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Mukesh Kumar Research Associate

Membrane fusion mediated cytosolic drug delivery through scFv targeted Sendai viral envelopes. Mukesh Kumar Research Associate 1. National Brain Research Centre, Manesar , Haryana

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Mukesh Kumar Research Associate

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  1. Membrane fusion mediated cytosolic drug delivery through scFv targeted Sendai viral envelopes Mukesh Kumar Research Associate 1. National Brain Research Centre, Manesar, Haryana 2. All India Institute of Medical Sciences, New Delhi

  2. Human Placental Alkaline Phosphatase (PAP) PAP »PAP is an isozyme of alkaline phosphatase » 513 amino acid peripheral membrane glycoprotein anchored to the outer surface of plasma membrane via a phosphatidylinositollinkage. PhosphatidylInositolAnchor »Dimer of two identical subunits with molecular weight of 64kDa each.

  3. »Expression is restricted to the second and third-trimester of human placenta. » It is a marker for placental cells as well as for tumors of reproductive organs like ovarian tumor, seminomas, and cervical tumors. Nearly universally seen in germ cell tumors. Characteristics:- Increased heat stability, differential inhibition by various uncompetitive inhibitors like L-Phenylalanine, L-Leucine, peptides like L-Phe-Gly-Gly.

  4. Features that make it an attractive immunotherapeuic target • Cell surface localization • Clathrinmediated endocytosis • Low shedding into circulation • Ectopic expression in various malignancies viz. choriocarcinoma, germ cells(>80%), seminomas,uterus and ovary,breast, colon, head and neck

  5. Human Alkaline Phosphatases

  6. Alkaline Phosphatase Isozymes PAP BONE INTESTINAL • L-Phe • L-Phe • L-Phe-gly-gly • L-Homo THESE INHIBITORS ARE : (i) Isozyme specific (ii) Bind to defined sites on the molecule

  7. Phagemid Filamentous Phage

  8. Bio-panning from Human Phage Display Antibody Library Tomlinson’s human antibody library was used for selection. Bio-panning »TG1 (E.coli) - “Suppressor strain”- recognizes stop codon (TAG) as glutamate- surface display of scFv » HB2151(E.coli) - “Non-suppressor” strain- recognize amber stop codon-for soluble expression of scFv.

  9. Selection of PAP binding monoclonal phage scFv Cross- reactivity profile of P4C8 in presence of varying amounts of PAP/IAP/BAP. PEG precipitated monoclonal phage ELISA . P4C8 is showing good binding to PAP consistently. P4C8 shows maximum inhibition in binding to PAP in the presence of 1.5µg of free PAP

  10. Soluble expression and binding of scFv SDS-PAGE of soluble expression of clones showing 29kD protein Binding of dialyzed soluble scFvs of monoclonals on PAP coated plate Binding of soluble scFv of P4C8 to PAP is maximum

  11. Cellular Internalization Three strategic categories to enhance endosomal escape :- Molecular ferries Leakage-inducing molecules Physico-chemical techniques

  12. Enveloped viral fusion The hemifusion model for bilayer fusion.

  13. SENDAI, A PARAMYXOVIRIDAE ( HEMOLYTIC VIRUS FAMILY ) • Enveloped animal virus, containing single stranded RNA as genetic material. • Discovered in early fifties in “Sendai” Japan, also known as “HVJ” • It requires physiological pH and temperature to enter/infect host cells. • Mechanisms of entry fairly resolved. However, the exact role of HN help to • F-protein in membrane fusion is yet to be deciphered. • It is highly expected to be non-pathogenic for human.

  14. F-protein • Its a glycoprotein which mediates pH-independent fusion of viral • envelope with the plasma membrane of host cell. • Synthesized as inactive precursor F0 (60 kD)– proteolytic cleavage – • F-protein - (disulfide-linked F1 (45kD) and F2 (15 kD) poly peptide) • Fusion peptide – Hydrophobic stretch of 20-25 amino acid at N-terminus • of F1 –essential for biological activity of the protein

  15. Preparation of Drug loaded Virosomes Ramani et. al . Proc. Natl. Acad. Sci. USA Vol. 95, pp. 11886-11890, September 1998

  16. Cloning Strategy of scFv with F-protein fragment ChimericscFv has been used with F-protein for virosomereconstitution.

  17. Choice of Cell Lines and Controls HeLa cells - have PAP; Controls – Heat inactivated CscFv-F-virosome F-virosome alone CscFv-F-virosome at 10°C SaOs cells - BAP only, no PAP SaOstransfected with PAP - have PAP with BAP CHO - no PAP Octadecylrhodamine B chloride (R18) Detection of Membrane Fusion Pictorial representation of a lipid-mixing assay based on fluorescence self-quenching.

