CD3xHER2 bsAb‐Mediated Activation of Resting T‐cells at HER2 Positive Tumor Clusters Is Sufficient to Trigger Bystander Eradication of Distant HER2 Negative Clusters Through IFNγ and TNFα

Bispecific antibodies (bsAbs) bridging CD3 on T-cells to tumor-associated antigens (TAA) on tumor cells can direct T-cell immunity to solid tumors. "Bystander killing", where T-cell targeting of TAA-positive tumor cells also leads to the eradication of TAA-negative cells, may overcome TAA heterogeneity. While bystander activity of activated, engineered T-cells has been shown to be robust and wide-reaching, spatiotemporal aspects of bsAb-mediated bystander activity are unresolved. Here, we developed a model where breast cancer tumoroids varying in HER2 expression were printed in to extracellular matrix (ECM) scaffolds. We generated (1) mixed tumors containing different ratios of HER2⁺ and HER2^- tumor cells, and (2) HER2⁺ and HER2^- tumoroids spaced at different distances within the ECM scaffold. Subsequently, tumors were exposed to peripheral blood-derived T-cells in the presence of CD3xHER2 bsAbs. We find that CD3xHER2 bsAb-mediated interaction of resting, nonactivated T-cells with HER2⁺ tumor cells is sufficient (1) to eliminate 50% HER2^- cells in mixed tumor areas, and (2) to eradicate distant HER2^- tumor areas. Such bystander killing involves paracrine IFNγ and TNFα activity but does not require T-cell accumulation in HER2^- areas. These findings indicate that bystander eradication of TAA-negative cells can significantly contribute to bsAb therapy for solid tumors.