Effective T cell responses not only require the engagement of T cell receptors (TCRs, "signal 1"), but also the availability of costimulatory signals ("signal 2"). T cell bispecific antibodies (TCBs) deliver a robust signal 1 by engaging the TCR signaling component CD3ε, while simultaneously binding to tumor antigens. The CD20-TCB glofitamab redirects T cells to CD20-expressing malignant B cells. While glofitamab exhibits strong single agent efficacy, adding costimulatory signaling may enhance the depth and durability of T cell-mediated tumor cell killing. We developed a bispecific CD19-targeted CD28 agonist (RG6333, CD19-CD28) to enhance the efficacy of glofitamab and similar TCBs by delivering signal 2 to tumor-infiltrating T cells. CD19-CD28 distinguishes itself from the superagonistic antibody TGN1412, as its activity requires the simultaneous presence of a TCR signal and CD19 target binding. This is achieved through its engineered format incorporating a mutated Fc region with abolished FcγR and C1q binding, CD28 monovalency, and a moderate CD28 binding affinity. In combination with glofitamab, CD19-CD28 strongly increased T cell effector functions in ex vivo assays using lymphoma patient-derived PBMC and spleen samples, and enhanced glofitamab-mediated regression of aggressive lymphomas in humanized mice. Notably, the triple combination of glofitamab with CD19-CD28 with the costimulatory 4-1BB agonist CD19-4-1BBL, offered substantially improved long-term tumor control over glofitamab monotherapy and respective duplet combinations. Our findings highlight CD19-CD28 as a safe and highly efficacious off-the-shelf combination partner for glofitamab, similar TCBs, and other costimulatory agonists. CD19-CD28 is currently in a Phase 1 clinical trial in combination with glofitamab.
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