Multi-component polymeric system for tumour cell-specific gene delivery using a universal bungarotoxin linker

Article abstract of DOI:10.1007/s11095-010-0088-8

Purpose: A new universal tool for specific, non-covalent and non-destructive attachment of a recombinant antibody fragment to a polymer-modified adenovirus has been utilised to regulate the tropism of adenoviral gene delivery vector. Methods: We have prepared a multivalent reactive N-(2-hydroxypropyl)methacrylamide-based copolymer (PHPMA) bearing an α-bungarotoxin-binding peptide (BTXbp). The copolymer was used for covalent surface modification of adenoviral vectors (Ad). The α-bungarotoxin protein (BTX) has a nanomolar binding affinity for BTXbp, allowing non-covalent linkage of BTX fusion proteins. A single chain variable fragment of anti-PSMA antibody bearing BTX (scFv-BTX) binding to the prostate-specific membrane antigen (PSMA) was conjugated with the copolymer-coated adenovirus to enable specific infection of prostate cancer cells via PSMA receptors. Results: As shown by ELISA, the copolymer-coated virus exhibited much reduced binding to anti-Ad antibodies. Infection of PC-3 and LNCaP prostate cancer cells was ～100-fold less efficient with copolymer-coated Ad than with un-modified Ad. Conjugation of scFv-BTX with Ad-PHPMA-BTXbp led to 5-10-fold restoration of infection in PSMA-positive LNCaP cells. In PSMA-negative PC-3 cells, the conjugation of scFv-BTX with Ad-PHPMA-BTXbp gave no enhancement of infection. Conclusions: We have shown that the presented Ad-PHPMA-BTXbp/scFv-BTX system can be used as a universal tool for a receptor-specific virotherapy.

Full text of DOI:10.1007/s11095-010-0088-8

Pharm Res (2010) 27:2274–2282
DOI 10.1007/s11095-010-0088-8
RESEARCH PAPER
Multi-component Polymeric System for Tumour Cell-Specific
Gene Delivery Using a Universal Bungarotoxin Linker
Ralph A. Willemsen & Michal Pechar & Robert C. Carlisle & Erik Schooten & Robert Pola & Amber J. Thompson &
Leonard W. Seymour & Karel Ulbrich
Received: 25 November 2009 /Accepted: 9 February 2010 /Published online: 19 March 2010
#
Springer Science+Business Media, LLC 2010
ABSTRACT
efficient with copolymer-coated Ad than with un-modified Ad.
Conjugation of scFv-BTX with Ad-PHPMA-BTXbp led to 5–
10-fold restoration of infection in PSMA-positive LNCaP cells.
In PSMA-negative PC-3 cells, the conjugation of scFv-BTX with
Ad-PHPMA-BTXbp gave no enhancement of infection.
Conclusions We have shown that the presented Ad-PHPMA-
BTXbp/scFv-BTX system can be used as a universal tool for a
receptor-specific virotherapy.
Purpose A new universal tool for specific, non-covalent and
non-destructive attachment of a recombinant antibody frag-
ment to a polymer-modified adenovirus has been utilised to
regulate the tropism of adenoviral gene delivery vector.
Methods We have prepared a multivalent reactive N-(2-
hydroxypropyl)methacrylamide-based copolymer (PHPMA)
bearing an α-bungarotoxin-binding peptide (BTXbp). The
copolymer was used for covalent surface modification of
adenoviral vectors (Ad). The α-bungarotoxin protein (BTX)
has a nanomolar binding affinity for BTXbp, allowing non-
covalent linkage of BTX fusion proteins. A single chain variable
fragment of anti-PSMA antibody bearing BTX (scFv-BTX)
binding to the prostate-specific membrane antigen (PSMA) was
conjugated with the copolymer-coated adenovirus to enable
specific infection of prostate cancer cells via PSMA receptors.
