Abstract 5121: TriTCE Co-stim, next generation costimulatory trispecific T cell engagers for the treatment of solid tumors
Authors: Newhook Lisa , Bhojane Purva P. , Repenning Peter W. , Escanda Diego Perez , Escalante Nichole K. , Zwierzchowski Patricia , Robinson Alec , Clifford Lauren , Pratap Harsh , Douda David N. , Piscitelli Chayne L. , Afacan Nicole J. , von Kreudenstein Thomas Spreter , Weisser Nina E. 
Published: April 2023

Abstract
Bispecific T cell engager (TCE) therapies have exhibited clinical utility against hematological cancers, but limited success in solid tumors. Treatment of solid tumors has additional challenges - including immunosuppressive environments and low T cell infiltration - limiting the antitumor activity of CD3-bispecific TCEs. Conventional T cell activation and sustained proliferation requires signaling via CD3 (signal 1) and costimulatory molecules (signal 2), such as CD28. The balance between signals 1 and 2 is critical for optimal T cell activation – signal 1 in the absence of signal 2 results in T cell anergy, while overactivation via signals 1 and 2 can lead to T cell dysfunction and cytokine release, as observed with toxicities associated with αCD28 superagonist antibodies (Abs). Optimal signal 2 costimulation via CD28 results in improved T cell fitness, activation and proliferation. To improve T cell responses in solid tumors, we developed costimulatory trispecific TCEs (TriTCE Co-stim) that engage CD3, CD28 and a tumor-associated antigen (TAA).
Our novel TriTCE Co-stim Abs were generated using the AzymetricTM and EFECTTM platforms to facilitate heterodimeric TriTCE Co-stim assembly and to knockout Fc gamma receptor interactions, respectively. To limit potential CD28-mediated toxicities, we evaluated a conventional αCD28 agonist paratope and generated a paratope library with varying affinities for CD28. TriTCE Co-stim Abs were engineered with various formats, geometries, paratope affinities, and TAA specificities. To understand the impact of TriTCE Co-stim Abs on T cell activation, we assessed in vitro cytotoxicity, cytokine production and proliferation of primary human CD3 T cells in co-culture with TAA-expressing cancer cell lines. A human PBMC-engrafted xenograft model was used to assess in vivo antitumor activity in a TAAhigh tumor model.
TriTCE Co-stim Abs exhibited a range of cytotoxic potency, with several formats exhibiting greater potency than bispecific TCEs, and induced greater cytotoxicity of tumor cells in long term co-cultures at low effector to target ratios. TriTCE Co-stim Abs exhibited TAA-dependent cytokine release and T cell proliferation, with enhanced IL-2 production and proliferation compared to that induced by bispecific TCEs. Tumor growth regression was observed in vivo following treatment with different TriTCE Co-stim Ab formats. In summary, we identified TriTCE Co-stim Ab formats that exhibit improved proliferation and antitumor activity against multiple TAA targets compared to bispecific TCE, which may translate to improved and more durable antitumor responses in solid tumors with low T cell infiltration. The evaluation of multiple formats, geometries and paratope affinities allowed optimization of activity and selectivity to promote maximal therapeutic index and efficacy, key factors that may contribute to improved clinical outcomes.
Citation Format: Lisa Newhook, Purva P. Bhojane, Peter W. Repenning, Diego Perez Escanda, Nichole K. Escalante, Patricia Zwierzchowski, Alec Robinson, Lauren Clifford, Harsh Pratap, David N. Douda, Chayne L. Piscitelli, Nicole J. Afacan, Thomas Spreter von Kreudenstein, Nina E. Weisser. TriTCE Co-stim, next generation costimulatory trispecific T cell engagers for the treatment of solid tumors. [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 5121.