Background T-cell engagers (TCEs) have established their therapeutic value in treatment of various cancers by harnessing the power of immune system against tumors in a targeted manner. IgM based TCEs may offer high avidity and safety advantages over other modalities because of its multivalent architecture and unique positioning of CD3 binder. We have built a costimulatory IgM TCE platform that engages both signal 1 and signal 2 on T-cells, with the goal of enabling their optimal activation and survival for more robust and durable cytotoxic activity. IgM platform is designed to allow this dual engagement while maintaining full avidity advantage for tumor targeting.
Methods IgM TCEs targeting CD20/solid tumor TAAs were generated by transient expression of heavy, light and J-chain cassettes in 293 cells. Optimized CD3 and CD28 binding scFvs were placed at either ends of the J-chain. Initial T-cell activation of various purified IgMs was evaluated by culturing Jurkat cells expressing IL2-promoter driven luciferase reporter with/without target cells. In vitro TDCC assays were done with co-culture of target cells and PBMCs/T-cells isolated from healthy donors at various E/T ratios to evaluate cytotoxicity. Cytokines were measured with multiplexed ELISA. For in vivo activity analyses, Hu-PBMC NSG xenograft mouse models were established with optimized injection and dosing regimens.
Results Costimulatory IgM TCEs engaged both CD3 and CD28 on T-cell surface, resulting in several fold enhancement of in vitro cytotoxicity and T-cell activation. Levels of IL-2 cytokine increased significantly in these assays with dual signal engagement promoting both proliferation and survival of T-cells. In absence of CD3 engagement, CD28 binding alone did not induce any activation, cytotoxicity or cytokines underscoring the safety profile of CD28 binder. Activation of costimulatory IgM TCEs was also target dependent. In long term culture up-to 14 days only costimulatory IgM TCEs promoted T-cell proliferation and survival, and this effect was even more pronounced in low E:T ratios. In Hu-PBMC xenograft tumor models, costimulatory IgM TCEs exhibited strong in vivo anti-tumor activity compared to only CD3 engaging TCEs. Anti-tumor activity of these molecules was maintained for much longer after dosing was stopped indicating the durability of responses.
Conclusions IgM based costimulatory TCEs provide enhanced cytotoxicity through optimal T-cell activation and proliferation/survival. This dual engagement of activation signals, along with high-avidity offered by IgM platform, could enable the creation of effective therapeutics targeting solid tumors that will maintain activity even in conditions with low T-cell counts in tumor microenvironment.
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