Checkpoint Antibodies but not T Cell-Recruiting Diabodies Effectively Synergize with TIL-Inducing γ-Irradiation.

T cell-recruiting bispecific antibodies (bsAbs) show promise in hematologic malignancies and are also being evaluated in solid tumors. In this study, we investigated whether T cell-recruiting bsAbs synergize with hypofractionated tumor radiotherapy (hRT) and/or blockade of the programmed death-1 (PD-1) immune checkpoint, both of which can increase tumor-infiltrating lymphocyte (TIL) numbers. Unexpectedly, large melanomas treated with hRT plus bsAb (AC133xCD3) relapsed faster than those treated with hRT alone, accompanied by massive TIL apoptosis. This fast relapse was delayed by the further addition of anti-PD-1. Mechanistic investigations revealed restimulation-induced cell death mediated by Bim and Fas as an additional cause of bsAb-mediated TIL depletion. In contrast, the double combination of hRT and anti-PD-1 strongly increased TIL numbers and even very large tumors were completely eradicated. Our study reveals the risk that CD3-engaging bsAbs can induce apoptotic TIL depletion followed by rapid tumor re-growth, reminiscent of tolerance induction by CD3 mAb-mediated T cell depletion, warranting caution in their use for the treatment of solid tumors. Our findings also argue that combining radiotherapy and anti-PD-1 can be quite potent, including against very large tumors.