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Target: gp100, N36RNA, Bead-based (His-tag), Fall 2012
September 4, 2012
Development of an RNA Aptamer for gp100 to enhance immunogenicity in malignant melanoma cells.
Melanoma, or skin cancer, is the most common form of cancer in the United States, accounting for nearly 50% of all cases . Cutaneous malignant melanoma (CMM), causes the majority of skin cancer deaths (9,000 out of the 12,000 per year in the U.S.)  yet only represents five percent of all skin cancers. Whereas the mortality rate for skin cancer has been decreasing due to early detection and removal, there is still a need for treatment, especially when the tumor has been discovered after metastasis [3,4].
Cancerous melanoma tissues over express the biomarker glycoprotein gp100, making it an effective diagnostic marker, though gp100 is also expressed at low levels on the surface of normal melanocytes . On it’s own, gp100 elicits a weak immune response; recognizable by tumor-infiltrating lymphocytes suggesting the target is accessible to the immune system . Current intravenously administered immune chemotherapies and radiation specifically target GP100 however, have they increased toxicity likely due to attack of healthy tissues .
Aptamers are oligonucleotides enriched through iterative rounds of nucleic acid in vitro selection from a starting library containing roughly 1013. The SELEX (Systematic Evolution of Ligands by Exponential Enrichment) method is designed to create greater specificity and binding affinity towards target (protein) for each subsequent round by getting rid of the species that don’t bind and amplifying those that do. 
An aptamer specific to gp100 has two main benefits over traditional therapies. First, drug delivery is relatively easy through subcutaneous methods against melanomas as opposed to intravenous administration mentioned above. The drug could be formulated in a rub on cream that penetrates the skin. Second, using an immune redirection technique , the aptamer could direct an immune response specifically to cells expressing targets over a certain threshold such as gp100 on melanoma compared to healthy melanocytes . It is hoped that this kind of aptamer therapy could greatly decrease the mortality rate associated with CMM skin cancer.
Specific Aim # 1 - To develop an aptamer through iterative rounds of nucleic acid enrichment that will specifically and tightly bind to human gp100. Working with Altermune Technologies, gp100 has been expressed and purified by GenScript (lot. No.: 163193S01/P20011204) with a poly-HIS tag for bead based selection.
Specific Aim # 2 - Once enrichment is detected, the selection will continue with gp100+ melanoma cells such as human gp100 transfected mouse melanoma B16 cells  and assayed using flow cytometry (FCM) to determine binding affinity (using a negative-gp100 B16 cell line as the control).
Specific Aim #3 - Next-generation sequencing will be utilized to interrogate the selected pool for specific binding variants.
Fig. 1. This figure outlines the specific aims of the project as well as the general outline of the overall procedure.
 "Skin Cancer Facts." Skin Cancer Facts. American Cancer
Society, 23 Jan. 2012. Web. 04 Sept. 2012.
A Cancer Journal for Clinicians.Jemal, A., et al. “Cancer Statistics, 2010.”
Advances in Experimental Medicine and Biology. 624:Ch. 8.Leiter, U. and Garbe, C.. (2008) “Epidemiology of Melanoma and Nonmelanoma Skin Cancer---The Role of Sunlight.”
 Salgaller, M., et al. (1996) “Immunization against Epitopes in the Human Melanoma Antigen gp100 following Patient Immunization with Synthetic Peptides.” Cancer Research. 56: 4749.
 Dudley, M. E., et al. (2005). “Adoptive Cell Transfer Therapy Following Non- Myeloblative but Lymphodepleting Chemotherapy for the Treatment of Patients With Refractory Metastatic Melanoma.” Journal of Clinical Oncology. 23(10):2346-2357.
 Brody, E.N., Gold, L. (2000). “Aptamers as therapeutic and diagnostic agents.”
Reviews in Molecular Biotechnology. 74(1):5-13.
 Popkov, M., Gonzalez, B., Sinha, S. C. & Barbas, C. F.(2009) “Instant immunity through chemically programmable vaccination and covalent self-assembly.” Proc Natl Acad Sci USA. 106, 4378–4383.
 Carlson, C. B., Mowery, P., Owen, R. M., Dykhuizen, E. C. & Kiessling, L. L. (2007) “Selective tumor cell targeting using low-affinity, multivalent interactions.” ACS Chem Biol. 2: 119–127.
 Chen, H. et al. (2012). “Shikonin enhances efficacy of a gene-based cancer vaccine via induction of RANTES.” Journal of Biomedical Science. 19(1):42.