Results As shown by ELISA, the copolymer-coated virus
exhibited much reduced binding to anti-Ad antibodies. Infection
of PC-3 and LNCaP prostate cancer cells was ∼100-fold less
KEY WORDS adenovirus . bungarotoxin . hydrophilic
polymers . scFv. tumour targeting
ABBREVIATIONS
Ad
adenovirus
BCA
bicinchoninic acid
BTX
α-bungarotoxin
BTXbp
HEPES
α-bungarotoxin-binding peptide
4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid
HINGS
HPMA
IPTG
heat-inactivated normal goat serum
N-(2-hydroxypropyl)methacrylamide
isopropyl β-D-1-thiogalactopyranoside
Ralph A. Willemsen, Michal Pechar and Robert C. Carlisle contributed
equally to the manuscript
:
R. A. Willemsen E. Schooten
Ma-GG-OH N-methacryloylglycylglycine
Ma-GG-TT
Laboratory of Experimental Tumor Immunology, Erasmus MC,
Rotterdam, The Netherlands
3-(N-methacryloylglycylglycyl)
thiazolidine-2-thione
poly(ethylene glycol)
N-(2-hydroxypropyl)methacrylamide-based
copolymer
:
:
M. Pechar (*) R. Pola K. Ulbrich
Institute of Macromolecular Chemistry,
Academy of Sciences of the Czech Republic,
Heyrovsky Sq. 2, 162 06,
PEG
PHPMA
Prague 6, Czech Republic
e-mail: pechar@imc.cas.cz
PBS
PSMA
scFv-BTX
phosphate buffered saline
prostate specific membrane antigen
single chain variable fragment of anti-PSMA
antibody bearing BTX
:
:
R. C. Carlisle A. J. Thompson L. W. Seymour
Department of Clinical Pharmacology, University of Oxford,
Oxford, UK
TMB
3,3′,5,5′-tetramethylbenzidine

Bungarotoxin Linker for Targeted Gene Delivery
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INTRODUCTION
non-covalent interaction between two peptides or proteins
represents a possible solution to this requirement.
Adenovirus-based gene therapy is a very promising ap-
proach for treatment of oncological diseases (1,2). The
transduction efficacy of adenoviruses is still several orders of
magnitude higher than that of any non-viral vectors.
However, clinical application of adenovirus vectors to treat
metastatic disease has been so far compromised by the poor
target cell selectivity, high immunogenicity and rapid
neutralisation by antibodies and clearance of the vectors
from the bloodstream (3–6).
In this paper, we introduce a new universal tool for
specific, high affinity and non-destructive attachment of
recombinant proteins to other macromolecules such as
synthetic or natural polymers or proteins. The principle of
the method is based on the strong and non-covalent
interaction between acetylcholine receptors and α-
bungarotoxin (BTX).
It has been reported in literature (13,14) that synthetic
peptide with amino acid sequence GSGGSGGTGYRS
WRYYESSLEPYPD (BTXbp) derived from the structure
of acetylcholine receptor binds BTX with high affinity, thus
inhibiting its interactions with acetylcholine receptor with
an IC50 of 2 nM. We linked the BTXbp to a reactive
HPMA-based hydrophilic copolymer and used the resulting
peptide-bearing copolymer (PHPMA-BTXbp) for surface
modification of an adenoviral gene delivery vector. A
recombinant anti-PSMA antibody scFv fragment contain-
ing the BTX binding region (scFv-BTX) was prepared and
added to the polymer-modified BTXbp-bearing adenovirus
(Ad-PHPMA-BTXbp) encoding luciferase as a reporter
gene (Ad) as shown in Fig. 1. The fusion protein scFv-BTX
served as a targeting ligand redirecting the natural viral
tropism to prostate-specific membrane antigen (PSMA) on
prostate cancer cell lines.
It has been demonstrated that modification of
adenoviruses with hydrophilic synthetic polymers based
on poly(ethylene glycol) (PEG) (7) or N-(2-hydroxypropyl)
methacrylamide copolymer (PHPMA) (8,9) significantly
prolongs plasma circulation times and ablates non-specific
cell entry. In addition, surface modification of the
adenovirus capsid with multivalent polymers provides a
platform for retargeting with receptor-specific ligands,
such as growth factors (10), tumour-specific peptides (11)
or folates (12). However, the covalent attachment of
targeting ligand to the coating polymer suffers from
several limitations. First, the reaction conditions required
lead to at least partial loss of the biological activity and
specificity of the ligand. The resulting organisation of the
polymer-ligand conjugate is usually not a chemically
homogenous structure but rather a heterogeneous mixture
of substances with various degrees of modification of the
targeting molecule. Second, such modification does not
necessarily present the ligand in the most effective
orientation for subsequent receptor binding. Third, the
use of amine reactive groups of the HPMA copolymer for
retargeting can diminish the efficiency with which the
polymer coats the adenovirus.
