Objective Although drastic responses to immune checkpoint inhibitors and targeted therapy occur in melanoma, many patients progress and have few effective alternatives. Our previous work has indicated avid and selective uptake of LLP2A, a peptidomimetic labeled with Cu-64 ([64Cu]-LLP2A), for positron emission tomography (PET) imaging in B16F10 murine melanoma. LLP2A binds strongly to very late antigen-4 (VLA-4, integrin α4β1), a transmembrane protein correlated with tumor growth and dissemination when overexpressed in melanoma and other tumor histologies. Yet B16F10 does not faithfully recapitulate clinical melanomas, as it lacks the BRAF mutation found in ≥50% of clinical specimens, among other aberrations. Here, we evaluated the PET tracer [64Cu]-CB-TE1A1P-PEG4-LLP2A ([64Cu]-LLP2A) in novel, translational BRAFV600E mutant melanoma models with distinct levels of VLA-4 expression - BPR (VLA-4-) and BPRα (VLA-4+). Methods To overexpress intact VLA-4, BPR cells were transduced with the α4 subunit (CD49d), generating the BPRα cell line. The binding metrics of [64Cu]-LLP2A to BPR and BPRα cells were determined by saturation binding assays. Plate-based assays were conducted to assess [64Cu]-LLP2A internalization into B16F10, BPR, and BPRα cells. Ex vivo biodistribution and PET/CT imaging were performed in mice bearing either subcutaneous BPR or BPRα tumors. Results BPRα bound [64Cu]-LLP2A with high affinity (Kd = 1.4 nM) but binding to BPR was undefined. The VLA-4+ lines (BPRα and B16F10) demonstrated similar time-dependent tracer internalization, but internalization by BPR was essentially undetectable. BPRα tumors displayed notably greater [64Cu]-LLP2A uptake vs. BPR tumors in vivo by PET/CT, which was corroborated by biodistribution analyses (p&lt;0.0001). Conclusions and Future Work As expected, [64Cu]-LLP2A differentiates BPRα (VLA-4+) vs. BPR (VLA-4-) melanomas in vivo, encouraging prospective imaging and theranostic studies with these cell lines - particularly BPRα. LLP2A may be especially apt to target clinically relevant and targeted therapy-resistant tumor variants towards eventual translation to patient care. As proof-of-concept towards individualized 67Cu-LLP2A therapy, longitudinal 64Cu-LLP2A PET images were acquired for BPRα tumor-bearing mice in a separate study for 67Cu-LLP2A dosimetry. Analysis of these images via dedicated 3D Monte Carlo software is ongoing.

Validation of VLA-4-targeted PET imaging in a new BRAFV600E mouse melanoma model

ACS Spring 2022

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