hnkyung yu, Bu-Nam Jeon, Hyunuk Kim, Youngeun Ha, Yunyeon Kim, Joo-Yeon Chung, Soojung Moon, Hyunseong Youn, Han-sol Kim, Kyung Mi Park, Hansoo Park. Novel immune checkpoint protein CNTN4 and the preclinical efficacy of affinity matured anti-CNTN4 antibody GENA-104A16 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2862.