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 1 a: 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 1 b: 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 1 c: 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 1 d: 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.
This application will develop an invention for targeting immunotherapy to the surface of melanoma cells and tumors that display a peptide receptor on the surface of melanoma cells. Immunotherapy can cure melanoma in a small number of metastatic melanoma patients and we believe that by targeting the immunotherapy to the melanoma rather treating systematically will decrease adverse immune reactions outside of tumor and allow more patients to be cured.
