Novel CD3 binders enable T cell engagers with potent tumor control, limited cytokine release, and safe pairing with co-stimulation

Introduction: T cell engagers (TCEs) have demonstrated clinical promise in hematologic malignancies, but their application in solid tumors has been held back by significant side effects and limited efficacy. TCEs that bind CD3 with SP34-derived clones exhibit marked polyreactivity, leading to off-target activation and narrow therapeutic windows. Leveraging a unique nanoparticle-based immunization platform, we discovered non-polyreactive, non-human primate (NHP) cross-reactive CD3 binders, many against novel CD3 epitopes. As exemplified by a CD3deltaε selective clone, SCRI-6, these binders drive potent anti-tumor activity with reduced cytokine production. Our data suggests that these novel binders are uniquely positioned to pair with TCE designs that incorporate costimulation strategies to improve T cell antitumor activity and persistence. Methods: Our antibody campaign used a novel immunization strategy in the OmniRat transgenic platform incorporating whole-cell and protein-nanoparticle immunogens. Identified hits from the campaign were produced as recombinant monoclonal antibodies and biochemically and biophysically characterized. Clinical benchmark TCEs incorporating our novel CD3 binders were evaluated for non-specific activation, antigen-dependent tumor-cell killing and cytokine release in vitro and in vivo. Results: We identified 12 distinct sequence families, 7 of which bound and activated human T cells. Among these, 5 showed no detectable polyreactivity. Four clones recognized both CD3deltaε and CD3gammaε subunits, whereas one clone (SCRI‑6) bound exclusively to CD3deltaε. Notably, 3 of these sequence families, including SCRI‑6, also activated NHP T cells. TCEs (targeting PD-L1, DLL3, and CD19) that incorporate our novel CD3 binders demonstrated antigen-dependent cytotoxicity comparable to SP34-based TCEs but with reduced cytokine release (e.g., IFNgamma, IL-2, TNFalpha, and IL-6). In an assay to assess tumor-independent activation of T cells, TCEs including SCRI-6 exhibit dramatically less non-specific T cell activation than matched-TCEs containing SP34. Adding CD28 or 4-1BB costimulation to this assay dramatically exacerbated the non-specific activation caused by SP34-containing TCEs; however, swapping the CD3 binder for SCRI-6 eliminated this activity. TCEs incorporating SCRI-6 demonstrate equivalent potency to clinical benchmark comparators in both solid and liquid in vivo tumor models, but with substantially lower cytokine release. Conclusion: Non-polyreactive CD3 engagers may be incorporated into TCEs to improve therapeutic index without sacrificing potency and allowing for therapeutic strategies that safely engage T cell costimulatory targets.