MATERIALS AND METHODS
Chemicals
Peptide BTXbp (GSGGSGGTGYRSWRYYESSLEPYPD,
−1
2728 gmol ) was prepared by standard Fmoc solid phase
peptide synthesis by peptide company Vidia (Vestec, Czech
Republic). All other reagents and solvents were purchased
from Sigma-Aldrich (Czech Republic). Methacryloyl chlo-
ride, 1-aminopropan-2-ol and dichloromethane were dis-
tilled immediately before use.
With regards to potential future applications and
regulatory approval of the new targeted therapeutics based
on polymer-coated adenoviruses, a more specific technique
of conjugating the targeting moiety to the vector to provide
better defined structures is desirable. A specific, strong,
Fig. 1 Schematic representation of a polymer-modified adenovirus targeted with a recombinant scFv antibody fragment attached via a bungarotoxin linker.

2
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Willemsen et al.
Synthesis of Monomers
acetonitrile in the presence of 0.1% TFA with a UV-VIS
diode array detector (Shimadzu, Japan). Determination of
molecular weights and polydispersity of the copolymers was
carried out by size exclusion chromatography on a HPLC
system (Shimadzu) equipped with refractive index, UV, and
multiangle light-scattering DAWN 8 EOS (Wyatt Technol-
ogy Corp., Santa Barbara, CA) detectors using Superose 6
column (Pharmacia) and 0.3 M acetate buffer, pH 6.5 at a
flow rate of 0.5 ml/min. The calculation of molecular
weights from the light-scattering detector was based on the
known injected mass presuming 100% mass recovery. The
content of TT groups was determined spectrophotometri-
cally on a UV/VIS spectrophotometer Helios Alpha
(Thermospectronic, UK) using the absorption coefficient
N-(2-hydroxypropyl)methacrylamide (HPMA) was prepared
by reaction of methacryloyl chloride with 1-aminopropan-
2
-ol in dichloromethane (15). N-Methacryloylglycylglycine
(
Ma-GG-OH) was prepared by Schotten-Baumann acyla-
tion of glycylglycine with methacryloyl chloride in aqueous
alkaline medium (16). 3-(N-Methacryloylglycylglycyl)
thiazolidine-2-thione (Ma-GG-TT) was prepared by the
reaction of Ma-GG-OH with 4,5-dihydrothiazole-2-thiol
in N,N′-dimethylformamide in the presence of N,N′-
dicyclohexylcarbodiimide (17).
Reactive Copolymer PHPMA
−
1
−1
for TT in methanol, ε305=10 280 l·mol ·cm .
The copolymer poly(HPMA-co-Ma-GG-TT) was prepared
by radical solution copolymerization of HPMA (90 mol %)
and Ma-GG-TT (10 mol %) performed in dimethyl
sulfoxide at 50°C for 6 h. The concentration of monomers
in the copolymerization mixture was 15 wt % and that of
Recombinant Protein Ligand scFv-BTX
To construct a PSMA-specific scFv-BTX fusion protein, the
scFv J591 (a kind gift from M. Sadelain, New York, USA)
was introduced into the pCES1 vector (18) via Sfi I and Not
I sites next to the BTX sequence (Gene-synthesis by
Baseclear, Leiden, The Netherlands), which was cloned
via Not I and Bam HI. After verification of the correct
sequence, the resulting pCES1-scFv-BTX vector was
introduced into TG1 bacteria via electroporation (Biorad
micropulser). One positive colony was then grown over-
night in 50 ml of 2×TY medium containing ampicillin
100 μg/ml and glucose 2% at 30°C. The next day, 7.5 ml
of the overnight culture was added to 750 ml of 2×TY
medium with ampicillin 100 μg/ml and glucose 1% and
grown for approximately 2 h until OD600 of 0.8–1.0 was
reached. Protein production was then induced by the
2
,2′-azobisisobutyronitrile initiator was 2 wt % (16). For
characteristics of resulting copolymer 1 see Table I.
