Preparation of LPD Nanoparticles Containing Her2 Peptides as Vaccine Against Breast Cancer
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Transcript of Preparation of LPD Nanoparticles Containing Her2 Peptides as Vaccine Against Breast Cancer
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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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