  18. PAP Expression Analysis of Transfected SaOs-2 Fold expression Real-Time analysis of PAP expression in transiently transfected SaOs-2 cell line. ~42 fold increase in the PAP RNA level was observed. Flow-cytometer analysis of the PAP transfectedSaOs cells. PAP expressed on the surface of the cells were detected with anti-PAP monoclonal as a primary Ab and anti-mouse alexa 596 as secondary Ab. 14% PAP transfected cells were positively stained for PAP.

  19. Fusion Kinetics of scFv Engineered Virosome with Cell Lines HeLa cells - Natural expression of PAP on surface SaOs-2 - No PAP but BAP expression on surface SaOs (T) - PAP transfectedSaOs CHO - No expression of PAP on surface Kinetics of scFv-F-virosome fusion with different cell lines. Dequenched fluorescence of R-18 shows that the initial membrane fusion is scFvdependent.

  20. FITC-lysozyme delivery by scFv-F-virosome HC = Heat treatment to virosomes abrogates fusion mediated delivery of the cargo without affecting scFv mediated endocytosis. C = Virosome without scFv. Fusion mediated cytosolic delivery of FITC-Lysozyme and fluorescence, Quantification by ImageJ, CTCF = Corrected Total Cell Fluorescence

  21. Doxorubicin delivery by scFv-F-virosome HC = Heat treatment to virosomes abrogates fusion mediated delivery of the cargo without affecting scFv mediated endocytosis. Fusion mediated cytosolic delivery of doxorubicin and fluorescence quantification by ImageJ, CTCF = Corrected Total Cell Fluorescence

  22. Cell Survival Analysis after Virosome Mediated Doxorubicin Treatment HeLA (HC) - Heat Control HeLa (control)- HeLa with F-virosome, no scFv SaOs(UT) - UntransfectedSaOs SaOs(T) - PAP Transfected SaOs CHO – Negative control Trypan blue exclusion assay for cell survival after Doxorubicin packaged virosomal delivery. (significance level of P-value is <0.05) Significant reduction in HeLa cells and PLAP transfectedSaOs cells viability

  23. Effect of Endocytotic Inhibitor on Cellular Internalization Delivery of FITC-lysozyme to PAP expressing HeLa cells by immuno-virosome with or without cytochalasin B (10µM). In presence of cytochalasinB (cyto(+)), the cargo is delivered by membrane fusion mediated pathway.

  24. Localization of Intracellular RITC-lysozyme Delivered by Immuno-virosome Yellow fluorescence indicates endocytotic route of internalization. Reduced yellow fluorescence in panel A in comparison to Panel B but no such change in red fluorescence confirms membrane fusion mediated cytosolic delivery by Immuno-virosome. Live cell confocal microscopy for intracellular localization of RITC-lysozyme in HeLa cells in presence and absence of cytochalasinB (10µM). Lysotracker dye was used for tracking endocytotic route of internalization. HC – Heat control in terms of endocytotic route of entry.

  25. Efficacy of scFv-F-virosome Efficacy of CscFv-F-virosome for RITC-lysozyme delivery to HeLa cells. Cells without primary antibody against PAP (H17E2) were negative control. App. 68% PAP positive cells were also positive for RITC-lysozyme .

  26. Graphical presentation of the modality scFv targeted Sendai viral membrane efficiently delivers the payload in cytosol via membrane fusion mediated process. Chimeric scFv- scFv fused with trans-membrane and cytosolic region of F-protein with a linker in between. PAP- Placental Alkaline Phosphatase

  27. Conclusions • scFv anchored on the virosome surface helps in binding of the virosome • specifically to PAP expressing cells and subsequent membrane fusion is • mediated by F-protein. • Most of the cargo (FITC-lysozyme/ doxorubicin) is delivered directly to • the cytosol. • Enhancement to endosomal escape may result in less immunogenicity of the • developed modality. The data is under review for publication.

  28. Acknowledgement Dr. Subrata Sinha, Director, National Brain Research Centre, Manesar, Haryana Dr. P. Chattopadhyay, Professor, Department of Biochemistry, AIIMS, N. Delhi Dr. D. P. Sarkar, Professor, Department of Biochemistry, South Campus, University of Delhi Dr. Kunzangchosdol, Additional Professor, Department of Biochemistry, AIIMS, N. Delhi PrashantMani, Department of Biochemistry, South Campus, University of Delhi Lab members and staff of the Department of Biochemistry, AIIMS, New Delhi Department of Biotechnology (DBT) and ICMR for funding UGC for fellowship

  29. Thanks

  30. Deglycosylatedimmunovirosome did not fuse with hepatic cells but retained its fusion ability with HeLa cells expressing PAP with greater intensity than the virosomes without PNGase treatment

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