A cyno cross reactive CD3 VHH antibody enables engineering of a potent CD20-targeted t cell engager with enhanced safety

Abstract Introduction: Due to the clinical successes of multiple B cell-depleting chimeric antigen receptor (CAR)-T cell and T cell engager (TCE) immunotherapies, great interest is being invested in extending these modalities into B cell-mediated autoimmune disorders; however, the tolerance for risk of adverse events caused by excessive cytokine release (e.g., cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS)) will be dramatically lower in autoimmune indications than it currently is for oncology use. While CAR-T therapies inherently pose a greater risk of CRS and ICANS due to more potent T cell activation resulting from avidity-based antigen binding and costimulatory signaling (e.g., CD28, 4-1BB) from within the CAR construct, multiple commercial TCEs have also demonstrated significant levels CRS (epcoritamab: 51%; glofitamab: 70%) and ICANS (epcoritamab: 6%; glofitamab: 5%). While cytokine release cannot be fully decoupled from T cell-mediated cytotoxicity, measures can be taken to reduce it while maintaining target cytotoxicity such as adjusting the binding valency or affinity of the CD3 or B cell-binding arms and optimizing the geometrical configuration of the overall TCE to maintain a proper immune synapse. Single domain, VHH antibodies simplify this process by allowing for more complex bispecific antibody (bsAb) geometries due to their smaller size, better stability, and making light chain mispairing irrelevant; therefore, we set forth to develop a CD3 VHH antibody to accelerate the development of a CD20-targeted TCE with optimal cytotoxicity and a reduced cytokine release profile. Additionally, we seek to make the CD3 binding arm to be cyno cross reactive so as to enable preclinical non-human primate (NHP) studies to more accurately assess the risk of CRS and ICANS. Methods: Following humanization and affinity maturation, a set of cyno cross reactive VHH antibodies with a range of CD3 affinities and T cell-activating potencies was developed from a camel-derived, parental clone. One of the CD3 VHH antibodies with moderate binding affinity (~20 nM) was selected and paired with rituximab and engineered into over 40 different bsAb configurations, differing in binding valency and geometry. The resulting CD20-targeted TCEs were evaluated in vitro and in vivo for their cytotoxic potency, antitumor efficacy, pharmacokinetics, cytokine release profile, and systemic toxicity. Results: Evaluation of multiple CD20 × CD3 TCE configurations led to the selection of four lead molecules with comparable cytotoxic potencies against CD20+ cells compared to glofitamab, while the CRS/ICANS-associated cytokine release (IL-2, IFN-γ, TNF-α) is lower upon incubation with PBMCs across multiple donors. Cell line-derived xenograft studies in mice revealed that one of the lead TCEs had similar antitumor efficacy as glofitamab at equivalent dose levels. No apparent systemic toxicities were observed across all tested TCEs and doses. Conclusions: Our affinity-optimized CD3 VHH antibodies, with a spectrum of affinities and potencies, allowed us to rapidly test multiple CD20 × CD3 TCE configurations and ultimately led us to identify a potentially safer, therapeutic, CD20-targeted TCE due to its ability to reduce cytokine release without sacrificing target cytotoxicity. The potentially reduced risk of CRS and ICANS would better position our lead TCE for use in B cell-mediated autoimmune indications, and we are currently conducting NHP studies to more accurately evaluate the safety benefit. As the limited homology of surface-exposed regions between human and cyno CD3 makes it exceptionally difficult to obtain an agonistic, cross reactive CD3 antibody, our cyno cross reactive CD3 VHH antibody serves as an ideal platform to develop better TCEs for autoimmunity and oncology.