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
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.
PAP
Phosphatidyl Inositol Anchor
» Dimer of two identical subunits with molecular weight of
64kDa each.
2
» 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.
3
Features that make it an attractive
immunotherapeuic target
• 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
4
Human Alkaline Phosphatases
Subunit mol. wt.
Tissue nonSpecific
(TNAP)
69 kDa
Subunit association
Dimer
dimer
Heat lability
Labile
70oC; 5min
Homology with PAP
60%
90%
Urea inhibition
2mol/L
70%
40%
Levamisole inhibition
10 µmol/L 50%
insensitive
insensitive
Amino – acid / peptide
inhibiton
L-Homoarginine
L-Phe
L-Phe, L-Leu,
L-Phe-gly-gly
Intestinal
(IAP)
68 kDa
Placental ( PAP)
64 kDa
dimer
70oC; 30 min
25%
Alkaline Phosphatase Isozymes
PAP
• L-Phe
• L-Phe-gly-gly
BONE
INTESTINAL
• L-Homo
THESE INHIBITORS ARE :
(i) Isozyme specific
(ii) Bind to defined sites on the molecule
• L-Phe
Phagemid
Filamentous Phage
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.
Selection of PAP binding monoclonal phage scFv
PLAP
IAP
BAP
Phage
Helper
Absorbance(492nm)
2
1.6
1.2
0.8
0.4
0
0.5
1
1.5
2
Phage
Helper
Enzyme(ug)
PEG precipitated monoclonal phage ELISA .
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
Soluble expression and binding of scFv
2
M
1.75
HB2151
28kD
Absorbance(492nm)
P1 P1 P4 P3
C6 C8 C8 C27
1.5
1.25
1
0.75
0.5
0.25
0
SDS-PAGE of soluble expression
of clones showing 29kD protein
neat periplasm
Binding of dialyzed soluble scFvs
of monoclonals on PAP coated plate
Binding of soluble scFv of P4C8 to PAP is maximum
Cellular Internalization
Three strategic categories to
enhance endosomal escape :(i) Molecular ferries
(ii) Leakage-inducing molecules
(iii)Physico-chemical techniques
Enveloped viral fusion
Sascha Martens & Harvey T. McMahon
(July 2008)
The hemifusion model for bilayer 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.

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
Chimeric scFv has been used with F-protein for virosome reconstitution.
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
SaOs transfected with PAP - have PAP with BAP
CHO - no PAP
Octadecyl rhodamine B chloride
(R18)
Detection of
Membrane Fusion
Pictorial representation
of a lipid-mixing assay
based on fluorescence
self-quenching.
18
Fold expression
PAP Expression Analysis of Transfected SaOs-2
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.
Fusion Kinetics of scFv Engineered Virosome with Cell Lines
HeLa cells
SaOs-2
SaOs (T)
CHO
- Natural expression of PAP on surface
- No PAP but BAP expression on surface
- PAP transfected SaOs
- 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 scFv dependent.
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
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
Cell Survival Analysis after Virosome
Mediated Doxorubicin Treatment
P<0.0014
P<0.001
P<0.069
P<0.0037
P<0.074
HeLA (HC)
- Heat Control
HeLa (control)- HeLa with F-virosome, no scFv
SaOs(UT)
- Untransfected SaOs
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 transfected SaOs cells viability
Effect of Endocytotic Inhibitor on Cellular Internalization
Delivery of FITC-lysozyme to PAP expressing HeLa cells by immunovirosome with or without cytochalasin B (10µM). In presence of cytochalasinB
(cyto(+)), the cargo is delivered by membrane fusion mediated pathway.
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.
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 .
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
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.
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
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