Abstract 4240: In vivo assessment of AICD and cytokine release upon treatment of OKT3 and CD3 bispecific antibody in homozygous CD3EDG HuGEMM

Introduction: CD3 bispecific antibodies have transformed the therapeutic landscape of immunotherapy for some hematological malignancies, however, the clinical therapeutic effects of CD3 bispecific therapies is still under investigation for solid tumors. Moreover, CD3 bispecific antibody-mediated tumor targeting as well as T cell engagement have resulted in cytokine release syndrome (CRS), increased on-target/off-tumor toxicities and poor T cell infiltration, which may affect safety and limit therapeutic efficacy. To better understand the relationship between T cell activation and systemic cytokine release in the context of CD3 bispecific antibody treatment, a homozygous human CD3EDG HuGEMM࣪ preclinical model was developed. Methods: The homozygous CD3EDG HuGEMM mice, which express human CD3ε/CD3δ/CD3γ on the surface of T cells, were subcutaneously inoculated with MC38 or MC38-hEpCAM HuCELL™ syngeneic tumors and treated with vehicle, anti-mPD-1 (RMP1-14), OKT3, and anti-hCD3×hEpCAM. For efficacy studies, mouse body weight and tumor volume were measured twice weekly and tumor growth inhibition (TGI%) was determined. To investigate OKT3 or CD3 bispecifics-induced cytokine release, mouse body weight was monitored daily and several inflammatory and proinflammatory cytokines in the serum were detected by MSD. Results: Firstly, anti-mPD-1 treatment (5 mg/kg and 15 mg/kg i.p.) of homozygous CD3EDG mice engrafted with MC38 syngeneic tumors led to 42% and 62% TGI, respectively. OKT3 treatment of MC38-hEpCAM bearing homozygous CD3EDG HuGEMM promoted tumor growth (TGI= -80%), likely due to activation-induced cell death (AICD) of T cells. In addition, CRS was induced within 24 hours of OKT3 dosing, with IL-2, IL-6, and TNF-α peaking 2 hr post the 1st dose, and IFN-γ, IL-10 and IL-5 peaking 6 hr post the 1st dose. In contrast, no significant cytokine release was observed upon anti-hCD3×hEpCAM treatment despite remarkable TGI, indicating that the delicate balance of in vivo T cell activation and T cell cytotoxicity is crucial for effective tumor killing by CD3 bispecific antibodies or engagers. Conclusions: Our CD3EDG HuGEMM and HuCELL models provide a predictive platform for in vivo efficacy and safety assessment of CD3 bispecific or trispecific antibodies. The uncoupling of systemic cytokine release and T cell cytotoxicity via CD3 bispecific or trispecific antibodies provides a biological rationale to clinically explore preventative treatment approaches to avoid or redu