PET Imaging of VLA-4 in a New BRAFV600E Mouse Model of Melanoma

Abstract

Purpose: Despite unprecedented responses to immune checkpoint inhibitors and targeted therapy in melanoma, a major subset of patients progresses and have few effective salvage options. We have previously demonstrated robust, selective uptake of the peptidomimetic LLP2A labeled with Cu-64 ([⁶⁴Cu]-LLP2A) for positron emission tomography (PET) imaging in subcutaneous and metastatic models of B16F10 murine melanoma. LLP2A binds with high affinity to very late antigen-4 (VLA-4, integrin α₄β₁), a transmembrane protein overexpressed in melanoma and other cancers that facilitates tumor growth and metastasis. Yet B16F10 fails to faithfully reflect human melanoma biology, as it lacks certain oncogenic driver mutations, including BRAF mutations found in ≥ 50 % of clinical specimens. Here, we evaluated the PET tracer [⁶⁴Cu]-CB-TE1A1P-PEG₄-LLP2A ([⁶⁴Cu]-LLP2A) in novel, translational BRAF^V600E mutant melanoma models differing in VLA-4 expression-BPR (VLA-4^-) and BPRα (VLA-4⁺).

Procedures: BPR cells were transduced with α₄ (CD49d) to overexpress intact cell surface VLA-4 (BPRα). The binding affinity of [⁶⁴Cu]-LLP2A to BPR and BPRα cells was determined by saturation binding assays. [⁶⁴Cu]-LLP2A internalization into B16F10, BPR, and BPRα cells was quantified via a plate-based assay. Tracer biodistribution and PET/CT imaging were evaluated in mice bearing subcutaneous BPR and BPRα tumors.

Results: [⁶⁴Cu]-LLP2A demonstrated high binding affinity to BPRα (K_d = 1.4 nM) but indeterminate binding to BPR cells. VLA-4⁺ BPRα and B16F10 displayed comparable time-dependent [⁶⁴Cu]-LLP2A internalization, whereas BPR internalization was undetectable. PET/CT showed increased tracer uptake in BPRα tumors vs. BPR tumors in vivo, which was validated by significantly greater (p < 0.0001) BPRα tumor uptake in biodistribution analyses.

Conclusions: [⁶⁴Cu]-LLP2A discriminates BPRα (VLA-4⁺) vs. BPR (VLA-4^-) melanomas in vivo, supporting translation of these BRAF-mutated melanoma models via prospective imaging and theranostic studies. These results extend the utility of LLP2A to selectively target clinically relevant and therapy-resistant tumor variants toward its use for therapeutic patient care.

Keywords: BRAF mutant melanoma; CD49d; LLP2A; PET imaging; Theranostic; VLA-4.