DENDRITIC CELLS VACCINES FOR TREATMENT OF MELANOMA Esha Lal Cluster 1: Biotechnology COSMOS What are Dendritic Cells? §  Antigen Presenting Cells are specialized white
blood cells that help fight off foreign substances
that enter the body
§  Main function is to present antigens to bring about
primary immune response in resting naïve T cells
§  To perform this function, DCs are capable of
capturing antigens, processing them, and presenting
them on the cell surface along with appropriate costimulation molecules.
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History of Dendritic Cells §  Dendritic Cells were discovered by Ralph Steinman in 1970 at Rockefeller University in New York §  In 2007 Steinman was diagnosed with pancreatic cancer ú  Participated in experimental trials and treated himself as an experiment ú  Lived four and a half years longer than he was supposed to ú  Died on September 30th 2011 of respiratory failure that was caused by pneumonia Being Treated with DC’s §  Dendritic Cells have already been used to treat renal cancer, §
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ovarian cancer, breast cancer, melanoma, myeloma, prostate cancer, colon cancer, and B Cell Lymphoma Clinical trials are currently being done in Kidney Cancer and Melanoma Dendritic Cells are an option for patients when all alternatives have been exhausted, or when the disease is stable and the patient is not currently receiving chemotherapy and radiation therapy Generally the percentage of heavily untreated, otherwise untreatable late stage patient in remission is 20% DC’s are also good for ensuring that remission continues A higher percentage of patients that do not achieve remission become stable with treatment Melanoma §  The skin is the largest organ in the body and as a result, cancer of the skin is the most prevalent §  Melanoma accounts for 4% of all cancer cases and 79% of all deaths-­‐ least common, most deadly §  Race is the primary risk factor for developing melanoma, fairer skins= higher risks §  US has experienced a dramatic increase in people suffering from Melanoma-­‐ it has more than doubled since 1973 ú  Sixth most common cancer in men ú  Seventh most common cancer in women Common Treatments and their Side-­‐effects Lymph node dissections §  Damage lymphatic systems and slow the flow of lymph in arm or leg §  Leads to lymphedema Chemotherapy/ Radiation Therapy §  Fatigue §  Hair Loss §  Affects cells that divide rapidly such as blood cells, cells in hair roots, and cells lining the digestive tract. ú  As a result, people are more likely to get an infection, bruise/bleed, experience shortness of breath, have less energy ú  Hair loss, loss of appetite, nausea, vomiting and sores Surgical Incisions §  Result in keloids Researchable Question §  Can the efficiency of dendritic cells as cancer vaccines for Melanoma be improved by exposing the dendritic cells to additional antigens prior to injection? Overview §
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1) Prepare Dendritic Cells 2) Monitor Melanoma 3) Take a Punch biopsy if Melanoma worsens or remains the same 4) Use a Western Blot on cancer cells to identify whether or not the tumors of the patient contain gp100, tyrosinase, and MAGE 5) If those proteins are not present complete a 2D gel electrophorese to separate proteins by properties 6) Complete a Mas-­‐Spectrometry in order to identify the amino acids present and thus conclude which protein is present in abundance in the tumor 7) Mass-­‐produce the proteins in CHO K1 cells 8) Complete a pilot study to see if the efficiency of Dendritic Cells improves Step 1:Prepare Dendritic Cells for use §  Pour ficoll-­‐hypaque into a 50ml column and mix in peripheral blood mononuclear cells §  Spin for 20 minutes at 2000 rpm §  Harvest the cloudy white layer and re-­‐suspend in PBS:EDTA §
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solution Spin at 1200 RPM for 12 minutes Remove excess supernatant Re-­‐suspend in PBS:EDTA and spin at 1200 RPM for 10 minutes Dispose of excess supernatant and suspend in phosphate saline buffer Repeat previous wash process Suspend in 5 ml of AIM-­‐V Plate cells and allow for incubation for 90 minutes at 37 degrees celscius Step 1: Continued §  Culture cells with GM-CSF and IL-4 ± TNF-α
