Combination immunotherapy targeting LAG-3, PD-1 and STING suppresses hepatocellular carcinoma as monitored by LAG-3 targeted PET imaging.

Background: The low response rate of anti-PD-1 monoclonal antibodies (mAbs) in hepatocellular carcinoma (HCC) requires the development of combination immunotherapy strategies to improve their efficacy. This study aimed to use LAG-3-targeted PET imaging to monitor the efficacy of anti-PD-1 mAb, a stimulator of interferon genes (STING) agonist, and anti-LAG-3 mAb, both individually and in combination. Furthermore, we evaluated the potential of a triple immunotherapy regimen (anti-PD-1 mAb, STING agonist, and anti-LAG-3 mAb) to improve HCC treatment.

Methods: The LAG-3 inhibitor C25 based on a cyclic peptide was chelated with NOTA, radiolabeled with [⁶⁸Ga]GaCl₃. The resulting [⁶⁸Ga]Ga-NOTA-C25 underwent in vivo PET imaging and ex vivo biodistribution examination in Hepa1-6 tumor-bearing mice. [⁶⁸Ga]Ga-NOTA-C25 PET was used to monitor the efficacy of monotherapy and dual immunotherapy with anti-PD-1 monoclonal antibody (mAb) and STING agonists. The tumor uptake of [⁶⁸Ga]Ga-NOTA-C25, tumor response, and survival rates were measured following different treatments. The therapeutic efficacy, molecular mechanisms, and safety of triple immunotherapy were validated using histopathological analysis and flow cytometry.

Results: [⁶⁸Ga]Ga-NOTA-C25 PET imaging effectively and noninvasively detected LAG-3⁺ tumor-infiltrating lymphocytes (TILs) in Hepa1-6 tumor-bearing mice. In mice treated with anti-PD-1 mAb, STING agonist, or a combination immunotherapy, [⁶⁸Ga]Ga-NOTA-C25 PET revealed significantly increased LAG-3⁺ TIL levels. At the treatment endpoint, the combination of the STING agonist with the anti-PD-1 mAb resulted in a significantly higher uptake (1.35 ± 0.191%ID/g) compared to the control group (0.402 ± 0.017%ID/g), the anti-PD-1 mAb group (0.647 ± 0.037%ID/g), and the STING agonist group (0.874 ± 0.089%ID/g). Uptake of [⁶⁸Ga]Ga-NOTA-C25 was positively correlated with tumor therapeutic effects and survival rates. Triple immunotherapy with anti-PD-1 mAb, a STING agonist, and anti-LAG-3 mAb further enhanced efficacy compared to any dual immunotherapy regimen, and treatment efficacy was linearly associated with [⁶⁸Ga]Ga-NOTA-C25 tumor uptake.

Conclusions: Anti-PD-1 mAb and STING agonists have shown notable synergy in upregulating LAG-3 expression on TILs in HCC, which can be successfully tracked by [⁶⁸Ga]Ga-NOTA-C25 PET imaging. Furthermore, integration of a triple immunotherapy regimen comprising an anti-PD-1 mAb, STING agonist, and anti-LAG-3 mAb demonstrated a significant improvement in therapeutic efficacy over dual immunotherapy approaches.