Bivalent bispecific CD28 antibodies reinforce T-cell responsiveness and revert anergy/quiescence in patients treated with bispecific CD3 antibodies

Bispecific T-cell engagers stimulating CD3 (TCEs) rapidly gain momentum in oncological therapy. We report that single-agent TCE-treatment of cancer patients induces T-cell hyporesponsiveness including abolished proliferative and lytic capacity. Single-cell RNA-sequencing identified transcriptional features of anergy/quiescence like upregulation of CBLB and BACH2, and suppression of AP-1-dependent activation programs due to isolated provision of "T-cell signal-1". To restore T-cell functionality, we engineered bispecific costimulators (BiCos) which deliver "signal-2" via highly efficient bivalent CD28-binding yet maintain strictly target-restricted activity. Preclinical analyses documented that BiCos potentiate efficacy of single-agent TCE treatment with activity exceeding that of univalent Knob-into-Hole CD28-costimulators and revert T-cell hyporesponsiveness. In humanized mice, BiCos induced elimination of established tumors in combination with very low doses of TCE. Finally, BiCos reversed molecular hallmark features of quiescence such as high expression of BACH2 and KLF2 in T-cells of TCE-treated patients resulting in complete restoration of cellular function, thereby reinstating durable antitumor immunity. Statement of significanceFirst clinical evidence that single-agent treatment with CD3-directed bsAbs induces T-cell hyporesponsiveness resulting in profoundly impaired functionality. Tumor-restricted CD28-costimulation with bsAbs can prevent and revert transcriptional features of anergy/quiescence, establishing conditional delivery of both, T-cell signal- 1 and -2 by combinatorial bsAb treatment as a strategy to improve cancer immunotherapy.