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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.

Gas6 and its receptors Axl, Mer and Tyro-3 (TAM) are highly expressed in human malignancy suggesting that signaling through this axis may be tumor-promoting. In pancreatic ductal adenocarcinoma (PDAC), Gas6 and the TAM receptor Axl are frequently co-expressed and their co-expression correlates with poor survival. A strategy was devised to generate fully human neutralizing antibodies against Gas6 using XenoMouse® technology. Hybridoma supernatants were selected based on their ability to inhibit Gas6 binding to the receptor Axl and block Gas6-induced Axl phosphorylation in human cells. Two purified antibodies isolated from the screened hybridomas, GMAB1 and GMAB2, displayed optimal cellular potency which was comparable to that of the soluble extracellular domain of the receptor Axl (Axl-Fc). In vivo characterization of GMAB1 was conducted using a pharmacodynamic assay that measured inhibition of Gas6-induced Akt activation in the mouse spleen. Treatment of mice with a single dose (100-1000 µg) of GMAB1 led to greater than 90% inhibition of Gas6-induced phosphorylated Akt (pAkt) for up to 72 hr. Based on the target coverage observed in the PD assay, the efficacy of GMAB1 was tested against human pancreatic adenocarcinoma xenografts. At doses of 50 µg and 150 µg, twice weekly, GMAB1 was able to inhibit 55% and 76% of tumor growth, respectively (p < 0.001 for both treatments vs. control Ig). When combined with gemcitabine, GMAB1 significantly inhibited tumor growth compared to either agent alone (p < 0.001). Together, the data suggest that Gas6 neutralization may be important as a potential strategy for the treatment of PDAC.

Drug discovery opportunities where loss-of-function alleles of a target gene link to a disease-relevant phenotype often require an agonism approach to up-regulate or re-establish the activity of the target gene. Antibody therapy is increasingly recognized as a favored drug modality due to multiple desirable pharmacological properties. However, agonistic antibodies that enhance the activities of the target enzymes are rarely developed because the discovery of agonistic antibodies remains elusive. Here we report an innovative scheme of discovery and characterization of human antibodies capable of binding to and agonizing a circulating enzyme lecithin cholesterol acyltransferase (LCAT). Utilizing a modified human LCAT protein with enhanced enzymatic activity as an immunogen, we generated fully human monoclonal antibodies using the XenoMouse(TM) platform. One of the resultant agonistic antibodies, 27C3, binds to and substantially enhances the activity of LCAT from humans and cynomolgus macaques. X-ray crystallographic analysis of the 2.45 Å LCAT-27C3 complex shows that 27C3 binding does not induce notable structural changes in LCAT. A single administration of 27C3 to cynomolgus monkeys led to a rapid increase of plasma LCAT enzymatic activity and a 35% increase of the high density lipoprotein cholesterol that was observed up to 32 days after 27C3 administration. Thus, this novel scheme of immunization in conjunction with high throughput screening may represent an effective strategy for discovering agonistic antibodies against other enzyme targets. 27C3 and other agonistic human anti-human LCAT monoclonal antibodies described herein hold potential for therapeutic development for the treatment of dyslipidemia and cardiovascular disease.

LCAT is intimately involved in HDL maturation and is a key component of the reverse cholesterol transport (RCT) pathway which removes excess cholesterol molecules from the peripheral tissues to the liver for excretion. Patients with loss-of-function LCAT mutations exhibit low levels of HDL cholesterol and corneal opacity. Here we report the 2.65 A crystal structure of the human LCAT protein. Crystallization required enzymatic removal of N-linked glycans and complex formation with a Fab fragment from a tool antibody. The crystal structure reveals that LCAT has an α/β hydrolase core with two additional subdomains that play important roles in LCAT function. Subdomain 1 contains the region of LCAT shown to be required for interfacial activation, while subdomain 2 contains the lid and amino acids that shape the substrate binding pocket. Mapping the naturally occurring mutations onto the structure provides insight into how they may affect LCAT enzymatic activity.

