Polymer therapeutics with a coiled coil motif targeted against murine BCL1 leukemia

Article abstract of DOI:10.1021/bm3019592

The specificity of polymer conjugates based on N-(2-hydroxypropyl) methacrylamide (HPMA) bearing cytostatic drugs for cancer cells could be significantly increased by the incorporation of a suitable targeting ligand, such as a monoclonal antibody (mAb). However, direct binding of the protein to the polymer carrier could cause considerable problems, such as decreasing the binding capacity of mAb to its target. Here, we introduce a novel strategy of joining a targeting moiety to a polymeric conjugate with cytostatic drug. The scFv of B1 mAb (specific for BCL1 leukemia cells) was tagged with peptide K ((VAALKEK)₄). Peptide E ((VAALEKE)₄), which forms a stable coiled coil structure heterodimer with peptide K, was assembled with the HPMA copolymers bearing doxorubicin. Such targeted polymeric conjugates possess very selective and high binding activity toward BCL1 cells. Similarly, targeted polymeric conjugates exert approximately 100 times higher cytostatic activity toward BCL1 cells in comparison to nontargeted conjugates in vitro. At the same time, the conjugates have comparable and rather low cytostatic activity for 38C13 cells, which are used as a negative control, in vitro.

Full text of DOI:10.1021/bm3019592

Article
pubs.acs.org/Biomac
Polymer Therapeutics with a Coiled Coil Motif Targeted against
Murine BCL1 Leukemia
Robert Pola,*,† Richard Laga,† Karel Ulbrich,† Irena Sieglova,‡ Vlastimil Kral,‡ Milan Fabry,‡
́
́
́
Martina Kabesova,§ Marek Kovar,§ and Michal Pechar†
̌
́
́
̌
†Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, v.v.i., Heyrovskeh
́
o nam
́
. 2, 162 06, Prague 6,
Czech Republic
‡Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, v.v.i., Flemingovo nam
Republic
§Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i., Víden
ska 1083, 142 20 Prague 4, Czech Republic
̌ ́
́
. 2, 166 10 Prague 6, Czech
ABSTRACT: The specificity of polymer conjugates based on
N-(2-hydroxypropyl)methacrylamide (HPMA) bearing cyto-
static drugs for cancer cells could be significantly increased by
the incorporation of a suitable targeting ligand, such as a
monoclonal antibody (mAb). However, direct binding of the
protein to the polymer carrier could cause considerable
problems, such as decreasing the binding capacity of mAb to
its target. Here, we introduce a novel strategy of joining a
targeting moiety to a polymeric conjugate with cytostatic drug.
The scFv of B1 mAb (specific for BCL1 leukemia cells) was
tagged with peptide K ((VAALKEK)4). Peptide E ((VAA-
LEKE)4), which forms a stable coiled coil structure
heterodimer with peptide K, was assembled with the HPMA
copolymers bearing doxorubicin. Such targeted polymeric conjugates possess very selective and high binding activity toward
BCL1 cells. Similarly, targeted polymeric conjugates exert approximately 100 times higher cytostatic activity toward BCL1 cells in
comparison to nontargeted conjugates in vitro. At the same time, the conjugates have comparable and rather low cytostatic
activity for 38C13 cells, which are used as a negative control, in vitro.
We have designed and characterized synthetic hydrophilic
copolymers based on N-(2-hydroxypropyl)methacrylamide
(HPMA) bearing multiple peptide sequences (VAALEKE)4,
called peptide E, attached to the polymer backbone via a
copper-catalyzed azide−alkyne cycloaddition, “click” chemis-
try.10 The copolymer formed a stable noncovalent complex
with a recombinant single-chain fragment (scFv) of antibody
M75 containing at the C-terminus the sequence (VAALKEK)4,
which is called peptide K. The formation of the complex was
mediated by a strong heterodimeric interaction between
peptides E and K, which form a coiled coil structure. This
noncovalent association of peptides represents a rapid, stable
and well-characterized binding of the targeting ligand to the
polymer conjugate. The antibody M75 specifically binds to
carbonic anhydrase IX (CA IX), a transmembrane protein
overexpressed in a wide variety of tumor cell types.11 The
specific binding of the polymer−scFv complex to CA IX was
confirmed by ELISA.
