Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA, 15213, USA.
Department of Medicine, University of Pittsburgh, Pittsburgh, PA, 15213, USA.
Mol Imaging Biol. 2022 Jun;24(3):425-433. doi: 10.1007/s11307-021-01666-1. Epub 2021 Oct 25.
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 ([Cu]-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 αβ), 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 [Cu]-CB-TE1A1P-PEG-LLP2A ([Cu]-LLP2A) in novel, translational BRAF mutant melanoma models differing in VLA-4 expression-BPR (VLA-4) and BPRα (VLA-4).
BPR cells were transduced with α (CD49d) to overexpress intact cell surface VLA-4 (BPRα). The binding affinity of [Cu]-LLP2A to BPR and BPRα cells was determined by saturation binding assays. [Cu]-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.
[Cu]-LLP2A demonstrated high binding affinity to BPRα (K = 1.4 nM) but indeterminate binding to BPR cells. VLA-4 BPRα and B16F10 displayed comparable time-dependent [Cu]-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.
[Cu]-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.
尽管免疫检查点抑制剂和靶向治疗在黑色素瘤方面取得了前所未有的进展，但仍有一部分患者出现进展，且有效挽救治疗选择有限。我们之前已经证明，在 B16F10 鼠黑色素瘤的皮下和转移性模型中，Cu-64 标记的肽模拟物 LLP2A([Cu]-LLP2A) 对正电子发射断层扫描(PET)成像具有强大、选择性的摄取。LLP2A 与非常晚期抗原-4(VLA-4，整合素 αβ)结合具有高亲和力，VLA-4 是一种在黑色素瘤和其他癌症中过度表达的跨膜蛋白，有助于肿瘤生长和转移。然而，B16F10 并不能真实反映人类黑色素瘤的生物学特性，因为它缺乏某些致癌驱动突变，包括在≥50%的临床标本中发现的 BRAF 突变。在这里，我们评估了新型转化 BRAF 突变黑色素瘤模型中 PET 示踪剂[Cu]-CB-TE1A1P-PEG-LLP2A([Cu]-LLP2A)的情况，这些模型在 VLA-4 表达上存在差异-BPR(VLA-4)和 BPRα(VLA-4)。
BPR 细胞被转导以过表达完整的细胞表面 VLA-4(BPRα)。通过饱和结合测定确定[Cu]-LLP2A 与 BPR 和 BPRα 细胞的结合亲和力。通过基于平板的测定定量测定 B16F10、BPR 和 BPRα 细胞内吞[Cu]-LLP2A 的情况。通过小动物 PET/CT 成像评估皮下 BPR 和 BPRα 肿瘤的示踪剂生物分布。
[Cu]-LLP2A 对 BPRα(K=1.4 nM)表现出高结合亲和力，但对 BPR 细胞的结合力不确定。VLA-4 BPRα 和 B16F10 显示出相似的时间依赖性[Cu]-LLP2A 内化，而 BPR 内化则无法检测到。PET/CT 显示 BPRα 肿瘤的示踪剂摄取增加，与体内 BPR 肿瘤相比，在生物分布分析中，BPRα 肿瘤摄取显著更高(p<0.0001)。
[Cu]-LLP2A 可区分体内 BPRα(VLA-4)与 BPR(VLA-4)黑色素瘤，支持通过前瞻性成像和治疗性研究转化这些 BRAF 突变黑色素瘤模型。这些结果扩展了 LLP2A 的用途，可选择性地针对临床相关且治疗耐药的肿瘤变体，以将其用于治疗患者的护理。