Gas6 and its receptors Axl, Mer and Tyro-3 (TAM) are highly expressed in human malignancy suggesting that signaling through this axis may be tumor-promoting. In pancreatic ductal adenocarcinoma (PDAC), Gas6 and the TAM receptor Axl are frequently co-expressed and their co-expression correlates with poor survival. A strategy was devised to generate fully human neutralizing antibodies against Gas6 using XenoMouse® technology. Hybridoma supernatants were selected based on their ability to inhibit Gas6 binding to the receptor Axl and block Gas6-induced Axl phosphorylation in human cells. Two purified antibodies isolated from the screened hybridomas, GMAB1 and GMAB2, displayed optimal cellular potency which was comparable to that of the soluble extracellular domain of the receptor Axl (Axl-Fc). In vivo characterization of GMAB1 was conducted using a pharmacodynamic assay that measured inhibition of Gas6-induced Akt activation in the mouse spleen. Treatment of mice with a single dose (100-1000 µg) of GMAB1 led to greater than 90% inhibition of Gas6-induced phosphorylated Akt (pAkt) for up to 72 hr. Based on the target coverage observed in the PD assay, the efficacy of GMAB1 was tested against human pancreatic adenocarcinoma xenografts. At doses of 50 µg and 150 µg, twice weekly, GMAB1 was able to inhibit 55% and 76% of tumor growth, respectively (p 0.001 for both treatments vs. control Ig). When combined with gemcitabine, GMAB1 significantly inhibited tumor growth compared to either agent alone (p 0.001). Together, the data suggest that Gas6 neutralization may be important as a potential strategy for the treatment of PDAC.

Growth arrest specific 6 (Gas6) is a vitamin-K dependent protein that is involved in the regulation of a wide array of cellular activities, including adhesion, migration, immune evasion, and apoptosis. Gas6 as well as its receptors Axl, Mer, and Tyro3 are highly expressed in multiple malignancies suggesting that signaling through this axis may be tumor promoting in human disease. A strategy was devised to generate fully human neutralizing antibodies against Gas6 using XenoMouse technology. Hybridoma supernatants were first screened for their ability to bind Gas6, then for their ability to inhibit Gas6 binding to the Axl receptor and block Gas6-induced Axl phosphorylation in human cells. Two purified antibodies isolated from the screened hybridomas, 1D9.1 and 1G9.1, maintained potency in all 3 assays and also inhibited Gas6-induced cellular migration and proliferation. In the scratch wound migration assay, the antibodies inhibited both basal and Gas6-induced wound closure to a greater extent than the soluble extracellular domain of the receptor Axl (Axl- Fc). In the proliferation assay, the antibody 1D9.1 completely inhibited Gas6-induced proliferation with a half maximal effective concentration (EC50) of 0.15nM. In vivo characterization of one of the antibodies, 1D9.1, was conducted using a pharmacodynamic (PD) assay that measured the ability of the antibody to inhibit Gas6-induced Akt activation in the mouse spleen. Treatment of mice with a single dose (100-1000 μg) of 1D9.1 led to greater than 90% inhibition of Gas6-induced phosphorylated Akt (pAkt) for up to 72 hours. Based on the target coverage observed in the PD assay, we tested the efficacy of 1D9.1 against Panc-1 human pancreatic adenocarcinoma xenografts implanted in athymic nu/nu female mice. At doses of 50 and 150 μg, twice weekly, the Gas6 neutralizing antibody 1D9.1 was able to inhibit 55% and 76% of tumor growth, respectively. (p Citation Format: Gordon E. Moody, Brian Belmontes, Chadwick King, Shuying Liu, Stephanie Masterman, Chris Murawsky, Trace Tsuruda, Wei Wang, Robert Radinsky, Pedro J. Beltran. Generation of a fully human Gas6 neutralizing antibody with anti-tumor activity in vivo. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5158. doi:10.1158/1538-7445.AM2013-5158

Passive therapy with neutralizing human monoclonal antibodies (mAbs) could be an effective therapy against severe acute respiratory syndrome coronavirus (SARS-CoV). Utilizing the human immunoglobulin transgenic mouse, XenoMouse®, we produced fully human SARS-CoV spike (S) protein specific antibodies. Antibodies were examined for reactivity against a recombinant S1 protein, to which 200 antibodies reacted. Twenty-seven antibodies neutralized 200TCID50 SARS-CoV (Urbani). Additionally, 57 neutralizing antibodies were found that are likely specific to S2. Mapping of the binding region was achieved with several S1 recombinant proteins. Most S1 reactive neutralizing mAbs bound to the RBD, aa 318–510. However, two S1 specific mAbs reacted with a domain upstream of the RBD between aa 12 and 261. Immunoglobulin gene sequence analyses suggested at least 8 different binding specificities. Unique human mAbs could be used as a cocktail that would simultaneously target several neutralizing epitopes and prevent emergence of escape mutants.