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