Targeting CD137L to MC1R-Expressing Melanoma Cells and Tumors.

 描述（由申请人提供）：在许多癌症上发现了特异性高亲和力受体配体，这些受体已作为成像探针进行了研究，我们认为我们应该尝试通过使用这些配体将免疫效应物递送至癌症微环境来增强免疫治疗。黑色素瘤是已知的免疫原性最强的肿瘤之一，因此这种疾病是这种新策略的一个很好的测试平台。黑皮质素1受体配体（MC 1 RL）是黑皮质素1受体的亚nM Ki结合剂，在黑色素瘤细胞和肿瘤的表面上以高水平存在。在本文中，我们建议使MC 1 RL与CD 137 L（一种重要的T细胞共刺激分子）缀合，以确定缀合物是否会归巢黑色素瘤微环境并增强免疫应答。我们的具体目标是：目标1a：合成MC 1 R特异性配体MC 1 RL，其通过三官能接头连接至IRDye 800和胺反应性位点，用于与市售鼠重组CD 137 L表面上的伯胺偶联。我们将使用LC-MS和MALDI-TOF MS实验表征前体和最终缀合物。使用体外荧光显微镜研究，与IRDye 800缀合的MC 1 RL在有和没有CD 137 L缀合物的情况下与MC 1 R阳性鼠B16-F10和MC 1 R siRNA敲低B16-F10细胞的结合和摄取时间将验证MC 1 RL结合亲和力和特异性。这些相同的细胞系将用于体外免疫活化试验。目的1b：进行PK研究，以确定C57 BL/6小鼠中非肿瘤和荷瘤小鼠中目的1a优化偶联物的半衰期。重要的是进行这些研究，以帮助确定最佳的剂量时间间隔。如果我们过量服用，因为所有可用的MC 1 R几乎饱和，我们可能会增加全身毒性的机会。目标1c：显示Aim 1a中开发的MC 1 RL-IRDye 800-CD 137 L在携带B16-F10黑色素瘤异种移植肿瘤的C57 BL/6小鼠中的靶向肿瘤摄取和保留，并测量这些缀合物的肿瘤摄取和全身清除率，以预测荷瘤动物的总体暴露量。目标1d：在上述使用的动物模型中测量MC 1 RL-IRDye-800-CD 137 L的体内活性，以获得相对于未处理动物的统计学显著的肿瘤生长减少，并确定在免疫活性鼠模型中是否耐受10个剂量并保持效力。

 DESCRIPTION (provided by applicant): There are specific high affinity ligands for receptors found on many cancers that have been investigated as imaging probes and we believe that we should try to enhance immunotherapy by using these ligands to deliver immune effectors to the cancer microenvironment. Melanoma is among the most immunogenic tumors known and so that disease is an excellent test bed for this new strategy. The melanocortin 1 receptor ligand (MC1RL) is a sub nM Ki binder of the melanocortin 1 receptor that is found at high levels on the surface of melanoma cells and tumors. Herein, we propose to make MC1RL conjugates with CD137L, an important T cell costimulator, to determine if the conjugate will home to the melanoma microenvironment and enhance the immune response. Our Specific Aims are: Aim 1a: Synthesize the MC1R specific ligand, MC1RL, connected via a trifunctional linker to IRDye800 and to an amine reactive site for coupling with primary amines on the surface of the commercially available murine recombinant CD137L. We will characterize the precursors and the final conjugates using LC-MS and MALDI-TOF MS experiments. The binding and the timing of uptake of the MC1RL conjugated to IRDye800 with and without the CD137L conjugate with MC1R positive murine B16-F10 and MC1R siRNA knock down B16-F10 cells using in vitro fluorescence microscopy studies will verify MC1RL binding affinity and specificity. Those same cell lines will be used for in vitro immune activation assays. Aim 1b: Perform PK studies to determine the half-life of optimized conjugates from Aim 1a in non-tumor and tumor bearing in C57BL/6 mice. It is important to have these studies performed to help determine optimal dose time intervals. If we overdose due to all of the available MC1R being nearly saturated, we will probably increase the chances of systemic toxicity. Aim 1c: Show targeted tumor uptake and retention of the MC1RL-IRDye800-CD137L developed in Aim 1a in C57BL/6 mice bearing B16-F10 melanoma xenograft tumors and measure the tumor uptake and systemic clearance rates of these conjugates to predict the overall exposure of the tumor-bearing animals. Aim 1d: Measure the in vivo activity of MC1RL-IRDye-800-CD137L in the animal models used above to obtain a statistically significant tumor growth reduction relative to untreated animals and determine if 10 doses are tolerated and remain efficacy in immune competent murine models.