ImmunoPET imaging of LAG-3 expression in tumor microenvironment with ⁶⁸Ga-labelled cyclic peptides tracers: from...

Lymphocyte activation gene 3 (LAG-3) has been considered as the next generation of immune checkpoint and a promising prognostic biomarker of immunotherapy. As with programmed cell death protein-1/programmed death-ligand 1 and cytotoxic T-lymphocyte antigen-4 inhibitors, positron emission tomography (PET) imaging strategies could benefit the development of clinical decision-making of LAG-3-related therapy. In this study, we developed and validated ⁶⁸Ga-labeled cyclic peptides tracers for PET imaging of LAG-3 expression in bench-to-bedside studies.

A series of LAG-3-targeted cyclic peptides were modified and radiolabeled with ⁶⁸GaCl₃ and evaluated their affinity and specificity, biodistribution, pharmacokinetics, and radiation dosimetry in vitro and in vivo. Furthermore, hu-PBL-SCID (PBL) mice models were constructed to validate the capacity of [⁶⁸Ga]Ga-CC09-1 for mapping of LAG-3⁺ lymphocytes infiltrates using longitudinal PET imaging. Lastly, [⁶⁸Ga]Ga-CC09-1 was translated into the first-in-human studies to assess its safety, biodistribution and potential for imaging of LAG-3 expression.

A series of cyclic peptides targeting LAG-3 were employed as lead compounds to design and develop ⁶⁸Ga-labeled PET tracers. In vitro binding assays showed higher affinity and specificity of [⁶⁸Ga]Ga-CC09-1 in Chinese hamster ovary-human LAG-3 cells and peripheral blood mononuclear cells. In vivo PET imaging demonstrated better imaging capacity of [⁶⁸Ga]Ga-CC09-1 with a higher tumor uptake of 1.35±0.33 per cent injected dose per gram and tumor-to-muscle ratio of 17.18±3.20 at 60 min post-injection. Furthermore, [⁶⁸Ga]Ga-CC09-1 could detect the LAG-3⁺ lymphocyte infiltrates in spleen, lung and salivary gland of PBL mice. In patients with melanoma and non-small cell lung cancer, primary lesions with modest tumor uptake were observed in [⁶⁸Ga]Ga-CC09-1 PET, as compared with that of [¹⁸F]FDG PET. More importantly, [⁶⁸Ga]Ga-CC09-1 delineated the heterogeneity of LAG-3 expression within large tumors.

These findings consolidated that [⁶⁸Ga]Ga-CC09-1 is a promising PET tracer for quantifying the LAG-3 expression in tumor microenvironment, indicating its potential as a companion diagnostic for patients stratification and therapeutic response monitoring in anti-LAG-3 therapy.