Radiobiological and immunomodulatory mechanisms of CAIX-targeted α therapy combined with immune checkpoint inhibition in hypoxic tumors

Abstract Purpose Tumor hypoxia is associated with tumor aggressiveness and therapy resistance. Targeted α therapy (TAT) offers a promising approach to eliminate hypoxic tumor cells, as α-particles induce complex DNA damage independent of oxygen availability. We previously demonstrated the therapeutic efficacy of the carbonic anhydrase IX (CAIX)-targeting radiopharmaceutical [ 225 Ac]Ac-DOTA-MSC3, in combination with immune checkpoint inhibition (ICI). Here, we investigated the radiobiological and immunomodulatory effects of [ 225 Ac]Ac-DOTA-MSC3 alone and combined with ICI. Methods B16F10-OVA tumor-bearing mice were treated with CAIX-TAT with or without anti-PD-1/anti-CTLA-4 ICI. Tumor and organ uptake of [ 225 Ac]Ac-DOTA-MSC3 was determined 5 and 8 days post injection. Radiobiological and immunological markers were quantified in CAIX + and CAIX − tumor areas. Results [ 225 Ac]Ac-DOTA-MSC3 selectively accumulated CAIX-expressing hypoxic regions in B16F10-OVA tumors. CAIX-TAT increased DNA damage (γH2AX) compared with PBS control, with stronger effects in CAIX + than CAIX − areas. No increase in apoptosis (caspase-3) was observed within the studied timeframe. Compared with ICI alone, CAIX-TAT with ICI increased cGAS foci and CD45 + immune cell infiltration in both CAIX + and CAIX − areas. The combination therapy also increased CD8 + T-cell and F4/80 + macrophage numbers, while PD-L1 + , FOXP3 + and Ly6G + cell numbers were unchanged. Conclusion CAIX-TAT induces DNA damage in hypoxic B16F10-OVA tumors and, when combined with ICI, promotes the infiltration of CD45 + and CD8 + immune cells. These findings provide a mechanistic rationale for combining CAIX-TAT with ICI to improve the treatment of hypoxic tumors.