Abstract 6366: The triple-specific antibody KA-3009 against EGFR, B7H3, and 4-1BB specifically enhances T cell activity within the tumor

Abstract Cancer immunotherapy with 4-1BB agonists faces limited further clinical development due to dose-limiting toxicity. Moreover, EGFR and B7H3 are highly expressed in various solid tumors, as well as in different normal tissues. In this study, we developed a trispecific antibody, KA-3009, targeting EGFR, B7-H3, and human 4-1BB, with the aim of restricting 4-1BB stimulation within tumors. KA-3009 was developed based on our common light chain bispecific antibody discovery platform and nanobody antibody discovery platform using knob-into-hole technology. It is in IgG1 format with mutations in the Fc domain to eliminate ADCC, ADCP, and CDC activity. Featuring two identical nanobodies at the C-terminal against 4-1BB, KA-3009 binds to EGFR and B7H3 respectively with two Fabs at the N-terminal, sharing an identical light chain. KA-3009 exhibits low avidity binding to tumor cells expressing only EGFR or B7H3, while binding to tumor cells co-overexpressing EGFR and B7H3 with increased avidity by more than ten times. Additionally, KA-3009 binds to 4-1BB expressed on 293T cells and activated human T cells. In the presence of a low concentration of CD3 antibody to stimulate T cell activation, co-culture of tumor cells co-expressing EGFR and B7H3 with primary T cells isolated from human PBMC enhances T cell activation and proliferation. In PBMC humanized immunodeficient mouse tumor models, KA-3009 effectively suppresses the growth of NCI-H1568 tumors expressing both EGFR and B7H3 but does not affect the growth of MS751 and NCI-H661 tumors, which only express EGFR or B7H3, respectively. Moreover, KA-3009 has a half-life of more than five days in the blood of Balb/c mice. Finally, we assessed the developability of this antibody with different treatments such as high concentration, low pH, repeated freezing and thawing, and high temperature, and KA-3009 shows neither aggregation nor degradation. In conclusion, the triple-specific antibody KA-3009, developed with our common light chain antibody discovery platform and nanobody antibody discovery platform, is a promising pre-clinical candidate drug for the treatment of different solid cancers that co-overexpress EGFR and B7H3. Citation Format: Guojin Wu, Tongtong Liu, Jiabei Liang, Hao Peng, Feng Hao, Jinying Ning. The triple-specific antibody KA-3009 against EGFR, B7H3, and 4-1BB specifically enhances T cell activity within the tumor abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 6366.