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

Cancers2008

Current Breast Cancer Treatment Modalities:Include: surgery, radiation, chemotherapy, endocrine therapies, and biological therapies.

Biological treatments:

Passive & active immunotherapy

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.

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.

Cont

In spite of these advantages, peptide vaccines against Her2 have produced variable results.

This variability is due :

Deliver system

Epitope specificity

Adjuvant

LPD nanoparticles(Lipid-protamine-DNA)

Methods

Peptide design Preparation of LPD Immunizationimmune response

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

Epitope prediction = Fishing

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

LPD pereparation

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

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

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

Cytokine Calcein AMWST-1

ELIspot assay

Cytoyoxicity assay

WST-1 proliferation assay

Results

Her2 expression

IL-4

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%

Tumor Size

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

Thank You

Liposome characterization

(Particle sizer)

The mean diameters: 151.8 nm 8.7 nm

Surface charges (zeta potential): 29.8 5.15 mV

**