Reactive Copolymer with BTXbp
The conjugation of BTX-peptide (20 mg, 7.3 μmol) to
reactive copolymer 1 (78 mg, 41 μmol TT) was performed
in 3% aqueous solution at constant pH 7.4. The reaction
course was monitored using reversed-phase HPLC. The
reaction was terminated after 1.5 h by addition of
CH COOH (20 μl, 0.33 mmol). The polymer conjugate
3
was purified by gel filtration on Sephadex G-25 in water
(
7
PD 10 column; Pharmacia) and freeze-dried, yielding
8 mg (80%) of reactive polymer-peptide conjugate 2.
o
addition of 1 mM IPTG and overnight incubation at 30 C.
For the characteristics, see Table I.
The next day, bacteria were spun down at 4,000 rpm for
15 min, and medium was stored for purification of the
Analytical Methods
fusion protein.
The fusion protein was purified by FPLC using a His-
trap crude column after adjustment of the immidazole
concentration to 5 mM. Protein eluted in PBS (10 mM
phosphate buffer saline) with 500 mM immidazole was
desalted using Zebra desalting Spin columns (Thermo
The monitoring of conjugation of the peptide to reactive
copolymer was performed by HPLC using column Chro-
molith Performance RP-18e, 100×4.6 mm (Merck, Ger-
many) and linear gradient water-acetonitrile, 0–100%
Table I Basic Characteristics of the Reactive Copolymers
a
b
c
Polymer
Structure
M
w
M
w
/M
n
Peptide
wt %
TT
−1
g∙mol
mol %
1
2
p(HPMA-co-Ma-GG-TT)
32 000
65 000
1.9
2.7
–
8.2
4.1
p(HPMA-co-Ma-GG-BTXbp–co-Ma-GG-TT)
16.7
a
Determined by SEC with light scattering detector
b
c
Determined by reverse-phase HPLC
Determined by UV/VIS spectrophotometry in methanol (ε305=10280 lmol−1 cm−1
)

Bungarotoxin Linker for Targeted Gene Delivery
2277
scientific). The final fusion protein preparation was checked
for concentration by BCA protein assay kit (Pierce) purity
and correct size by SDS-PAGE and western blotting.
sealed (greiner Bio-one, 676001). After incubation over-
night at 4°C, the plate was washed 10× with PBS using a
Nunc Immuno Wash12. Fiftyµl of blocking buffer (coating
buffer+5% HINGS) was added to each well, the plate
sealed and incubated at 37°C for 1 h, then washed twice
Modification of the Adenoviruses with Polymers
8
with PBS. Ad samples were diluted to 5×10 particles per
LNCap and PC-3 carcinoma cells were obtained from
ATCC; PSMA status was confirmed using an anti-PSMA
antibody (AbCam) and flow cytometric analysis. E1, E3-
deleted Ad5 expressing CMV IE promoter driven luciferase
was purchased from Hybrid Systems Ltd. (Oxford, UK)
and is denoted “Ad” throughout.
well in diluent (PBS +5% HINGS) and 50µl added to each
well. The samples were sealed and incubated overnight at
4°C before the plate was washed 10 times in PBS.
Polyclonal goat anti-adenovirus-Biotin conjugate (AbD
Serotec, 0151-9104) was diluted 1/100 in diluent, then
50µl was added to each well, sealed, and incubated at room
temperature for 1 h. After washing 10× in PBS, 50µl of
Avidin-HRP (eBioscience, 18-4100-94) diluted 1/1000 in
diluent was added to each well and sealed and incubated at
room temperature for 30 min. After washing 10× in PBS,
50µl of TMB substrate (ADI Inc, 80091) was added to each
well and left to develop at room temperature for 10 min.
