Combined VLA-4-Targeted Radionuclide Therapy and Immunotherapy in a Mouse Model of Melanoma
Combined VLA-4-Targeted Radionuclide Therapy and Immunotherapy in a Mouse Model of Melanoma
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DOI：
10.2967/jnumed.118.209510
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发表时间：
2018-12-01
影响因子：
9.3
通讯作者：
Anderson, Carolyn J.
中科院分区：
文献类型：
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作者：
Choi, Jaeyeon;Beaino, Wissam;Anderson, Carolyn J.
非常晚期抗原-4 (VLA-4，也称为整合素α (4) β(1))在侵袭性和转移性黑色素瘤中高水平表达，可能为成像和靶向放射性核素治疗（TRT）提供理想的靶标。Lu-177-DOTA-PEG4-LLP2A （Lu-177-LLP2A）是一种对VLA-4具有高亲和力的TRT，在B16F10小鼠黑色素瘤体内具有高摄取性。在这里，我们报告了（177）LuLLP2A单独或与免疫检查点抑制剂（ICIs）（抗pd -1、抗pd - l1和抗ctla -4抗体）联合在B16F10荷瘤小鼠中的疗效研究。方法：将1 × 10(6)个肿瘤细胞植入C57BL/6小鼠皮下。8 ~ 10 d后，随机分为8组。Lu-177-LLP2A于第8天或第9天静脉注射（单剂量），ICI抗体分3次腹腔注射。通过卡尺监测肿瘤的生长情况。对固定肿瘤进行末端脱氧核苷酸转移酶dUTP镍端标记（TUNEL）染色。在另一项研究中，将Cy3-LLP2A或cy3 -重组LLP2A注射到荷瘤小鼠体内，注射后4 h收集肿瘤，使用不同的免疫细胞标记物进行流式细胞术和免疫荧光显微镜分析。结果：单独TRT的疗效与双ici抗pd -1 + antitla -4或抗pd - l1 +抗ctla -4相当，而TRT + ICIs可显著提高生存率。TUNEL染色显示TRT + ICI组细胞凋亡水平最高。除了靶向肿瘤细胞外，TRT还结合肿瘤微环境中的免疫细胞。流式细胞术数据显示，肿瘤由约77%的肿瘤细胞和成纤维细胞（cd45阴性/ cd49d阳性）和约23%的免疫细胞（cd45阳性/ cd49d阳性）组成，免疫细胞表达更高水平的VLA-4。Cy3-LLP2A和CD49d与巨噬细胞（CD68）、T细胞（CD8、CD4）和B细胞（CD19）共定位。免疫组织化学分析发现Cy3-LLP2A和CD68显著共定位。结论：TRT + ICIs联合治疗可同时靶向肿瘤细胞和免疫细胞，具有作为转移性黑色素瘤治疗药物的潜力。
Very late antigen-4 (VLA-4; also known as integrin alpha(4)beta(1)) is expressed at high levels in aggressive and metastatic melanoma tumors and may provide an ideal target for imaging and targeted radionuclide therapy (TRT). Lu-177-DOTA-PEG4-LLP2A (Lu-177-LLP2A) is a TRT that shows high affinity for VLA-4 and high uptake in B16F10 mouse melanoma tumors in vivo. Here, we report efficacy studies of (177)LuLLP2A, alone and combined with immune checkpoint inhibitors (ICIs) (anti-PD-1, anti-PD-L1, and anti-CTLA-4 antibodies), in B16F10 tumor- bearing mice. Methods: Tumor cells (1 x 10(6)) were implanted subcutaneously in C57BL/6 mice. After 8-10 d, the mice were randomized into 8 groups. Lu-177-LLP2A was injected intravenously on day 8 or 9 (single dose), and ICI antibodies were administered intraperitoneally in 3 doses. Tumor growth was monitored over time via calipers. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining for apoptosis was performed on fixed tumors. In a separate study, Cy3-LLP2A or Cy3-scrambled LLP2A was injected in tumor-bearing mice, and tumors were collected 4 h after injection and then analyzed by flow cytometry and immunofluorescence microscopy using different immune cell markers. Results: TRT alone showed efficacy comparable to the dual-ICI anti-PD-1 + antiCTLA-4 or anti-PD-L1 + anti-CTLA-4, whereas TRT + ICIs significantly enhanced survival. TUNEL staining showed that the highest levels of apoptosis were in the TRT + ICI groups. In addition to targeting tumor cells, TRT also bound immune cells in the tumor microenvironment. Flow cytometry data showed that the tumors consisted of about 77% tumor cells and fibroblasts (CD45-negative/CD49d-positive) and about 23% immune cells (CD45-positive/CD49d-positive) and that immune cells expressed higher levels of VLA-4. Cy3-LLP2A and CD49d colocalized with macrophages (CD68), T cells (CD8, CD4), and B cells (CD19). Immunohistochemical analysis identified a significant colocalization of Cy3-LLP2A and CD68. Conclusion: Combination treatment with TRT + ICIs targets both tumor cells and immune cells and has potential as a therapeutic agent in patients with metastatic melanoma.