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presence(granulocyte/macrophage-colony
stimulating factor ) for 7 days
Load dendritic cells with keyhole limpet
hemocyanin and allow to sit for 2 hours
Pulse the Dendritic Cells with tumor lysates for 4
hours at room temperature
Wash 3 times in PBS
Re-suspend in 0.5 ml of PBS solution
Inject immediately
Step 2: Monitoring Melanoma §  LDH (Lactate dehydrogenase) is very useful in identifying tissue damage ú  Consists of taking a sample of blood from the vein behind the elbow and analyzing the LDH level §  Positron Emission Tomography Scans identifies areas that appear to be “metabolically active” §  Computed Tomography Scans searches for other areas of the body where Melanoma may have spread Step 3: Punch Biopsy §  Prepare the skin with alcohol §  Anesthetize the skin by injecting Lidocaine until a bubble §
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beneath the skin’s surface is formed that is greater than 3mm in diameter Test for numbness in the area surrounding the bubble 3 mm skin punch pierces the skin moving in a drilling motion until the section of the skin has been carved out Clean off the blood from the area before beginning to excise the biopsy Use forceps to lift up the cored skin and use the scalpel to cut it free Step 4:Western Blot Step 4: Western Blot §  Separate proteins using a Polyacrylamide Gel §
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Electrophoreses (by size) Transfer protein pattern onto nitrocellulose paper Submerge paper in a solution of generic proteins to bind to any sticky parts Add antibodies for Tyrinosase, Mage,and MelonA into the solution that are attached to the enzyme Alkaline Phosphatase Mix in Bromochloroindoyl phosphate/nitro blue tetrazolium (the substrate for Alkaline Phosphatase) into the solution to indicate if any of the previous proteins are present Step 5: Mas-­‐Spectrometry part 1: 2d Gel Electrophorese §  Load the proteins onto a polyacrilamide gel that separates them by two factors: §  First, on the basis of their net charge which occurs when exposed to isoelectric focusing §  Secondly, on the basis of molecular mass by electrophoreses while exposed to sodium dodecyl sulfate Step 5:Continued Finally, they are measured and classified by their lengths which determines the amino acid sequence §  Send in sequences to NCBI to identify present proteins §  Identify the most abundant protein in the tumor that is not as prevalent in the body §
Step 6: Transfection of CHO-­‐
K1 CELLS §  Begin by growing the CHO-­‐cells on a dish §  ·∙ Dilute rB2m and r144 in a medium §  ·∙ Mix in 40 microliters of superfect with the DNA of §
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the protein sequence desired ·∙ Allow the solution to sit for 5-­‐10 minutes ·∙ Wash the solution in PBS ·∙Mix serum and medium together to put the new solution into the cells ·∙ Ensure that the solution incubates for 3 hours ·∙ Wash in PBS ·∙ Mix in a 10 ml medium to re-­‐suspend the solution Step 7: Pilot Study Create an experiment using in C57BL/6 and C3H mice to see if dendritic cells trained to recognize the new protein can treat the cancer in mice Improved Effectiveness When Dendritic Cells are Coupled With: §  Immunomodulatory drugs – agents that suppress the immune system and bring about additional function in lymphocytes §  Chemotherapy Bibliography §
Wieder, Eric. "Dendritic Cells: A Basic Review." International Society for Cellular Therapy. ICST, May 2003. Web. 06 July 2012. <http://www.celltherapysociety.org/files/PDF/Resources/OnLine_Dendritic_Education_Brochure.pdf>. §
O’Neill, David W., and Nina Bhardwaj. "Exploiting Dendritic Cells for Active Immunotherapy of Cancer and Chronic Infections." Molecular Biotechnology 36.2 (2007): 131-­‐41. Print. §
Avignan, David. "Dendritic Cell-­‐Tumor Fusion Vaccines for Renal Cell Carcinoma." Dendritic Cell-­‐Tumor Fusion Vaccines for Renal Cell Carcinoma. Clinical Cancer Research, n.d. Web. 19 July 2012. <http://