Fiftyµl of stop solution (ADI Inc. 80100) was added to each
well and the plate read at 450 nm, 1 s on Wallac Victor2
1420 multilabel counter. Data are represented as the Abs
Polymer coating was performed by mixing Ad with
PHPMA or PHPMA-BTXbp in 10 mM HEPES buffer
(
1 h, 10 mg/ml, pH 7.4) before purifying away un-reacted
polymer using S400 columns 27-5140-01 (GE Healthcare).
Recoveries were calculated using a Picogreen assay as
previously reported for Ad-PHPMA using Oligreen (19).
9
Mixing of 17µl (10 copies) of polymer-coated Ad with
0
.05 mg/ml solution of scFv in a total volume of 60µl was
then performed.
450 nm value after subtraction of the value obtained for the
Anti-Ad Antibody ELISA to Assay Polymer Coating
negative control.
Polyclonal rabbit anti-adenovirus antibody (AbCam,
ab6982-100) was diluted 1/500 in coating buffer (Alpha
Diagnostics International Inc. [ADI], 80050) and 50µl
added to each well of a Nunc Maxisorb flat-bottom
microplate (Fisher Scientific, 439454), and the plate was
Cancer Cell Line Infections
4
Cells were plated (96 well plate, 10 per well) and 24 h later
Ad samples (500 copies / cell) added. After 90 min, infection
media were removed and washing in 150µl PBS performed
Scheme 1 Synthesis of the Reactive Copolymers

2
278
Willemsen et al.
Fig. 2 HPLC chromatogram of the reaction mixture containing polymer 1 and BTXbp at the beginning of reaction and after 1.5 h using C18 column
Chromolith, linear gradient water–acetonitrile, 0–100% acetonitrile, 0.1% TFA and UV detection at 220 nm.
before the addition of fresh media. Twenty-four h later,
media were removed, washing in PBS performed, lysis
solution was added and then luciferase levels measured using
the luciferase reporter system E151A (Promega) and a
luminometer (LB9507, Berthold). Uptake studies were
performed in suspension by incubating 200,000 cells with
7
1
0 copies of Ad sample for 90 min with constant agitation.
Cells were then spun (2,000 g, 5 min), supernatant removed
and a PBS wash performed. After re-suspension in 400µl
fresh media, half of the sample was analysed for Ad genome
content by QPCR as in (5), and half the sample was re-
plated and assayed at 24 h for luciferase expression.
RESULTS
Synthesis of the Polymer-Peptide Conjugates
Radical copolymerization of HPMA and Ma-GG-TT
afforded reactive polymer precursor 1. Polymer precursor
1
reacted with BTXbp in aqueous solution at constant pH
7
.4 to yield the target reactive polymer-peptide conjugate 2
as shown in Scheme 1. The content of reactive TT groups
decreased during 1.5 h of the reaction from 8.2 mol % to
final 4.1 mol %. The amount of BTXbp bound to the
polymer corresponded to 1.1 mol %; the remaining TT
groups were hydrolyzed during the coupling reaction. The
HPLC profile is shown in Fig. 2, and the molecular weights
and polydispersity values of the polymer precursor and the
polymer peptide conjugate are summarized in Table I.
Fig. 3 Production and purification of the scFv-BTX fusion protein. The
recombinant protein scFv J591-BTX was produced in E. Coli as described
in Materials and Methods. The fusion protein was purified from medium
using a His-trap crude column and analyzed by SDS-PAGE (left picture)
and western blotting (right picture) for purity and correct size. By SDS-
PAGE, a pre-dominant band of +/− 37 KD was obtained (>90% purity).
This was confirmed by western blotting using anti-c Myc antibody 9E10 (s:
non-purified medium, p: purified). M = molecular weight marker lane.

Bungarotoxin Linker for Targeted Gene Delivery
2279
Recombinant Protein Ligand scFv-BTX
The recombinant PSMA-specific scFv-BTX fusion protein
was cloned into the pCES1 vector and produced by
standard protein expression in E-Coli, essentially as
described in the Materials and Methods section. As shown
in Fig. 3, the scFv-BTX protein was produced and could be
purified (>90% purity).
