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

2. 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

3. Cancers 2008

4. Current Breast Cancer Treatment Modalities: • Include:surgery, radiation, chemotherapy, endocrine therapies, and biological therapies. • Biological treatments: Passive & active immunotherapy

5. EGFR family • The 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.

6. 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.

7. Cont… • In spite of these advantages, peptide vaccines against Her2 have produced variable results. • This variability is due : • Deliver system • Epitope specificity • Adjuvant

8. LPD nanoparticles(Lipid-protamine-DNA)

11. Methods

12. immune response Immunization Preparation of LPD Peptidedesign

15. Epitope prediction = “Fishing”

18. LPD pereparation Lipid films(DOTAP, chol 1:1 molar ratio) Evaporating the chloroform Hydrated with an aqueous solution of Peptide Vortex (MLV), sonication Extrusion 100 nm membrane filters Adding protamine, CpG

19. 2 1 3 9 6 8 7 5 4 Groups LPD- Pep C- CpG LPD- Pep E- CpG LPD- Pep C- non- CpG LPD- Pep E- non-CpG Pep E Pep C LPD LP PBS

20. TUBO: A cloned cell line over-expressing the Her2(neu)protein, was established from a BALB/neu-T transgenic mouse 8 week old Balb/c (n=10) Peptide encapsulated in LPD TUBO Challenge (n=6) Harvest spleen (n=4) Analyse the production of cytokines (IFN-γ , IL-4) Proliferation assay Monitoring of tumor growth for 2 months Analysis of CTL cytotoxicity

21. Her2 Expression in TUBO cells (qRT-PCR) Tissue homogenization (tumoral and normal tissues) Homozenizer RNA Isolation Trizol Production of cDNA by RT-PCR Reaction RT enzyme, primer PCR (Her2 and GAPDH gene) Run in gel Determination size Analyse density bands by Kodak software

22. Proliferation Cellular response Cytotoxicity ELISpot WST-1 Calcein AM Cytokine

23. ELIspot assay

24. Cytoyoxicity assay

25. WST-1 proliferation assay

27. Results

28. Her2 expression

29. Number of Spot/ 105 Cells LP PBS LPD-Pep C-CpG Pep C LPD-Pep E-CpG Pep E LPD LPD-Pep C-nonCpG LPD-Pep E-nonCpG

30. PBS LP LPD-Pep C-CpG Pep C Pep E LPD-Pep E-CpG LPD-Pep C-nonCpG LPD LPD-Pep E-nonCpG

31. IL-4 Pep C LPD-Pep E-CpG LPD-Pep C-CpG PBS LPD LP LPD-Pep C-nonCpG Pep E LPD-Pep E-nonCpG

32. Cytotoxicity%

34. LP PBS LPD-Pep C-CpG Pep C LPD Pep E LPD-Pep E-CpG LPD-Pep C-nonCpG LPD-Pep E-nonCpG

35. Tumor Size

36. Conclusions • In 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

37. Thank You

39. Liposome characterization (Particle sizer) • The mean diameters: 151.8 nm ± 8.7 nm • Surface charges (zeta potential): 29.8 ± 5.15 mV