INTRODUCTION
■
Polymer drug conjugates that are targeted using monoclonal
antibodies belong to the most advanced category of nano-
medicines in the field of tumor therapy.1 Unfortunately, the
covalent attachment of an antibody (or generally any protein/
glycoprotein molecule) to a polymer carrier is accompanied by
several significant problems. First, modification of the protein
with a multivalent reactive polymer precursor rarely leads to a
well-defined product. Second, the biological activity of the
protein is very likely altered after such modification.
Consequently, eventual regulatory approval of the resulting
heterogeneous polymer−protein conjugates becomes a formi-
dable and complicated task for any pharmaceutical company
trying to get their product through clinical trials. Hence, the
demand for a new technology that is suitable for the
preparation of site-specific polymer−protein conjugates is
quite urgent.
Recently, we described2 a new method of conjugating two
macromolecules based on a strong and specific interaction
between two peptides to form a heterodimeric coiled coil
structure. A similar approach based on the coiled coil
heterodimers was described also by several other investigators
in the field of biomedicinal polymers.3−9
In this paper, we introduce a more advanced system intended
for affinity therapy with a polymer-bound cytostatic drug
Received: December 19, 2012
Revised: January 31, 2013
Published: February 1, 2013
© 2013 American Chemical Society
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of 1.0 mL/min (solvent A, 0.05 M sodium acetate buffer, pH 6.5;
solvent B, 300 mL of 0.17 M sodium acetate and 700 mL of
methanol). The molecular mass of the peptide products was
determined using mass spectrometry performed on an LCQ Fleet
mass analyzer with electrospray ionization (ESI MS) (Thermo Fisher
Scientific, Inc., Waltham, MA). The determination of the molecular
weights and polydispersity of the copolymers was carried out by size
exclusion chromatography (SEC) on a Shimadzu HPLC system
(Shimadzu) equipped with refractive index, UV, and multiangle light
scattering (LS) DAWN 8 EOS (Wyatt Technology Corp., Santa
Barbara, CA) detectors using either a Superose 6 column (Pharmacia)
(0.3 M acetate buffer, pH 6.5) or a TSK 3000 SWXL column (Tosoh
Bioscience, Japan) (50% methanol, 0.1% TFA) at a flow rate of 0.5
mL/min. The calculation of molecular weights from the light-
scattering intensity was based on the known injected mass, assuming
100% mass recovery. The content of thiazolidine-2-thione (TT)
groups and Dox was determined spectrophotometrically on a Helios
Alpha UV/vis spectrophotometer (Thermospectronic, U.K.) using the
absorption coefficients for TT in DMSO (ε306 = 10 280 L·mol−1·cm−1)
and for Dox in methanol (ε488 = 9900 L·mol−1·cm−1). Solid-phase
peptide synthesis and solid-phase fragment condensation of protected
peptide fragments were performed on an AVSP-2 multiple automatic
peptide synthesizer (Development Workshops of the Institute of
Organic Chemistry and Biochemistry, Academy of Sciences of the
Czech Republic).
consisting of HPMA copolymers with the antitumor drug
doxorubicin (Dox) and the recombinant scFv of B1 mAb,
which recognizes the idiotype of surface IgM expressed on
mouse BCL1 leukemia cells. The B1 mAb very specifically
binds to murine BCL1 leukemia cells; thus, they have high
potential to be used as a targeting moiety in the polymer−Dox
conjugates designed for selective leukemia-cell-targeted therapy.
The major aim of this paper was to verify the binding activity of
the B1 mAb-targeted polymer conjugates to BCL1 cells and the
selectivity of such binding using a 38C13 cell line (a negative
control), which is a mouse B-cell lymphoma closely resembling
BCL1 cells. These cells also express surface IgM but with a
different epitope not recognized by the B1 mAb. Verification of
the effect of the specificity of such targeting to BCL1 cell
receptors on the accumulation of doxorubicin in the cells and
the determination of the cytostatic activity of the B1 mAb-
targeted polymeric conjugates and their specificity in vitro are
important aims of this study. Nontargeted polymer conjugates
with peptide E-bearing doxorubicin and HPMA copolymer-
bound doxorubicin (PK1) were selected as negative controls to
evaluate the improvement of the cell-specific cytostatic activity.