Modification of Ad with PHPMA-BTXbp Polymers
The binding of anti-adenovirus antibodies to Ad-PHPMA-
BTXbp was assessed by ELISA (see methods). Fig. 4 shows
that antibody binding to Ad was higher than the level of
binding to Ad-PHPMA-BTXbp or Ad-PHPMA. Indeed,
the Abs 450 nm reading achieved with Ad-PHPMA-
BTXbp or Ad-PHPMA was equivalent to the signal
8
achieved using 1.25×10 copies of Ad, indicating the
binding of antibodies had been reduced 75%. The level
of antibody binding to Ad-PHPMA or Ad-PHPMA-
BTXbp was equivalent. This indicates that the presence
of the BTXbp does not diminish the ability of the PHPMA
to coat and protect Ad.
Fig. 5 Luciferase expression in PSMA-positive LNCap cells and PSMA-
negative PC-3 cells after incubation with unmodified or polymer-modified
Ad. Ad, polymer-coated Ad or polymer-coated, retargeted Ad was
prepared as described in the Materials and Methods and used to infect
LNCaP (panel A) or PC-3 cells (panel B). Luciferase reporter gene
expression was assayed at 24 h (see Materials and Methods). N=4,
standard deviation shown, **=p<0.005.
Retargeting with scFv-BTX
Having verified the ability of the PHPMA-BTXbp to coat
Ad, fluorimetry/SDS-PAGE analysis of the binding of Ad-
PHPMA-BTXbp to BTX-Alexa488 confirmed that Ad-
PHPMA-BTXbp could interact with BTX (data not
shown). Infections in prostate cancer cell lines were then
used to test whether retargeting of Ad-PHPMA-BTXbp
could be achieved using scFv-BTX (Fig. 5). In PSMA-
positive LNCaP cells (Panel A) luciferase transgene expres-
sion was knocked down ∼100-fold upon coating Ad with
either PHPMA-BTXbp or conventional PHPMA. Howev-
er, after incorporation of the targeting scFv-BTX ligand,
activity of Ad-PHPMA-BTXbp was restored between 5-
and 10- fold (p<0.001), whereas the activity of Ad-PHPMA
was not altered. In PSMA-negative PC-3 cells, infection
activity was not rescued by the incorporation of the scFv-
BTX targeting moiety (Panel B).
To verify that the presence of scFv-BTX instigated
improved infection by enhancing the level of binding of Ad-
PHPMA-BTXbp to cells, an association assay was per-
formed and quantitative polymerase chain reaction
(
QPCR) used to measure the number of Ad genomes in
the cells (see Materials and Methods). Fig. 6 demonstrates
that, in LNCaP cells, coating of Ad with PHPMA-BTXbp
leads to a decrease in cell association from 15% of the input
dose to less than 5% (Panel A). However, incorporation of
targeting scFv-BTX moiety leads to a significant (p<0.05)
2-fold restoration of cell association. It is notable that this
small increase in cell association leads to a much greater
increase in transgene expression (9-fold). In PSMA negative
PC-3 cells, neither the cell association nor the resulting
transgene expression was enhanced by the inclusion of scFv-
BTX.
Fig. 4 PHPMA-BTXbp provides efficient coating of Ad. Ad, Ad-PHPMA-
BTXbp or Ad-HPMA were exposed to an anti-Ad antibody-based
8
8
sandwich ELISA (see methods). 5×10 or 1.25×10 copies per well,
N=4, standard error shown.

2
280
Willemsen et al.
Fig. 6 Percentage of the dose of
the vector associated with the cells
after their incubation with un-
modified or polymer-modified Ad.
Sample was added to LNCaP
(
panels A and B) or PC-3 (panels
C and D) cells in suspension, and
the amount of Ad genomes asso-
ciated with cells after 90 min was
assayed by QPCR (see Materials
and Methods). Half the sample
was maintained to assay the
resulting transgene expression.
Data is represented as % of the
input dose associated with cells.
N=3, standard deviation shown,
*
=p<0.05, **=p<0.005.
