mukesh kumar research associate
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Membrane fusion mediated cytosolic drug delivery through scFv targeted Sendai viral envelopes. Mukesh Kumar Research Associate 1. National Brain Research Centre, Manesar , Haryana - PowerPoint PPT PresentationTRANSCRIPT
Mukesh Kumar Research Associate 1. National Brain Research Centre, Manesar, Haryana 2. All India Institute of Medical Sciences, New Delhi
Membrane fusion mediated cytosolic drug delivery through scFv targeted Sendai viral envelopes
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Human Placental Alkaline Phosphatase (PAP)
» PAP is an isozyme of alkaline phosphatase
» 513 amino acid peripheral membrane glycoprotein anchored to the outer surface of plasma membrane via a phosphatidylinositol linkage.
Phosphatidyl Inositol Anchor
PAP
» Dimer of two identical subunits with molecular weight of 64kDa each.
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» 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.
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• Cell surface localization
• Clathrin mediated endocytosis
• Low shedding into circulation
• Ectopic expression in various malignancies viz. choriocarcinoma,
germ cells(>80%), seminomas, uterus and ovary, breast, colon, head
and neck
Features that make it an attractive immunotherapeuic target
Human Alkaline Phosphatases
Tissue non- Specific (TNAP)
Intestinal (IAP)
Placental ( PAP)
Subunit mol. wt. 69 kDa 68 kDa 64 kDa
Subunit association Dimer dimer dimer
Heat lability Labile 70oC; 5min 70oC; 30 min
Homology with PAP 60% 90%
Urea inhibition 2mol/L
70% 40% 25%
Levamisole inhibition 10 µmol/L 50% insensitive insensitive
Amino – acid / peptide inhibiton
L-Homoarginine L-Phe L-Phe, L-Leu, L-Phe-gly-gly
PAP BONE INTESTINAL
• L-Phe• L-Phe-gly-gly
• L-Phe• L-Homo
THESE INHIBITORS ARE : (i) Isozyme specific (ii) Bind to defined sites on the molecule
Alkaline Phosphatase Isozymes
Filamentous Phage
Phagemid
Bio-panning from Human Phage Display Antibody Library
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.
Tomlinson’s human antibody library was used for selection.
PEG precipitated monoclonal phage ELISA .
Selection of PAP binding monoclonal phage scFv
0
0.4
0.8
1.2
1.6
2
0.5 1 1.5 2 Phage Helper
Enzyme(ug)
Abs
orba
nce(
492n
m)
PLAP IAP BAP Phage Helper
Cross- reactivity profile of P4C8 in presence of varying amounts of PAP/IAP/BAP. 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
SDS-PAGE of soluble expression of clones showing 29kD protein
0
0.25
0.5
0.75
1
1.25
1.5
1.75
2
Ab
sorb
an
ce(4
92
nm
)
neat periplasm
Binding of dialyzed soluble scFvs of monoclonals on PAP coated plate
Soluble expression and binding of scFv
Binding of soluble scFv of P4C8 to PAP is maximum
P1C8
P1C6
P4C8
P3C27 M HB2151
28kD
Cellular Internalization
Three strategic categories toenhance endosomal escape :-
(i) Molecular ferries
(ii) Leakage-inducing molecules
(iii)Physico-chemical techniques
Sascha Martens & Harvey T. McMahon(July 2008)
The hemifusion model for bilayer fusion.
Enveloped viral fusion
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.
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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
F-protein
16 Ramani et. al . Proc. Natl. Acad. Sci. USAVol. 95, pp. 11886-11890, September 1998
Preparation of Drug loaded Virosomes
Cloning Strategy of scFv with F-protein fragment
Chimeric scFv has been used with F-protein for virosome reconstitution.
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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 PAPSaOs transfected with PAP - have PAP with BAP
CHO - no PAP
Detection of Membrane Fusion
Pictorial representation of a lipid-mixing assay based on fluorescence self-quenching.
Octadecyl rhodamine B chloride (R18)
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 transfected SaOs 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.
Fold
exp
ress
ion
PAP Expression Analysis of Transfected SaOs-2
Kinetics of scFv-F-virosome fusion with different cell lines.
Dequenched fluorescence of R-18 shows that the initial membrane fusion is scFv dependent.
HeLa cells - Natural expression of PAP on surfaceSaOs-2 - No PAP but BAP expression on surfaceSaOs (T) - PAP transfected SaOsCHO - No expression of PAP on surface
Fusion Kinetics of scFv Engineered Virosome with Cell Lines
Fusion mediated cytosolic delivery of FITC-Lysozyme and fluorescence, Quantification by ImageJ, CTCF = Corrected Total Cell Fluorescence
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 doxorubicin and fluorescence quantification by ImageJ, CTCF = Corrected Total Cell Fluorescence
Doxorubicin delivery by scFv-F-virosome
HC = Heat treatment to virosomes abrogates fusion mediated delivery of the cargo without affecting scFv mediated endocytosis.
HeLA (HC) - Heat Control
HeLa (control)- HeLa with F-virosome, no scFv
SaOs(UT) - Untransfected SaOs
SaOs(T) - PAP Transfected SaOs
CHO – Negative control
Cell Survival Analysis after Virosome Mediated Doxorubicin Treatment
0
20
40
60
80
100
120 P<0.0037P<0.0014P<0.001
P<0.069 P<0.074
0
20
40
60
80
100
120 P<0.0037P<0.0014P<0.001
P<0.069 P<0.074
Trypan blue exclusion assay for cell survival after Doxorubicinpackaged virosomal delivery. (significance level of P-value is <0.05)
Significant reduction in HeLa cells and PLAP transfected SaOs cells viability
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.
Effect of Endocytotic Inhibitor on Cellular Internalization
Localization of Intracellular RITC-lysozyme Delivered 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.
Yellow fluorescence indicatesendocytotic route of internalization. Reduced yellow fluorescence in panel A in comparison to Panel B but no such change in red fluorescence confirms membrane fusionmediated cytosolic delivery by Immuno-virosome.
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 .
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
Graphical presentation of the modality
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.
Conclusions
The data is under review for publication.
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. Kunzang chosdol, Additional Professor, Department of Biochemistry, AIIMS, N. Delhi
Prashant Mani, 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
Thanks
Deglycosylated immunovirosome 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