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In this study, we describe the characterization and validation of a diverse panel of fully human CD3-binding antibodies, including hundreds of human and cyno cross-reactive binders. We used two proof-of-concept TCE targets to demonstrate that this panel streamlines CD3 T-cell engager (TCE) development, enabling identification of optimal tumor cell-killing and cytokine-release profiles. CD3 TCEs have potential to be powerful cancer treatments, but the small number of available CD3-binding antibodies and limited multispecific engineering technologies have been barriers to development. Identifying TCEs that balance anti-tumor potency with potential toxicities, such as cytokine release syndrome, requires simultaneous tuning of both the CD3- and tumor-binding arms. Pairs of antibodies that achieve this balance are rare, creating a need for diverse panels of developable antibodies that can be combined and tested to identify optimal clinical candidates. To streamline TCE development, we discovered a diverse panel of CD3-binding antibodies. We screened over 5 million single cells from humanized mice and identified 585 unique CD3-specific antibody sequences. Of these, over 170 were identified as cross-reactive to human and cyno CD3 in primary screening. We then used high-throughput characterization to curate a panel of diverse and developable antibodies. We found a wide range of CD3εδ and CD3εγ binding specificities, affinities, and kinetics. Epitope binning analysis revealed multiple bins containing human and cyno cross-reactive binders, some of which are distinct from previously described cross-reactive antibodies, such as SP34-2. We assessed their biophysical properties and identified antibodies with good developability properties, including high thermal stability and low hydrophobicity, self-association, polyspecificity, and aggregation. To validate these antibodies, we used OrthoMab™ to generate proof-of-concept TCE panels with fixed tumor-binding arms. We identified CD3 x EGFR TCEs with high potency, low cytokine release, functional cross-reactivity in a cyno T cell-mediated tumor killing assay, and good pharmacokinetic properties in Tg32 mice. A second proof-of-concept CD3 x PSMA panel further validated our antibodies in bispecific formats. Together, these studies demonstrate that starting with diverse CD3-binding antibodies streamlines identification of developable TCEs with optimal potency and cytokine release. We leveraged data from our extensive characterization of CD3-binding antibodies in mono- and bispecific formats to develop a strategy for down-selection and pairing of CD3- and tumor-binding antibodies, and a high-throughput method for analysis of resulting TCEs. By categorizing antibodies based on functional properties, we are able to rapidly pinpoint optimal potential clinical candidates for specific tumor targets. Citation Format: Juntao (Matt) Mai, Kate Caldwell, Lindsay DeVorkin, Grace P. Leung, Karine Herve, Yuri Hwang, Cristina Faralla, Wei Wei, Emma Lathouwers, Valentine de Puyraimond, Lauren Clifford, Rhys S. Chappell, Stefan Hannie, Katherine J. Lam, Harveer Dhupar, Tran N. Tran, Melissa Cid, Lena M. Bolten, Tova Pinsky, Ping Xiang, Courteney Lai, Ahn Lee, Vivian Z. Li, Patrick Chan, Jasmine Chin, Steve Booth, Amy C. Lee, Stephanie Masterman, Sherie Duncan, Aaron Yamniuk, Kush Dalal, Tim M. Jacobs, Raffi Tonikian, Bryan C. Barnhart. Identifying T-cell engagers with optimal potency and cytokine-release profiles with a diverse panel of CD3-binding antibodies [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 1886.

