Discovery of CD28 targeting single domain antibodies to facilitate the development of costimulatory T cell engager

Optimal T-cell activation is a multistep process requiring T-cell receptor engagement by peptide-MHC complexes (Signal 1) coupled with costimulatory signals (Signal 2). The lack of Signal 2 increases the TCR activation-induced cell apoptosis and decreases T cell proliferation, which may lead to compromised therapeutic activity of anti-PD1 therapy or CD3 T cell engagers (TCEs) in the clinic. The purpose of this study is to identify CD28 single domain antibodies to develop co-stimulatory T cell engager to improve T cell proliferation, survival and response durability, and therefore further expand the utility of checkpoint blockade and/or CD3 TCE therapies for treatment of a broad range of solid tumors. The agonistic propriety of CD28-targeting VHH or CD28-based bi-specific TCE (BiTCE) or tri-specific TCE (TriTCE) was evaluated by analysis of IL-2 secretion in PBMC/T cell activation assay. T cell survival was measured by the expression of anti-apoptosis marker Bcl-xl after treatment of CD28-based antibody. Tumor cells were incubated with T cells together with CD28-based bi- or tri-TCE in the presence of CD3 TCE or anti-PD-1 mAb. Viability of target cells were then measured to reflect T cell killing activity. Xenograft mice models were further used to confirm the benefit of integrating CD28-targeting agonist into CD3 TCE. A series of CD28-targeting agonistic VHHs binding to CD28 with different binding affinities in a non-superagonistic manner were obtained. The co-stimulatory effect of CD28-targeting T cell engagers were demonstrated: (1) CD28xB7-H3 BiTCE significantly improved T cell killing potency and durability on OVCAR-3 cells when combined with CD3 TCE and strongly activated T cells and enhanced T cell killing potency of A375 cells when combined with anti-PD-1 mAb; (2) CD3/CD28/TAA TriTCE were generated by screening of the most suitable CD28 agonist and delicate format design. Both CD3xROR1xCD28 and CD3xMUC16xCD28 significantly improve T cell survival which in turn allows more persisted T cell killing of target tumor cells in vitro and in vivo, compared to CD3xROR1 and CD3xMUC16, respectively. Therefore, including CD28 signaling during CD3 TCE and immune checkpoint blockade treatment may improve the therapeutic benefit in patients with solid tumors.