The 38C13 cells were used as a receptor negative control to
verify the specificity of the cytostatic activity of the targeted
conjugates.
The major advantage of the described “coiled coil” self-
assembly method over standard covalent modification of
antibodies with polymers is the well-defined structure of the
complex. The presented drug delivery system is also ideal for
bivalent (or multivalent) targeting. The bivalent targeting of
polymer therapeutics is one of the topics of our current
investigations. Moreover, in situ self-assembly of independently
stored polymer-drug and targeting antibody enabling prepara-
tion of the conjugate “on request” for personalized therapy
could be another advantage of the described system in future.
Azide Derivative of Peptide E (1). The linear coiled coil peptide
consisting of four repeating heptads terminating with an azide group
was assembled using solid phase fragment condensation of fully
protected heptapeptides, as described earlier.2
Trt-GFLG-OH. The Fmoc protecting group was removed from 1 g
of 4-Fmoc-hydrazinobenzoyl AM Novagel resin with a piperidine−
DMF solution (1:4). The loading of the resin was determined by
spectrophotometric measurement of the Fmoc group (0.46 mmol/g).
The linear tetrapeptide Gly-Phe-Leu-Gly was assembled using a solid-
phase synthesis starting from the C-terminus using standard Fmoc
procedures, including the consecutive addition of the N-Fmoc-
protected amino acid (3 equiv), PyBOP (3 equiv), HOBt (3 equiv),
and DIPEA (6 equiv) in DMF. Fmoc groups were removed using
piperidine−DMF (1:4). After removal of last Fmoc group, the N-
terminus of the peptide was reacted with TrtCl (3 equiv) in the
presence of DIPEA (3 equiv) in DCM for 4 h. The peptide was
cleaved from the resin (350 mg, 0.16 mmol of peptide) with a 0.05 M
aqueous solution of Cu (II) acetate (16 mg, 0.08 mmol), pyridine
(130 μL, 1.6 μmol) and dioxane (5.25 mL), while the resin was
bubbled with air vigorously for 4 h. The resin was removed by
filtration and washed with DCM. The combined filtrates were washed
with a 1 M aqueous solution KHSO4 and water; the organic phase was
dried with anhydrous Na2SO4 and evaporated to dryness, yielding 28
mg (28%) of white solid. The product was characterized by reversed-
phase HPLC and ESI MS (calculated 634.3; found 633.2, M − H).
H2N-GFLG-Dox (2). H2N-GFLG-Dox was prepared as described
earlier.12 The yield was 125 mg (68%) of red solid 2. The product was
characterized by reversed-phase HPLC and ESI−MS (calculated
918.0; found 916.8, M − H).
Synthesis of Monomers. N-(2-Hydroxypropyl)methacrylamide
(HPMA) was prepared by the reaction of methacryloyl chloride with
1-aminopropan-2-ol in DCM.13 N-Methacryloylglycylglycine (Ma-GG-
OH) was prepared by the Schotten−Baumann acylation of
glycylglycine with methacryloyl chloride in an aqueous alkaline
medium. 3-(N-Methacryloylglycylglycyl)thiazolidine-2-thione (Ma-
GG-TT) was prepared by the reaction of Ma-GG-OH with H-TT in
DMF in the presence of N,N′-dicyclohexylcarbodiimide.14 N-
Methacryloyl glycylphenylalanylleucylglycine (MA-GFLG-OH) was
assembled by automatic solid phase peptide synthesis on 2-chlorotrityl
chloride resin starting from the C-terminus using standard Fmoc
procedures, including the consecutive addition of the N-Fmoc-
protected amino acid (2.5 equiv), PyBOP (2.5 equiv), HOBt (2.5
equiv), and DIPEA (5.0 equiv) in DMF.