In this study, we describe the discovery of antibodies against a MAGE-A4 peptide-major histocompatibility complex (pMHC). These antibodies will form the basis for the tumor-binding arm of T-cell engagers (TCEs) against this target. Bispecific CD3 TCEs have the potential to transform cancer treatment by redirecting T cells to tumor targets, but technological barriers have limited their development for solid tumors. Targets for TCEs have generally been limited to surface-expressed proteins, however, access to intracellular proteins that are mutated and/or differentially expressed in cancer cells would expand the target pool. Peptides of these intracellular proteins presented on MHC class I (MHC-I) provide opportunities for TCE development. Technologies powering discovery of rare antibodies that are ultra-specific, high-affinity pMHC binders are needed to expand this promising class of tumor targets. We have developed a technology platform for the discovery of optimal TCEs, including a diverse panel of CD3-binding antibodies and an antibody discovery and development engine that includes multispecific engineering capabilities, powered by OrthoMabTM. We are applying this platform to develop TCEs against MAGE-A4, an intracellular tumor target expressed by many solid tumors, but not by healthy tissues. Using proprietary immunization technologies, we triggered robust, diverse antibody responses against a complex of a human MAGE-A4 peptide presented on MHC-I. We used high-throughput microfluidic technology to screen single B cells using a multiplexed bead-binding assay to identify antibodies specific to the target, but not closely-related pMHCs. We then expressed and purified antibodies for downstream validation and characterization. Antibody specificity was initially validated using a panel of related pMHC complexes, and developability properties were assessed, including hydrophobicity, self-association, polyspecificity, stability, and aggregation. With complex data integration and analysis, we identified a panel of diverse and developable antibodies that bind with high affinity to a human MAGE-A4 peptide sequence of 10 amino acids presented on MHC-I (HLA:02*01). Strategic selection and pairing of these target-binding antibodies with our large and diverse panel of fully human CD3-binders will power the discovery of ultra-specific MAGE-A4 TCEs with optimal potency and cytokine release. Citation Format: Davide Tortora, Peter Bergqvist, Grace P. Leung, Elena Vigano, Antonios Samiotakis, Harveer Dhupar, Wei Wei, Shirley R. Zhi, Yukiko Sato, Allison Goodman, Cindy-Lee Crichlow, Melissa Cid, Jessica Fernandes Scortecci, Ping Xiang, Ahn Lee, Vivian Li, Stephanie Masterman, Sherie Duncan, Aaron Yamniuk, Kush Dalal, Tim Jacobs, Raffi Tonikian, Bryan C. Barnhart. Breaking barriers to access intracellular targets with T-cell engagers: Discovery of diverse, developable, and ultra-specific antibodies against a MAGE-A4 pMHC [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 1891.

Bispecific antibodies that redirect cancer-killing T cells towards tumors are promising next-generation cancer therapies. While there are hundreds of T cell engagers (TCEs) in development, there is only one approved and marketed CD3-binding TCE. The high rate of attrition is largely attributable to dose-limiting toxicities, including cytokine release syndrome, due in part to the small pool of high affinity CD3-binding antibodies that are commonly used. The discovery of safe and effective TCEs is limited because diverse panels of parental CD3 antibodies are hard to produce, the pairing of parentals is hard to perfect, and the sheer complexity and volume of data is hard to action. In this study, we will present a panel of functionally diverse, fully human CD3-binding parental antibodies. We will present data characterizing the diversity of our panel across multiple parameters, including sequence diversity, CD3 affinity, epitope binding, T cell activation, cytokine release, and tumor cell killing. Using OrthoMab࣪, our clinically-validated bispecific engineering platform, allows this panel to be tested with a series of tumor-associated antigens (TAAs) in a matrix format. Results from high-throughput production and characterization of bispecific antibodies will be presented. These multidimensional datasets of TCE composition and function allow for the identification of pairs that are optimal candidates for clinical development. This work will show how the diversity of our CD3-binding panel, combined with a robust bispecific protein engineering technology, can be used to quickly assess large and diverse TAA-binding panels discovered through our technology stack. An integrated workflow that doubles the data with diverse panels of parentals, assembles stable, safe, and manufacturable TCEs, and visualizes multidimensional datasets are critical to successfully identifying lead therapeutic candidates to bring the next generation of cancer therapies to patients sooner. Citation Format: Lindsay DeVorkin, Tim M. Jacobs, Raffi Tonikian, Karine Hervé, Kate Caldwell, Yuri Hwang, Cristina Faralla, Wei Wei, Katherine J. Lam, Harveer Dhupar, Tran NT Tran, Melissa Cid, Lena M. Bolten, Tova Pinsky, Kush Dalal, Kevin A. Heyries, Bryan C. Barnhart. Redirecting T cells to tumor targets with functionally diverse CD3-binding antibodies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 312.