Abstract C001: Rapid and efficient generation of format-diverse co-stimulatory CD28 and CD3 multispecific antibody panels for T cell engagement via complementary technologies

Abstract Background: Currently approved monospecific antibody checkpoint inhibitors are often insufficiently effective for all patients and/or indications. Thus, investigators are interested in targeting multiple signaling pathways and/or cell types to enhance clinical outcomes, often in the form of multispecific antibody treatments (‘multispecifics’). Many of these multispecifics target the T cell as T cell engagers (TCEs), with the majority targeting CD3 and a tumor-associated antigen (TAA) to activate T cell-dependent cellular cytotoxicity. Co-stimulation by targeting CD28 or CD137 in addition to CD3 has recently shown promise. However, the large number of possible topologies and added complexity for manufacturing necessitate the development and application of a robust set of complementary technologies that facilitate generation and allow for functional screening of large multispecific panels. Methods: Co-stimulatory antibodies against CD28 were identified from synthetic IgG (kappa & lambda) and heavy chain-only antibody (HCAb) libraries using a yeast-based antibody platform. Discovered CD28 antibodies were subsequently affinity optimized to generate a panel of leads with varied epitopic e breadth, affinity, and good developability. Bispecific CD28 x HER2 leads were subsequently generated using a number of internally developed chain pairing technologies1,3. Bispecific CD28 antibodies were then assessed for their ability to stimulate T cell-dependent cellular cytotoxicity when combined with a broad affinity panel of CD3 antibodies2. Results: Mutation sets have been identified that drive greater than 95% desired HC-LC pairing and HC-HC heterodimerization when producing bispecific antibodies in a single transfection system. An alternative set of mutations is amenable to producing large arrays of bispecific antibodies using a Cchain Eexchange (ChEx) reaction. A format-diverse panel of anti-CD28 antibodies have has been discovered and used to generate bispecific antibodies using our pairing mutation sets. The resulting bispecific antibodies have been shown to provide costimulatory signal and enhance tumor cell killing in redirected T cell cytotoxicity (RTCC) assays when paired with an anti-CD3 bispecific antibody. Conclusion: We have developed an exemplary set of technologies that demonstrate the ability to direct desired antibody chain pairing (HC-HC and HC-LC), isolate and engineer single-domain antibodies, as well as generate large panels of multispecific antibodies with diverse topologies from a limited number of input molecules. Specifically, we illustrate these capabilities in the context of TCE cell engaging (TCE) multispecifics that leverage our affinity- and developability-optimized anti-CD3 and anti-CD28 antibody panels. References [1] Barlow, K. A. et al, MABs 2025, 17(1): 2479531 [2] Liu, CY et al, MABs 2023, 15(1): 2189974 [3] Pejchal R. et al, Antibodies 2023, 12(3): 54 Citation Format: Ross Connor. Rapid and efficient generation of format-diverse co-stimulatory CD28 and CD3 multispecific antibody panels for T cell engagement via complementary technologies [abstract]. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr C001.