Adoptive transfer of membrane-restricted IL-12-TCR T cells promotes antigen-spreading and elimination of antigen-negative tumor variants

Adoptive T-cell therapy has shown promising results with high response rates and frequent complete responses in B-cell malignancies, offering hope for its application to a broad spectrum of cancers. However, a significant portion of patients with solid tumors experience primary or secondary resistance to this treatment modality. Known resistance mechanisms to this therapy include tumor heterogeneity and target antigen loss resulting from defects in antigen processing and presentation machinery. Constitutively expressed membrane-anchored interleukin-12 (caIL-12) has demonstrated low systemic exposure and antitumor activity in multiple preclinical adoptive T-cell treatment models. In this study, we assess the therapeutic impact of caIL-12 on target antigen-negative variants within a heterogeneous tumor. Target antigen-positive tumors were generated by engineering OVA-SIINFEKL peptide to B16 melanoma (B16-U-OVA), while B16 tumors served as antigen-negative variants. C57BL mice bearing heterogeneous tumors were treated with OT-I TCR-T cells engineered with or without caIL-12. Our results demonstrated that TCR-T cells (OT-I) could effectively deliver caIL-12 to the B16-U-OVA tumor sites and induce robust tumor control and survival benefits even in mice bearing OVA-negative B16 tumor variants. CaIL-12 was found to exert its effect on OVA-negative B16 variants within heterogeneous tumors primarily by activating endogenous antitumor CD8 T cells via antigen spreading. In addition, antigen spreading induced by OT-I-ca12 resulted in controlling OVA-negative tumors implanted at distant sites. This therapeutic effect required antigen-specific TCR-T cells and caIL-12 to co-localize at the tumor site, along with endogenous CD8 T cells capable of recognizing shared tumor antigens. These findings highlight the potential of caIL-12 to address challenges of antigen escape and tumor heterogeneity that may limit the efficacy of T-cell therapy against solid tumors.