PPT On Synthetic Vaccines

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Immunologic and Therapeutic Evaluation of a synthetic peptide vaccine for the treatment of patients with metastatic melanoma

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    Metastatic Melanoma Melanoma is a type of skin cancer. When it spreads to other places in your body, it's called metastatic, or advanced. You may also refer to it as stage IV melanoma. Melanoma often spreads to: Tissue under the skin Lymph nodes Lungs Liver Brain @MyFreePPT 0)
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    Table 1. Frequency of melanoma metastases to other organs9 Site of metastasis Skin (other areas of the skin), subcu- taneous tissue, and lymph nodes Lung Liver Brain Bone Gastrointestinal tract Heart Pancreas Adrenals Kidneys Thyroid Frequency (%) 50-75 70-87 36-54 26-58 40—45 36-54 35-48 25-39
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    SYNTHETIC VACCINES-- "POSSIBLE SOLUTION" Identification of genes encoding Melenoma- associated antigens. Synthetic peptide vaccines based on the genes encoding cancer Novel cancer immunotherapies @MyFreePPT
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    METHOD WB(Ecapeo www-medscape.com Source: Cancer Control 2005 H. Lee Moffitt Cancer Center and Research Institute. Inc. To identify melanoma antigens involved in tumor rejection in humans, we utilized tumor-infiltrating lymphocytes (T ILS) grown from metastatic melanoma nodules and adoptively transferred to the autologous melanoma patient. Those TILS associated with in vivo tumor regression were used to screen tumor-derived cDNA libraries to identify the antigens they recognized. Two antigens, MART-I and gp100, whose recognition was re} d by HLA-A2*0201 (HLA-A2) were initially identified, an o were shown to be non-mutated differentiation antigens ex ed cells of melanocytic lineage including melano melanocytes and pigmented retinal cells, but not in tissues or non-melanoma tumors. @MyFreePPT ormal normal
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    By screening large numbers of peptides from these two molecules conforming to known HLA-A2 binding motifs, a single, immunodominant, nine amino acid peptide was identified in the MART-I molecule, and five different epitopes recognized by TILS were identified in the gp100 molecule. The MART-I epitope, m27-35, and two gp10 epitopes, g209—217 and g280—288, bound to the HL A2 molecule with intermediate affinity and di t have optimal amino acids at one of the known binding anchor residues. @MyFreePPT
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    METHOD A large number of synthetic peptides in which single- and double-amino acid substitutions were introduced at HLA- A2 binding positions. A modified g209—217 peptide (referred to as g209-2M), in which a methionine replaced the natural threonine at position 2, bound to the HLA-A2 molecule with gre r affinity than the unmodified peptide and was shown to an increased ability to generate melanoma cytotoxic T lymphocytes (CTLs) in vitro when or sensitization of peripheral blood mononu cells (PBMCs) from patients. @MyFreePPT
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    ISOLATION OF PERIPHERAL BLOOD MONONUCLEAR CELLS FROM PATIENTS Platelet rich plasma (pRp) Buffy ring (mainly white blood cells, WBCs) Red blood cells (RBCs) Whole blood after first centrifugation step @MyFreePPT percoll Diluted buffy ring 1:1 with TNC set over Percoll Plasma/Platelets Layer of PBMCs percoll RBCs and granulocytes Layer distribution after centrifugation through Percoll density gradient
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    Clinical protocol Identification of peptides by MS & HPLC Modification of Peptides Emulsification of Peptides + IFA Generation of Melenoma Reactive Cytotoxic T Lymphocytes Sensitization of PBMCs every 3 weeks. @MyFreePPT
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    Clinical protocol Cell-mediated Immunity Pre Clinical Trials Clinical Trials Cell lines Mouse Models Human Trials @MyFreePPT
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    ADVANTAGES OF PEPTIDE VACCINES Readily synthesized and purified at low cost Stable in many storage conditions Safe Very effective at inducing CD8 or CD4 T cell responses in vivo in humans Enables direct monitoring of T cell responses induced by the vaccine. Using defined epitopes avoids use of uncharacterized antigens th nontherapeutic autoimmune activity. Repeated booster vaccines feasible @MyFreePPT have
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    LIMITATIONS Class I MHC restriction limits relevance of individual peptides to certain HLA types. Rapidly degraded by serum/tissue peptidases. Peptides with low affinity for MHC may be poorly immunogenic. Patients have variable repertoires for melanoma antigens: a pepti vaccine may have to include a large number of peptides to be ul across a wide range of patients. Immune responses may be transient and/or of low mag @MyFreePPT
