470 Towards optimizing clinical performance of anti-cancer immunotherapies: Anti-CD3epsilon Mono-Fab enhances T cell anti-tumor responses without inducing immune related adverse effects

<h3>Background</h3> Despite the clinical success of immune checkpoint inhibitors (ICIs) against several cancers, the overall rates of about 20–40% response in patients are not satisfying. While the toxicity of ICI’s treatments relates with the heterogeneous mechanisms they exert on the immune system, curtailing clinical utility and patient’s outcome, several studies suggest a positive correlation between ICI’s immune-related toxicities and their anti-tumor efficacy. Thus, the conundrum of how to optimize performance of immunotherapies against cancer without enhancing their toxicity remains unsolved. <h3>Methods</h3> We have developed a novel anti-tumor immunotherapy. This strategy uses an anti-CD3ε Monovalent Fab fragment (Mono-Fab) that binds to the TCR/CD3 complex on T cells, enhancing T cell capacity to recognize and target tumor antigens. Our objective for this study is to determine the therapeutic efficacy and immune-toxicity of anti-CD3ε Mono-Fabs in comparison with ICIs such as anti-CTLA-4 and anti-PD-1 mAbs that are already used in the clinic. To compare anti-tumor efficacy of anti-CD3ε Mono-Fab with ICIs we utilized a collection of YUMM melanoma cell lines of increasing immunogenicity (YUMM1.7 &lt; YUMMER1.7) implanted in B6 mice. To compare their drive of autoimmune pathologic responses, we used an established experimental autoimmune encephalomyelitis (EAE) B6 mouse model. <h3>Results</h3> Anti-CD3ε Mono-Fab, alongside with anti-PD-1 as a single therapy were less efficient than anti-CTLA-4 mAb against the more immunogenic melanoma cell line YUMMER1.7 when implanted in B6 mice. However, none of these therapies used in isolation were effective against the less immunogenic melanoma variant YUMM 1.7. Yet, combining anti-CD3ε Mono-Fab with ICIs; anti-CTLA-4 and/or anti-PD-1 mAbs delivered superior therapeutic effects against the less immunogenic YUMM 1.7 variant. Furthermore, treatments including anti-CTLA-4 mAb (alone or combined) increased EAE severity while altering both innate and adaptive immune profiles, increased cytokine production, as well as increased recruitment of myeloid infiltrating cells into the CNS. Strikingly, treatment with anti-CD3ε Mono-Fab did not increase EAE severity in B6 mice, neither when used in isolation nor when combined with ICIs, anti-CTLA-4 and/or anti-PD-1 mAbs. <h3>Conclusions</h3> Anti-CD3ε Mono-Fab is an effective immunotherapy against melanoma in mice that lacks capacity to induce immune-toxicities as observed with anti-CTLA-4 and anti-PD-1 mAbs, and that can be used to enhance anti-tumor efficacy of these ICIs.