preparation of lpd nanoparticles containing her2 peptides as vaccine against breast cancer
DESCRIPTION
IN THE NAME oF GOD. Preparation of LPD Nanoparticles Containing Her2 Peptides as Vaccine Against Breast Cancer. Supervisors: Dr. Jaafari MR. Dr. Tavakol Afshari J. Nanobiotechnology Lab, Bu-Ali Research Institute, Mashhad, Iran. By: Jalali SA. Cancers. - PowerPoint PPT PresentationTRANSCRIPT
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Preparation of LPD Nanoparticles Containing Her2 Peptides as Vaccine Against Breast Cancer Supervisors: Dr. Jaafari MR. Dr. Tavakol Afshari J.
IN THE NAME oF GODBy: Jalali SA Nanobiotechnology Lab, Bu-Ali Research Institute, Mashhad, Iran
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Cancers2008
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Current Breast Cancer Treatment Modalities:Include: surgery, radiation, chemotherapy, endocrine therapies, and biological therapies.
Biological treatments:
Passive & active immunotherapy
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EGFR familyThe epidermal growth factor (ErbB or Her) receptors
Upon ligand binding, receptors dimerize and are activated and initiate signaling cascades resulting in regulation of cell growth, proliferation, and division.
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Active immunotherapeutic (Vaccines ):Various vaccination approaches: protein based, DNA based and peptide based.
Several advantages: Peptides are simple, economical to produce, lack infectious and can induce a very epitope specific response.
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Cont
In spite of these advantages, peptide vaccines against Her2 have produced variable results.
This variability is due :
Deliver system
Epitope specificity
Adjuvant
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LPD nanoparticles(Lipid-protamine-DNA)
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Methods
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Peptide design Preparation of LPD Immunizationimmune response
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ProgramURLService available
ProPredhttp://www.imtech.res.in/raghava/propred147 MHC alleles
nHLAPredhttp://www.imtech.res.in/raghava/nhlapred67 MHC alleles
SYFPEITHIhttp://www.syfpeithi.de> 200 MHC alleles
RANKPEPhttp://mif.dfci.harvard.edu/Tools/rankpep.html>40 MHC alleles
BIMAShttp://bimas.dcrt.nih.gov/molbio/hla_bind/>46 MHC alleles
MAPPPhttp://reiner.bu.edu/zhiping/lppep.html>50 MHC alleles
TAPPredhttp://www.imtech.res.in/raghava/tappred/TAP prediction
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Epitope prediction = Fishing
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PeptidesAmino acid sequenceSolubility of peptidesPi(PH isoelectric) AP 5-25ELAAWCRWGFLLALLPPGIAG21.86.05CP 373-381KIFGSLAFL14.46.26DP 435-455IRGRILHDGAYSLTLQGLGIH3.76.39EP 1209-1229SPPHPSPAFSPAFDNLYYWDQ-18.15.7GP 346-354 +P 373-381+P 911-919 CYGLGMEHL RR KIFGSLAFL RR SYGVTVWEL56.47
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LPD pereparation
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Groups
LPD- Pep E- CpG
LPD- Pep E- non-CpG
Pep C
LPD- Pep C- non- CpG
LP
LPD- Pep C- CpG
LPD
Pep E
PBS
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8 week old Balb/c (n=10) Peptide encapsulated in LPDHarvest spleen (n=4)TUBO Challenge (n=6)Analyse the production of cytokines (IFN- , IL-4)Proliferation assay Analysis of CTL cytotoxicity
TUBO:
A cloned cell line over-expressing the Her2(neu) protein, was established from a BALB/neu-T transgenic mouse
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Her2 Expression in TUBO cells (qRT-PCR)
Homozenizer RT enzyme, primer TrizolTissue homogenization (tumoral and normal tissues)Production of cDNA by RT-PCR Reaction PCR (Her2 and GAPDH gene)Run in gelDetermination sizeAnalyse density bands by Kodak software RNA Isolation
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Cytokine Calcein AMWST-1
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ELIspot assay
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Cytoyoxicity assay
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WST-1 proliferation assay
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Results
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Her2 expression
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IL-4
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Cytotoxicity%
CytotoxicityE/T ratio 3/1 TUBOE/T ratio 6/1 TUBOE/T ratio 3/1 Con negLPD-Pep C-CpG9.3%13.6%-11.5%LPD-Pep C-non CpG8.07%18.4%-7.3%Peptide C11.5%12%-15.7%LPD-Pep E-CpG9.3%13.6%-11.5%LPD-Pep E-non CpG20%23%-0.7%Peptide E16.38%19.77%-2.6%LPD-CpG-10%-13%-21%PBS-14%-12%-66%
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Tumor Size
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ConclusionsIn our studies, peptides C, E were able to induce T-cell responses and also the responses against the Her2-expressing tumor cells were effective.
Peptide C, E alone as an antigen weakly induces an immune response.
LPD as a peptide antigen carrier induced stronger immune response
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Thank You
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Liposome characterization
(Particle sizer)
The mean diameters: 151.8 nm 8.7 nm
Surface charges (zeta potential): 29.8 5.15 mV
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