clincancerres.aacrjournals.org/content/10/18/6347S.full>. "Stable Transfection of CHO Cells." Caltech.edu. California Institute of Technology, n.d. Web. <http://
www.its.caltech.edu/~bjorker/Protocols/Stable%20transfection%20of%20CHO%20.pdf>. Harmon, Katherine. "How Ralph Steinman Raced to Develop a Cancer Vaccine-­‐-­‐And Save His Life: Scientific American." How Ralph Steinman Raced to Develop a Cancer Vaccine-­‐-­‐And Save His Life: Scientific American. Scientific American, 20 Dec. 2011. Web. 01 Aug. 2012. <http://www.scientificamerican.com/article.cfm?id=the-­‐patient-­‐
scientist>. Wierecky, Jan, Martin Mueller, Stefan Wirth, Edith Halder-­‐Oelher, Donna Dorfel, and Susanne Schmidt. "Immunologic and Clinical Responses after Vaccinations with Peptide-­‐Pulsed Dendritic Cells in Metastatic Renal Cancer Patients." Immunologic and Clinical Responses after Vaccinations with Peptide-­‐Pulsed Dendritic Cells in Metastatic Renal Cancer Patients. American Association for Cancer Research, 2006. Web. 19 July 2012. <http://
cancerres.aacrjournals.org/content/66/11/5910.short>. §
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Lucas, Brain, Lynne Giere, Richard DeMarco, Amy Shen, Vanessa Chisholm, and Craig Crosley. "Nucleic Acids Research." High-­‐Level Production of Recombinant Proteins in CHO Cells Using a Dicistronic DHFR Intron Expression Vector. Oxford Journal, 14 Mar. 1996. Web. 01 Aug. 2012. <http://nar.oxfordjournals.org/content/24/9/1774.full>. Zhao, David, Richard Fike, Borka Naumovich, and Mark Stramagalia. "Improving Protein Production in CHO Cells." BioPharm International. Invitrogen Corporation, June 2008. Web. 30 July 2011. <http://www.invitrogen.com/etc/
medialib/en/filelibrary/bioproduction/pdfs.Par.34443.File.dat/Biopharm-­‐article-­‐Improving-­‐Protein-­‐Production-­‐in-­‐
CHO.pdf>. Bibliography §
"Melanoma Center -­‐ Melanoma Basics -­‐ Melanoma Statistics." Melanoma Center -­‐ Melanoma Basics -­‐ Melanoma Statistics. Melanoma Center, Jan. 2010. Web. 01 Aug. 2012. http://www.melanomacenter.org/basics/statistics.html. §
"Melanoma." Cancer.net. American Society of Clinical Society, 16 Feb. 2012. Web. 7 July 2012. <http://
www.cancer.net/patient/Cancer+Types/Melanoma?sectionTitle=Treatment>. §
"FAQ." Denndritic Cell Therapy For Cancer. Wordpress, n.d. Web. 7 July 2012. <http://dendritic.info/faq/>. Pinzon, Cristina. "Protocol for Dendritic Cell Preparation from Monocytes." Dendritic Cell Preparation from Monocytes. Greenburg Lab, 1 June 2011. Web. 14 July 2012. <http://labs.xcrc.org/warren/protocols/
Dendritic_Cell_Preparation_from_Monocytes.pdf>. §
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Strome, Scott. "Strategies for Antigen Loading of Dendritic Cells to Enhance the Antitumor Immune Response 1." Strategies for Antigen Loading of Dendritic Cells to Enhance the Antitumor Immune Response. American Association of Cancer Reseach, 2012. Web. 01 Aug. 2012. <http://cancerres.aacrjournals.org/content/62/6/1884.full>. Nestle, Frank. "Vaccination of Melanoma Patients with Peptide or Lysate Pulsed Dendritic Cells." Nature Publishing Group. Nature, 24 Aug. 2012. Web. 1998. <http://www.nature.com/nm/journal/v4/n3/pdf/nm0398-­‐328.pdf>. Zuber, Thomas. "Punch Biopsy of the Skin -­‐ March 15, 2002 -­‐ American Family Physician." Punch Biopsy of the Skin -­‐ March 15, 2002 -­‐ American Family Physician. American Academy of Physicians, 15 Mar. 2002. Web. 01 Aug. 2012. http://www.aafp.org/afp/2002/0315/p1155.html §
DiChaira, Timothy. "Guide to the LDH Test For Melanoma." About.com Skin Cancer. CNN, 21 May 2009. Web. 01 Aug. 2012. <http://skincancer.about.com/od/diagnosis/a/LDH.htm>. §
"Overview of Western Blotting." Overview of Western Blotting. Thermo Fisher Scientific, 2012. Web. 01 Aug. 2012. <http://www.piercenet.com/browse.cfm?fldID=8259A7B6-­‐7DA6-­‐41CF-­‐9D55-­‐AA6C14F31193>. §
"Overview of Mass Spectrometry." Overview of Mass Spectrometry. Thermo Fisher Scientific, 2012. Web. 01 Aug. 2012. <http://www.piercenet.com/browse.cfm?fldID=33C6C4ED-­‐4B0D-­‐49FA-­‐ABD2-­‐23BCB0FADEC0>. Pictures §  http://en.wikipedia.org/wiki/
Follicular_dendritic_cell_sarcoma §  http://www.google.com/imgres?imgurl=http://
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