EXPERIMENTAL SECTION
■
Chemicals. 2,2′-Azobis(isobutyronitrile) (AIBN), N,N′-dicyclo-
hexylcarbodiimide (DCC), dichloromethane (DCM), 4-
(dimethylamino)pyridine (DMAP), dimethyl sulfoxide (DMSO),
1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), 1-hydroxybenzotriazole
( H O B t ) , N - h y d r o x y s u c c i n i m i d e ( H O S u ) ,
pentamethylcyclopentadienyl(cyclooctadiene)ruthenium(II) chloride
(Ru(COD)), triphenylchloromethane (TrtCl), and thiazolidine-2-
thione (H-TT) were purchased from Sigma-Aldrich, Czech Republic.
Ethyldiisopropylamine (DIPEA), N,N-dimethylformamide (DMF),
9-fluorenylmethoxycarbonyl-amino acids (Fmoc-aa), and (benzotria-
zol-1-yloxy)trispyrrolidinophosphonium hexafluorophosphate
(PyBOP) were purchased from Iris Biotech, GmbH, Germany. N-
[2-(2-[2-(2-Azidoethoxy)ethoxy]ethoxy)ethyl]biotin amide (azide-
Peg4-biotin) was purchased from Click Chemistry Tools, Scottsdale,
AZ.
All other solvents and reagents were purchased from Sigma-Aldrich,
Czech Republic. All chemicals and solvents were of analytical grade.
Solvents were purified and dried using standard procedures.
Methods. The homogeneity of the peptide derivatives and the
progress of the conjugation reactions were monitored by reversed-
phase HPLC using Chromolith Performance RP-18e columns, 100 ×
4.6 mm (Merck, Germany), with a linear gradient of water−
acetonitrile (0−100% acetonitrile) in the presence of 0.1% TFA
with a UV−vis diode array detector (Shimadzu, Japan). The amino
acid analysis of the hydrolyzed samples (6 M HCl, 115 °C, 18 h in a
sealed ampule) was performed on a reversed-phase Chromolith
Performance RP-18e column, 100 × 4.6 mm (Merck, Germany), using
precolumn derivatization with phthalaldehyde (OPA) and 3-
sulfanylpropanoic acid (excitation at 229 nm, emission at 450 nm)
and a gradient elution of 0−100% solvent B over 18 min at a flow rate
The Fmoc groups were removed using piperidine−DMF (1:4).
After removal of the last Fmoc group, the methacrylic acid (2.5 equiv)
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Scheme 1. Synthesis of Polymer−Peptide Conjugates Prepared by Free Radical Polymerization
was attached to the N-terminus of the tetrapeptide in the presence of
PyBOP (2.5 equiv), HOBt (2.5 equiv), and DIPEA (5.0 equiv) in
DMF. Cleavage of the unprotected monomer from the resin was
performed using a solution of 30% HFIP in DCM (20 mL/g of resin)
in the presence of 4-(1,1,3,3-tetramethylbutyl)pyrocatechol as a
polymerization inhibitor for 2 h. The resin was filtered off and rinsed
with TFA. The filtrate was concentrated under vacuum and
precipitated with diethyl ether. The precipitate was isolated by
filtration and dried in vacuum.
OH with 4,5-dihydrothiazole-2-thiol in the presence of DCC and
DMAP.14
Reactive Copolymers with TT Groups. The copolymer
poly(HPMA-co-Ma-GFLG-TT) (3) was prepared by solution radical
copolymerization of HPMA (87.5 mol %) and Ma-GFLG-TT (12.5
mol %) in DMSO at 60 °C for 6 h (Scheme 1). The concentration of
comonomers in the polymerization mixture was 13% (w/w), and that
of AIBN was 2% (w/w).14
The copolymer poly(HPMA-co-Ma-GG-TT) (4) was prepared by
reversible addition−fragmentation chain transfer (RAFT) polymer-
ization of HPMA (90 mol %, 200 mg) and Ma-GG-TT (10 mol %, 47
3-(N-Methacryloylglycyl-phenylalanylleucylglycyl)thiazolidine-2-thi-
one (Ma-GFLG-TT) was synthesized by the reaction of Ma-GFLG-
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Scheme 2. Synthesis of Polymer−Peptide Conjugates Prepared by RAFT Polymerization
mg) using AIBN (0.85 mg) as an initiator and 4-cyano-4-
Table 1. Characteristics of Copolymers 3−8
thiobenzoylsulfanylpentanoic acid (2.29 mg) as a chain transfer
agent. The polymerization mixture was dissolved in tert-butyl alcohol
(1.724 mL, 0.9 M solution of monomers), transferred into a glass
ampule, bubbled with Ar and sealed. After 6 h at 70 °C, the product
was isolated by precipitation with acetone; the precipitate was then
washed with diethyl ether and dried under vacuum.
