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

Abstract

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.