DISCUSSION
physiological conditions and hardly available for the acyla-
tion at pH 7.4 used for the reaction. We also assumed that the
eventual product of acylation of the phenolic group in
tyrosine would be quickly hydrolyzed back as a result of
general reactivity of phenyl esters with nucleophiles. Relative
stability of the TT groups towards hydrolysis allowed us to
bind most of the peptide to polymer in aqueous solution
during 1.5 h and still preserve half of the TT groups for the
subsequent modification of the adenovirus. The course of the
binding reaction was monitored by HPLC (Fig. 2).
Synthesis of the Polymer–Peptide Conjugates
Copolymers of HPMA with 4-nitrophenyl ester (ONp) or
thiazolidine-2-thione (TT) derivatives of N-methacryloylated
oligopeptides have been reported numerous times as reactive
polymer precursors for synthesis of various biologically active
compounds (BAC), such as cytostatics, peptides, enzymes
and targeting antibodies. In most cases, the modification
reaction between the polymer and BAC was based on the
acylation of a primary amino group of BAC with reactive ester
Nevertheless, the increase in polydispersity (from 1.9 to
2.7) of the polymer after the reaction with the peptide
indicates that a very low degree of Arg modification on the
guanidino group could not be ruled out completely.
(ONp) or amide (TT) groups of the polymer. In the case of
proteins or peptides containing more than one nucleophilic
group susceptible to the acylation, the resulting polymer–
peptide conjugate is at least partially branched. Consequently,
its biological activity is compromised by the modification re-
action. Careful choice of the reaction conditions is therefore
crucial for minimization of these undesired reactions.
The advantage of using reactive polymer precursors with
TT instead of ONp groups is their much slower rate of
undesired hydrolysis in aqueous solutions. Here we report
the use of this approach for attachment of BTXbp peptide
to reactive copolymer 1 via the N-terminus of the peptide.
This work was performed with the deprotected peptide due
to the extreme difficulties with purification of the corre-
sponding fully protected peptide derivative. Our initial
attempts to bind the deprotected BTXbp to polymer 1 in
organic solvent always led to highly branched or even in-
soluble cross-linked product, probably due to acylation of the
side chain of the two Arg and four Tyr residues in the amino
Recombinant Protein Ligand scFv-BTX
The production of scFv-BTX fusion proteins in bacteria
provides a fast and simple means to obtain retargeting ligands
of choice. The method is easily expandable to other ligands,
not limited to scFv, but also other antibody fragments or
complete proteins. In addition to the PSMA-specific scFv J591
we have now also successfully expressed scFv-BTX fusion
proteins specific for PSCA, HLA-A1/MAGE-A1, single
domain antibody fragments (VHH) specific for EGFR, CEA
and HLA-A2/multi-MAGE-A (data not shown).
Cell Transfection Studies
Successful systemic application of adenoviruses as gene
delivery vectors is still associated with many serious hurdles,
such as immunogenicity, low target selectivity and short
blood circulation times of the vectors. Surface modification
acid sequence of BTXbp. As the pK value of the guanidino
group of arginine is 12.5, it remains protonated under
a

Bungarotoxin Linker for Targeted Gene Delivery
2281
of the viruses with hydrophilic synthetic polymers based, e.g.,
on PEG or HPMA is a possible promising strategy over-
coming at least to some extent the mentioned obstacles (9).
The first step in changing the undesirable natural Ad
tropism is detargeting of the virus from its natural cell entry
mechanism via coxsackievirus and adenovirus receptor
PHPMA-BTXbp with scFv-BTX ligand, the number of Ad
genomes recovered from LNCaP cells increased just 2-fold.
This increase instigated a significant 10-fold enhancement
of reporter gene expression. These data indicate that in the
case of non-specific cell entry of non-targeted polymer-
modified Ad-PHPMA-BTXbp, a large proportion of the
Ad is incapable of efficient transgene expression compared
with Ad-PHPMA-BTXbp/scFv-BTX conjugate targeted to
PSMA receptor. The fact that anti-PSMA retargeted Ad
does not match the transgene expression obtained with
unmodified Ad despite achieving similar cell association
emphasises the improved efficiency of intracellular process-
ing and routing of the Ad upon binding to its natural
receptor (CAR) rather than PSMA. This indicates that
although PSMA provides impressive selectivity of retarget-
ing, for applications where higher efficiency is needed,
alternate cell surface receptor targets may be preferable.