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    FUTURE ASPECTS Autologous immune enhancement therapy cells mainly target cancer cells, while chemotherapy and Radiotherapy destroy healthy cells as well. T cells transduced with chimeric antigen receptors composed of hybrid immunoglobulin light chains with endodomains of T-cell signaling molecules. TILS together with high-dose interleukin 2 have durable clinical response rates near 50% or more. Significantly higher cancer regression rates. The emulsification of peptide in adjuvant is thought to provide pr on exposure to antigen and to activate nonspecific inflammatory possible recruitment of antigen-presenting cells to the site of i @MyFreePPT tion.
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    Mass Spectrometry ' Mass spectrometry (MS) is an analytical technique that ionizes chemical species and sorts the ions based on their mass to charge ratio Mass spectrometry is used in many different fields and is applied to pure samples as well complex mixtures MALDI-TOF for protein analysis @MyFreePPT
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    Principle of MALDI A focused laser beam, either in infra-red ranges, can "evaporate" from the solid phase A focused laser beam, either in infra-red ranges, can "evaporate" from the solid phase @MyFreePPT the UV or compounds the UV or compoun
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    Matrix-Assisted Laser Desorption Ionization (MALDI) Sli01t laser pulse Flight tube and drift region to measure the time-of-flight (TOF) 0 detectOr Accelerating pulse
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    MALDI-TOF IONIZATION N2 laser pulse (337 nm) r_M+lJ- matrix ions Protein/peptide/nucleotide/saccharide deposited on crystal surface
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    How TOF instrument works? Linear flight tube (1.0 • 1.5 m) Source ccelerating pulse Oscilloscope to time ion arrival Oscilloscope to time ion arrival (1 gsec) Reflectron Effective flight tube (3.0 m)
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    destain trypsin 1:20 Water Bath 370c MALDI plate 0000 Desalting Ziptip Eppendorf tube Incubate overnight 15000 0 10000 5000 Speed-Vac 00 1000 1500 3 2000 2500 Mass (m/z) 3000
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    H p LC HPLC Coumn gniectoc AutoSatnpicf Samplo Morvvjof Solvent {Mobdo Phase) P'.rnp Solvent Manager Oe&very System Oetoctcy Cctnputer Data Statui Waste
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    Single Phase Column ' It contains the reverse-phase resin C 18, which interacts with the hydrophobic moieties of the peptides ' Resulting peptides are loaded onto the single- phase column and eluted into the mass analyzer using an increasing gradient of an organic solvent ' Peptides resulting from the digestion of o e complex mixtures of proteins are resolved us biphasic column. @MyFreePPT
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    Single-phase column ' Peptides initially interact with the SCX resin and are eluted into the C 18 resin by ammonium acetate that competes for the peptide-binding sites ' Peptides are then eluted from the C 18 resin into the mass analyzer This process is repeated using increasi concentrations of ammonium acetate differentially elute peptides in a stepwise f @MyFreePPT
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    Procedure ' Load the digested protein sample of interest onto the column. For single-phase columns: ' i. Load the digested protein sample directly onto the equilibrated C 18 5-gm tip. ' ii. Wash the protein-loaded column for 5-10 minutes with buffer A. ' iii. Elute peptides from the HPLC with the refe gradient @MyFreePPT
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    1. 2. 3. 4. 5. 6. For integrated biphasic columns Load the digested protein sample onto the equilibrated desalting column. Desalt the loaded sample by washing for 5-10 minutes with 100% buffer A. At this point, the sample is loaded onto the C 18 resin of the filter column setup. Take the equilibrated 5-gm tip and cut at the bottom of the SCX to remove the unloaded portion of the column. Replace the waste line of the desalting column with the pm tip to yield the integrated biphasic column. Degas peptide elution buffer C. Start elution of peptides from the HPL ammonium-acetate-free step, followed by gra with buffer B an lution @MyFreePPT
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    For integrated biphasic columns Continue elution with a series of increasing ammonium- acetate steps (where X = % buffer C), each followed by gradient elution with buffer B The number of ammonium-acetate steps depends on the complexity of the sample, where more complex samples require more ammonium-acetate steps. Typically, four salt steps of 10%, 25%, 50%, and 80% of buffer C ar sufficient. Conclude the elution with a salt step of 100% buf and gradient elution with buffer B @MyFreePPT
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    Analysis with Maldi-TOF

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