TT
Dox
c
a
a
b
polymer
structure
Mw
Mw/Mn mol % wt %
3
p(HPMA-co-Ma-GFLG-
TT)
41000
1.6
11.1
-
4
p(HPMA-co-Ma-GG-
TT)-DTB
31000
1.2
n.d.
-
Copolymer 4 was then reacted with AIBN (10 molar excess) in
DMSO (15% w/w solution of polymer) under Ar for 3 h at 70 °C in a
sealed ampule to remove dithiobenzoate (DTB) end groups. The
reaction mixture was isolated by precipitation with acetone; the
precipitate was washed with diethyl ether and dried under vacuum to
yield copolymer 5 (Scheme 2).
Copolymers 6−8 with Dox and/or Propargyl Groups.
Reactive copolymer 3 (100 mg, 58.6 μmol TT/g of the polymer)
was dissolved in DMF (0.8 mL) and propargylamine (7.6 mg, 117.2
μmol) was added to the solution. After 30 min, the polymer was
isolated by precipitation with acetone/diethyl ether (3:1) followed by
centrifugation; the precipitate was washed with diethyl ether and dried
under vacuum to form polymer 6 (Scheme 1).
5
6
p(HPMA-co-Ma-GG-TT)
32500
41000
1.2
1.7
9.6
-
-
-
p(HPMA-co-Ma-GFLG-
propargyl)
7
8
p(HPMA-co-Ma-GFLG-
propargyl-co-Ma-GFLG-
Dox)
43000
36000
1.6
1.4
-
-
8.5
5.1
p(HPMA-co-Ma-GG-
propargyl-co-Ma-
GGGFLG-Dox)
a
Molecular weights were determined by SEC using RI and LS
b
detection. TT determined by UV/vis spectrophotometry in DMSO
c
(ε306 = 10 280 L·mol−1·cm−1). Dox determined by UV/vis
spectrophotometry in methanol (ε488 = 9900 L·mol−1·cm−1).
Reactive copolymer 3 (100 mg, 58.6 μmol TT/g of the polymer)
was dissolved in DMF (0.8 mL); Dox.HCl (10 mg, 17 μmol) and
DIPEA (3.2 μL, 18.7 μmol) were then added. After 45 min,
propargylamine (3.8 mg, 58.6 μmol) was added to the reaction
mixture. After another 30 min, the polymer conjugate was isolated by
precipitation with acetone/diethyl ether (3:1) followed by centrifuga-
tion; the precipitate was washed with diethyl ether and dried under
vacuum to form polymer 7.
Reactive copolymer 5 (16.6 mg, 10.1 μmol TT/g of the polymer)
and H2N-GFLG-Dox (2) (3.4 mg, 3.7 μmol) were dissolved separately
in a total volume of 0.5 mL of DMF and mixed together. The progress
of the reaction was monitored with HPLC. After the coupling was
completed, propargylamine (6.5 mg, 101 μmol) was added to the
reaction mixture to end-cap the remaining reactive groups. After 30
min, the polymer was isolated by precipitation with acetone/diethyl
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Table 2. Characteristic of polymer-peptide conjugates 9-13
a
a
b
c
d
polymer
structure
Mw
Mw/Mn
peptide wt %
Dox wt %
biotin wt %
9
p(HPMA-co-Ma-GG-peptide E-co-Ma-GG-biotin)
p(HPMA-co-Ma-GFLG-peptide E)
56000
46500
48000
39000
49000
1.7
2.1
1.8
1.5
2.0
29.0
15.0
13.3
14.9
13.5
-
1.8
10
11
12
13
-
-
p(HPMA-co-Ma-GFLG-peptide-E-co-Ma-GFLG-Dox)
p(HPMA-co-Ma-GG-peptide E-co-Ma-GGGFLG-Dox)
8.3
5.1
8.8
-
-
p(HPMA-co-Ma-GFLG-peptide E-co-Ma-GFLG-Dox-co-Ma-GFLG-Peg4-biotin)
2.0
a
b
Molecular weights were determined by SEC using RI and LS detection. The peptide content (without Peg spacer) determined by amino acid
c
d
analysis. Dox determined by UV/vis spectrophotometry in methanol (ε488 = 9900 L·mol−1·cm−1). Determined from HPLC.