This serves to highlight a great advantage of this system, in
that an array of vectors and retargeting ligands can be
tested using the same PHPMA-BTXbp molecule, and so
the laborious direct conjugation of each new test ligand to
PHPMA is not required.
(
CAR). In this study, this was achieved by surface coating
of Ad with multivalent highly hydrophilic HPMA-based
copolymers 1 or 2. ELISA studies confirmed that high
efficiency of coating and protection of the capsid had been
achieved. The finding was further confirmed by the fact
that in cancer cell line infection studies, the polymer
modification of Ad caused a knock-down in transgene
expression of more than 100-fold. We assume that this
decrease of infectivity is a result of polymer-shielding of
the knob domain of Ad which is responsible for CAR
receptor-mediated cell entry. Moreover, efficient coating of
the adenovirus with hydrophilic HPMA copolymer protects
it from interaction with anti-adenovirus antibodies. This
result is in accordance with previously published data
showing significant decrease in immunogenicity of proteins
after their modification with HPMA copolymers (by two
orders of magnitude) (20,21).
The second important step is retargeting of the polymer-
coated Ad to a specific cell receptor. Prostate-specific
membrane antigen (PSMA) expressed on LNCaP cells was
chosen in this work as a model target. To achieve PSMA
targeting, a recombinant fusion protein scFv-BTX consist-
ing of anti-PSMA antibody scFv fragment and BTX
binding region was prepared. After the addition of scFv-
BTX to Ad-PHPMA-BTXbp, the recombinant protein
non-covalently bound to the BTXbp sequences on the
polymer-coated virus forming PSMA-targeted viral gene
delivery vector. This resulted in partial restoration of the
luciferase transgene expression. Notably, no such restora-
tion of luciferase expression was observed when scFv-BTX
ligand was added to Ad-PHPMA with no BTXbp attach-
ment handle. The results of a control experiment with
PSMA-negative PC-3 cells showed no response to the
addition of the targeting ligand, demonstrating the impres-
sive selectivity and specificity of the system.
CONCLUSIONS
We have prepared and characterized a multivalent reactive
HPMA copolymer bearing an α-bungarotoxin-binding
peptide (BTXbp). The polymer was used for covalent
surface modification of adenoviral vectors containing
luciferase reporter gene (Ad). The peptide BTXbp is known
to have extremely high binding affinity to bungarotoxin
(
+/− 2 nM).
A recombinant protein consisting of a PSMA-specific
antibody scFv fragment and the BTX binding region (scFv-
BTX) was used for retargeting of the polymer-modified Ad
to prostate-specific membrane antigen. While thepolymer-
modifiedAdexhibitedapproximately100-foldlowerinfectivity
than the unmodified Ad, the addition of scFv-BTX targeting
ligand to the polymer-coated Ad led to 5–10-fold restoration of
luciferase expression in PSMA-positive LNCaP cells. No such
increase of transduction activity was observed in PSMA-
negative PC3 cells.
The data presented here demonstrate the impressive
activity and flexibility of this novel PHPMA-BTXbp/scFv-
BTX linker system that can provide a universal tool for
achieving receptor-specific adenovirus gene transfer.
Qualitatively similar results were also obtained from a
QPCR study designed to assess the degree to which the
scFv-BTX influenced cell association. While the polymer-
modified Ad without targeting ligand associated with cells
>3 times less efficiently than unmodified Ad, the difference
in the level of transgene expression of the modified versus
unmodified viruses was almost 100-fold. This indicates that
the polymer coating does not only protect the vector from a
non-specific internalization but to a much larger extent
compromises successful transgene expression. This empha-
sises the importance of the correct intracellular routing of
the virus to its eventual activity. After retargeting of Ad-
ACKNOWLEDGEMENTS
Financial support of this project by EU grant GIANT No.
512087, the Grant Agency of the Czech Republic, grant No.

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