Targeting CD137L to MC1R-Expressing Melanoma Cells and Tumors.

将 CD137L 靶向表达 MC1R 的黑色素瘤细胞和肿瘤。

• 批准号: 8905409
• 负责人: MARK L MCLAUGHLIN
• 金额: $ 24.48万
• 依托单位: MODULATION THERAPEUTICS, INC.
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-24 至 2016-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8905409
• 关键词: Affinity; Amines; Animal Model; Animals; Applications Grants; Area; Awareness; Beds; Binding; Binding Sites; Biological Assay; Breast; C57BL/6 Mouse; Cell Line; Cells; Companions; Coupling; Development; Diagnosis; Disease; Dose; Dose-Limiting; Drug resistance; Dyes; Fluorescence Microscopy; Half-Life; Home environment; Human; Image; Immune; Immune Targeting; Immune response; Immune system; Immunotherapy; In Vitro; Incidence; Interleukin-2; Lesion; Ligand Binding; Ligands; Lung; MHC antigen; Malignant Neoplasms; Measurement; Measures; Melanocortin 1 Receptor; Melanoma Cell; Metabolic Clearance Rate; Metastatic Melanoma; Modeling; Mus; Mutate; Neoplasm Metastasis; Overdose; Patients; Peptide Receptor; Phase; Process; Prostate; Proteins; Reaction; Recombinants; Relative (related person); Resistance development; Risk Factors; Skin Cancer; Small Interfering RNA; Specificity; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Sun Exposure; Surface; Survival Rate; Symptoms; T-Lymphocyte; Technology; Testing; Therapeutic; Time; Tissues; Toxic effect; Variant; chemotherapy; imaging agent; imaging probe; immune activation; immunogenic; in vivo; knock-down; melanoma; mouse model; novel strategies; overexpression; public health relevance; receptor; receptor expression; research study; response; scaffold; targeted imaging; time interval; tumor; tumor growth; tumor microenvironment; tumor xenograft; uptake

 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.

 描述（适用提供）：在许多癌症上发现的受体有特定的高亲和力配体，这些配体已被研究为成像问题，我们认为我们应该尝试通过使用这些配体对癌症微环境提供免疫疗法来增强免疫疗法。黑色素瘤是已知的免疫原性肿瘤之一，因此疾病是这种新策略的出色测试床。黑色素皮质1受体配体（MC1RL）是黑色素瘤皮质1受体的亚NM Ki粘合剂，在黑色素瘤细胞和肿瘤表面高水平上发现。本文中，我们建议将MC1RL与重要的T细胞costimulator CD137L共轭，以确定结合物是否会归结为黑色素瘤微环境并增强免疫反应。我们的具体目的是：AIM 1A：合成MC1R特异性配体MC1RL，该配体通过三功能连接器连接到IRDYE800，并与胺反应性位点连接，以与在市售鼠重组CD137L表面与原代胺相结合。我们将使用LC-MS和MALDI-TOF MS实验表征前体和最终共轭物。有或没有MC1R阳性鼠B16-F10和MC1R siRNA使用体外荧光显微镜研究将B16-F10细胞与MC1R阳性鼠B16-F10和MC1R siRNA相结合，MC1RL的结合和摄取时间与IRDYE800结合到IRDYE800中，将验证MC1RL的结合亲和力和特定性。这些相同的细胞系将用于体外免疫激活测定。 AIM 1B：进行PK研究，以确定C57BL/6小鼠中非肿瘤和肿瘤轴承的AIM 1A优化结合物的半衰期。重要的是要进行这些研究以帮助确定最佳剂量时间间隔。如果由于所有可用的MC1R几乎饱和，我们过量服药，我们可能会增加系统性毒性的机会。 AIM 1C：在AIM 1A中，在C57BL/6小鼠的AIM 1A中，靶向肿瘤的摄取和保留，轴承B16-F10黑色素瘤异种移植肿瘤并测量这些结合物的肿瘤吸收率和全身清除率，以预测肿瘤的整体暴露于肿瘤的动物。 AIM 1D：在上面使用的动物模型中，测量MC1RL-ERDYE-800-CD137L的体内活性相对于未经治疗的动物，并确定是否耐受10剂的肿瘤生长，并在不合时宜的鼠模型中保持有效。