LAUSR

T-cell exhaustion is defined as T-cell dysfunction leading to loss of effector T-cells function. Restoring exhausted T-cells with agonistic anti-4-1BB monoclonal antibody is a promising strategy for cancer treatment. Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) is a classical target with specific expression profiles in tumors. It has limited expression in normal adult tissues, but is overexpressed in multiple types of cancer, especially in colorectal carcinoma and non-small cell lung cancer. Here, we reported a novel bispecific antibody, LM-24C5 that specifically activates 4-1BB in a CEACAM5-dependent manner with localized T cell activation and reduced systemic toxicity. LM-24C5 was developed by introducing qualified 4-1BB heavy-chain variable into a human IgG1 CEACAM5 monoclonal antibody with disabled FcgR-mediated function. LM-24C5 was evaluated for its binding activity to human CEACAM5 and 4-1BB through protein and cell-based assays. The binding activity of LM-24C5 to tumor cells endogenously expressing CEACAM5 and activated primary T cells was confirmed. LM-24C5 was further evaluated in 4-1BB signalling reporter and co-culture assay with human peripheral blood mononuclear cells (PBMC). In vivo anti-tumor activity of LM-24C5 was assessed in hu4-1BB knock-in mice implanted with colon cancer cell line MC38 overexpressing huCEACAM5. Percentage of tumor infiltrating T cells and central memory CD8+ T cells were evaluated by FACS analysis. In addition, 2-week repeated dose toxicity study of LM-24C5 was conducted in hu4-1BB/4-1BBL double-transgenic mice. LM-24C5 efficiently bound to huCEACAM5-postitive cells (EC50: 3.78 nM) and activated human primary T cells. It induced superior 4-1BB activity (EC50: 1.07 nM) than benchmark antibody Urelumab (EC50: 3.38 nM) in the presence of cells expressing CEACAM5. The strength of 4-1BB activation induced by LM-24C5 was correlated with CEACAM5 expression levels. LM-24C5 was also found to stimulate T-cell activation (CD4+ EC50: 0.471, CD8+: 0.482) in PBMCs in a CEACAM5-dependent manner leading to significant IFN-γ production. Injection of LM-24C5 led to complete tumor regression around one month post treatment. Moreover, the tumor-free mice were resistant to tumor rechallenge, suggesting that LM-24C5 can induce long-term protective immunological memory. We have also observed that LM-24C5 increased tumor infiltrating lymphocytes and percentage of central memory CD8+ T cells in spleens. LM-24C5 was well tolerated in hu4-1BB/4-1BBL double-transgenic mice at a dose of 100mg/kg given once weekly for 2 weeks. In summary, LM-24C5 is a novel CEACAM5 dependent 4-1BB bispecific agonist antibody that could redirect and activate T cells to CEACAM5 positive tumor cells by engaging 4-1BB antigen, thus positioned as a potential novel therapy for colorectal carcinoma and other CEACAM5 positive tumors. Citation Format: Wei Cao, Jianjian Liu, Wentao Huang, Junwei Yang, Lei Shi, Yuan Li, Te Du, Xia Qin, Da Fei, Runsheng Li. Pre-clinical efficacy and toxicity profile of LM-24C5: A novel CEACAM5 x 4-1BB bispecific antibody in cancer immunotherapy [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 2650.