Abstract 6068: CT111, a novel trispecific PD-1 x CTLA-4 x VEGF single-domain antibody, synergistically targets exhausted T cells and promotes cooperative antitumor effects

Abstract Background: Dual PD-1 and CTLA-4 targeting immunotherapy shows enhanced therapeutic efficacy and is approved across multiple advanced solid tumor indications. In recent clinical trials, combined treatment of anti-PD-(L)1 with anti-VEGF have also been shown to improve clinical outcomes and overcome treatment resistance to either drugs alone. Bispecific antibodies targeting PD-(L)1/CTLA-4 and PD-(L)1/VEGF synergistically potentiates antitumor activity in preclinical models, and more recently, demonstrated enhanced clinical efficacy and tolerability. We developed a de novo, multi-targeting PD-1 x CTLA-4 x VEGF single domain trispecific antibody with enhanced synergistic anti-tumor activity by combining the effects of dual immune checkpoint and VEGF blockade. Methods: Novel function-blocking anti-PD-1, anti-CTLA-4 and anti-VEGF single-domain antibodies generated from protein-immunized camelids were screened, optimized, and humanized using the VHHMAb® AI-powered platform. Multiple VHH leads were combinatorially screened in different Fc-backbone formats for optimal engagement of all three targets. Binding affinity and specificity were evaluated by protein-based ELISA, cell-based FACS and Octet BLI, and ligand blocking activities were measured by competitive ELISA, cell-based FACS and reporter assays. Multitarget interactions and avidity were measured by competitive ELISA, Octet BLI, and cell-based FACS. Bioactivity of PD-1 and CTLA-4 blockade on primary immune cells was evaluated in CMV-recall and SEB assays for cytokine release, and anti-VEGF bioactivity was determined in a HUVEC proliferation assay. Anti-tumor activity of CT111 was assessed in multiple hPBMC-engrafted humanized xenograft tumor models. Results: All three de novo VHH antibodies used as building blocks for CT111 were selected based on strong functional activities that were superior to clinical benchmarks in cell-based assays. CT111 exhibited strong binding and optimal ligand blocking activity to human PD-1, CTLA-4 and VEGF alone and simultaneously in different assay formats. In CMV-recall and SEB stimulation assays, CT111 mediated potent IFN-γ and IL-2 secretion compared to clinical mono-targeting Ab combinations and bispecific benchmarks across multiple donors, and CT111 effectively blocked VEGF-mediated signaling and attenuated HUVEC proliferation. CT111 showed significant antitumor activity compared to clinical benchmarks, including AK112 and AK104, in PBMC humanized tumor models. Conclusion: CT111 is a novel, potential first-in-class single-domain trispecific antibody that can simultaneously target PD-1 and CTLA-4 immune checkpoints on exhausted T cells, while also targeting VEGF-mediated tumor angiogenesis to overcome immune suppression synergistically within solid tumors. Citation Format: John Lee, Huiyuan Tang, Jia Yu, Weihong Wang, Leo Ng, Jing Yu, Yong Wang, Toya N. Baral, Jiaguo Li, Xinhao Wang, Qijun Qian, Wenfeng Xu. CT111, a novel trispecific PD-1 x CTLA-4 x VEGF single-domain antibody, synergistically targets exhausted T cells and promotes cooperative antitumor effects [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 6068.