ether (3:1) followed by centrifugation; the precipitate was washed with
diethyl ether and dried under vacuum to form polymer 8 (Scheme 2).
For characteristics of copolymers 3−8, see Table 1.
cell lymphoma of C3H/HeN origin without an epitope recognized by
B1 mAb. Both cell lines were cultured in RPMI 1640 medium enriched
with L-glutamine (4 mM), sodium pyruvate (1 mM), 2-mercaptoe-
thanol (0.05 mM), HEPES (10 mM), penicillin (100 Units/mL),
streptomycin (100 μg/mL) and 10% (v/v) heat inactivated fetal
bovine serum (FBS).
Copolymers (9−13) with Peptide E. Biotinylated polymer 9 was
prepared as described previously.2
Polymer 7 (7.5 mg, 4.4 μmol of propargyl groups) and peptide 1
(1.3 mg, 0.4 μmol) were dissolved in DMF (150 μL). The solution
was thoroughly bubbled with Ar to remove oxygen and Ru(COD)
(0.15 mg, 0.4 μmol) was added to the reaction mixture. The progress
of the reaction was monitored by HPLC; all peptide azide was bound
to the polymer within 6 h. The polymer−peptide conjugate was
separated by SEC on a Sephadex G-25 column in water and
lyophilized to yield 7 mg of polymer−peptide conjugate 11.
Conjugates 10 and 12 were prepared analogously starting with
polymers 6 and 8, respectively.
Conjugate 13 with biotin was prepared starting with polymer 7
(12.3 mg, 7.2 μmol of propargyl groups) and peptide 1 (2.2 mg, 0.66
μmol) in DMF (180 μL). The solution was thoroughly bubbled with
Ar to remove oxygen and Ru(COD) (0.25 mg, 0.66 μmol) was added
to the reaction mixture. After 30 min, azide-Peg4-biotin (0.3 mg, 66
μmol) and Ru(COD) (0.25 mg, 0.66 μmol) dissolved in 15 μL of
oxygen-free DMF were added. The progress of the reaction was
monitored by HPLC. All peptide 1 and azide-Peg4-biotin were bound
to the polymer within 6 h. The polymer−peptide conjugate was
separated by SEC on a Sephadex G-25 column in water and
lyophilized to yield 10.3 mg of polymer−peptide conjugate 13
(Scheme 1). For characteristics of copolymers 9−13, see Table 2.
Construction of the Expression Vector for the Protein with
Fusion Sequence (scFv-K). The scFv fragment derived from the
monoclonal antibody B1, scFv B1, has been obtained using a
procedure similar to the previously described procedure for obtaining
scFv M75 fragment.15 The scFv B1 molecule, in the format (VH)-
(Gly4Ser)4-(VL), contains 118 N-terminal residues of the heavy chain
linked to 108 N-terminal residues of the light chain followed by the C-
myc tag sequence EQKLISEEDL. The peptide K, i.e., (VAALKEK)4,
was introduced at the C-terminus of the scFv fragment, as described
previously.2 The final construct thus codes for scFv B1 mAb in the
format VH-(Gly4Ser)4-VL-myc-K peptide-His5.
Proliferation Assay in Vitro. The cytostatic activity of the
conjugates was assessed using the [3H] thymidine incorporation assay.
