Abstract 3212: Development of a Trop2/CD3 bispecific antibody with modulated Trop2 binding affinity as a novel immunotherapy for advanced gastrointestinal cancer treatment

Abstract Trophoblast cell surface antigen 2 (Trop2) is considered as a potential therapeutic target because its over-expression in tumors is found to be associated with poor prognosis of patients with different cancers. Trop2-directed antibody-drug conjugates have been approved for the treatment of solid tumors, however, the expression of Trop2 in a broad range of epithelial tissues, even in low expression, would limit their clinical benefits. The present work generated a novel Trop2/CD3 bispecific antibody with its binding affinity towards Trop2 being well-modulated to mitigate the chance of on-target off-tumor effect. By linking a humanized anti-Trop2 monoclonal antibody with a CD3-binding scFv, a Trop2/CD3 bispecific antibody was produced. In order to adjust the bispecific antibody's affinity to the tumor target, ProABC-2 was used for in-silico paratope prediction (Ambrosetti F et al. , 2020). A complementarity-determining residue (CDR) variant was produced and its binding to Trop2 antigen was analyzed using biolayer interferometry. The binding of both wild type (WT) and variant antibodies to Trop2-positive cancer cells, the activation of T cells upon target engagement and the redirection of T cell-mediated killing on cancer cells expressing high level of Trop2 were examined. The binding affinity of CDR variant to Trop2-expressing tumor cells was significantly reduced, which led to a slower induction of cytokines and T-cell proliferation, less acute T-cell activation, and a milder cytotoxic effect. This allowed the bispecific antibody to have a higher level of safety by restricting the cytotoxicity only to cancer cells that expressed high level of Trop2. Our novel Trop2/CD3 bispecific antibody not only showed potent anti-tumor activity, but also demonstrated a lower risk of on-target off-tumor toxicity. Monkey toxicity and other IND-enabling studies are beginning to further bring the drug candidate into clinical trial. Table showing EC50 values of Trop2/CD3 bispecific WT and CDR variant antibodies in different assays WT CDR variant Binding affinity (M) Trop2 4. 62E-11 2. 14E-10 Cytotoxicity (M) AsPC1 (low Trop2 level) 1. 24E-11 No killing effect SW480 (medium Trop2 level) 1. 25E-12 2. 25E-11 NCI-H596 (high Trop2 level) 5. 65E-12 5. 89E-10 Receptor occupancy (%) Trop2 0. 65 2. 75 CD3 0. 08 5. 83 Cytokine induction (M) IL-2 4. 82E-11 5. 42E-11 IFNγ 6. 46E-13 2. 59E-11 T cell activation (M) CD25+ marker 2. 02E-12 2. 39E-10 CD69+ marker 3. 81E-13 7. 40E-11 T cell proliferation (M) Ki-67 marker 3. 54E-12 8. 80E-11 Citation Format: Chui Yee O, Po Yee Wong, Kronos Chow, Kwan Wa Wong, Dennis Wong, John Moon Luk, Kwong Fai Wong. Development of a Trop2/CD3 bispecific antibody with modulated Trop2 binding affinity as a novel immunotherapy for advanced gastrointestinal cancer treatment 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 3212.