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

96 Objectives: About 50% of melanomas harbor a mutation in the serine/threonine kinase BRAF, resulting in constitutive activation of the MAPK/ERK signaling pathway; most of these are a single nucleotide substitution at codon 600 (BRAFV600E). However, treatment with BRAF inhibitor (BRAFi) monotherapies such as dabrafenib and vermurafenib are not durable, with a median time to progression of less than 6 months. Therefore, there is a substantial need to develop novel therapeutic modalities and combination immunotherapy for effective treatment of advanced-stage BRAF-mutant melanoma. Very late antigen-4 (VLA-4, α4β1) is a transmembrane non-covalent heterodimer overexpressed in melanoma tumors that plays an important role in tumor growth, angiogenesis, and metastasis by promoting adhesion and migration of cancer cells. In this study, we evaluated a VLA-4 targeted PET tracer, 64Cu-CB-TE1A1P-LLP2A (64Cu-LLP2A), in a novel pair of BRAFi-resistant melanoma cell lines (BPR and BPRα4).\n Methods: BPR cells were transduced to express the α4 subunit of VLA-4 to generate BPRα4. To verify the α4 transduction and isolate clones, flow sorting was conducted. LLP2A was conjugated with PEG4 and a cross-bridged copper chelator (CB-TE1A1P) and radiolabeled with Cu-64, as previously described (Beaino et al. JNM 2014; 55:1856-63). To determine the 64Cu-LLP2A binding to BPR and BPRα4 cells, a saturation binding assay was conducted. 64Cu-LLP2A was injected via tail vein in BPR and BPRα4 subcutaneous tumor bearing C57BL/6 mice (7.4 MBq per animal, 57.2 MBq/nmol. PET/CT imaging was performed at 4 and 24 h post-injection (p.i.), and the biodistribution was evaluated at 24 h p.i. by gamma-counting.\n Results: The saturation binding cell assay indicated that 64Cu-LLP2A had a higher binding affinity for the BPRα4 cells (Kd= 48.23 nM) compared to the BPR cells (Kd= 1.400 x 103 µM). The BPRα4 tumors showed increased tracer uptake, with SUVmean of 1.4 ± 0.3 and 0.6 ± 0.2 at 4 and 24 h, respectively. The SUVmean in the BPR tumors was 0.9 ± 0.3 at 4 h and 0.3 ± 0.06 at 24 h) (Figure 1). Additionally, the biodistribution at 24 h showed a higher tumor uptake in BPRα4 tumors (2.80 ± 0.18 %ID/g) compared to 1.70 ± 0.22 %ID/g for BPR tumors. Uptake in VLA-4 negative BPR tumors is likely due to VLA-4 expression in immune cells in the tumor microenvironment.\n Conclusions: These data demonstrate that 64Cu-LLP2A could be used as an imaging agent in BRAFV600E mutant melanoma tumors that are VLA-4 positive. Future experiments will explore dosimetry towards targeted radiotherapy with 177Lu-LLP2A.\nAcknowledgments: This research was supported by NIH R01 CA204018 to CJA. UPMC Hillman Cancer Center shared resources (In Vivo Imaging Facility) were supported in part by NIH P30 CA047904.