BCL1 (0.5 × 104/well) or 38C13 (0.25 × 104/well) cells in the
standard cultivation medium were seeded into 96-well flat-bottom
microtiter plates. ScFv-K/polymer complexes, polymer conjugates
without targeting structure or free doxorubicin were then added to the
wells (in triplicate) to achieve the desired Dox concentrations
(0.0016−0.4 μg/mL). The controls were incubated in standard
cultivation medium only. The plates were incubated in 5% CO2 at 37
°C for 72 h. Next, 18.5 kBq (0.5 μCi) of [3H]thymidine was added to
each well for the last 6 h of incubation. The cells were then collected
onto glass fiber filters using a cell harvester and the radioactivity was
measured in a scintillation counter. All IC50 values (concentration of a
drug that inhibits proliferation to 50% of controls) are means of at
least three independent experiments.
Flow Cytometry Analysis. The scFv-K/polymer complexes were
added to the cells (BCL1 or 38C13 as negative control). Cells were
incubated with either the complexes, the scFv-K alone or biotinylated
B1 mAb for 20 min on ice in the dark and afterward washed three
times with FACS solution (PBS with 2% FBS and 2 mM EDTA). The
cells were then incubated with 1:1000 streptavidin-APC, streptavidin-
FITC or with antimyc antibody conjugated with Alexa Fluor 647 for
20 min on ice in the dark. The cells were washed three times with
FACS solution and then immediately analyzed on a BD LSR II flow
cytometer. The mean fluorescence intensity (MFI) of APC/FITC/
Alexa Fluor 647 was determined in live (Hoechst33258-negative) cells,
and GateLogic software was used for analysis of the data. The
concentrations of conjugates are expressed as the concentration of
scFv fragment in the sample.
Statistical Analysis. The results demonstrate an average
standard deviation or representative result of at least three
independent experiments. The unpaired Student’s t test was used to
assess if the differences observed between the various experimental
groups were statistically significant (p < 0.05).
Expression and Purification of the Fusion Protein scFv-K.
For expression in Escherichia coli BL21(DE3) cells, a modified pET-
22(b) vector was used in which the scFv coding sequence is preceded
by the PelB signal sequence, allowing for translocation of the product
into the periplasmic space. The His5 tag at the C-terminus of the
polypeptide is used for product isolation and purification by IMAC
chromatography on Ni-CAM (Sigma). Final purification was achieved
by ion exchange chromatography on a MonoS column.
RESULTS AND DISCUSSION
■
Peptide Synthesis. The coiled coil peptide E was prepared
by solid phase condensation of protected heptapeptide
fragments, as described previously.2
Synthesis of tetrapeptide-Dox derivative 2 was based on
reaction of Trt-GFLG with doxorubicin followed by removal of
the trityl group as described earlier.12 According to the original
work, Trt-GFLG was prepared using a laborious multistep
solution synthesis that required lengthy purifications of the
intermediate products after every reaction step. Therefore, we
decided to prepare the protected tetrapeptide on a solid phase
support. However, use of the most common resins with acid-
labile linkers does not allow for cleavage of N-tritylated peptide
without loss of the trityl group. Instead, we synthesized Trt-
GFLG on 4-Fmoc-hydrazinobenzoyl AM Novagel resin,
Preparation of the Polymer−scFv Complexes Using Coiled
Coil Interactions. Polymer−peptide conjugates 9−13 were dissolved
in PBS buffer (1 mg/mL) and mixed with the recombinant protein
scFv-K (0.84 mg/mL) to obtain molar ratios of 1:1, 2:1, and 4:1
between peptide K and peptide E. Polymer−peptide conjugates and
scFv-K were incubated for 20 min at room temperature to prepare the
coiled coil complexes. Formation of the complexes was verified by
SEC and by sedimentation analysis as described in our previous
paper.2
Cell Lines. The following cell lines of mouse origin were used:
BCL1, a spontaneous B cell leukemia of BALB/c origin, which
expresses epitope IgM recognized by B1 mAb, and 38C13, a murine B
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Figure 1. Binding activity of the recombinant protein scFv-K and B1 mAb to BCL1cells evaluated by flow cytometry. BCL1 cells were incubated
with biotinylated B1 mAb or recombinant protein scFv-K for 20 min. BCL1 cells were then incubated with streptavidin-FITC or antimyc-Alexa Fluor
647 antibody to detect bound B1 mAb or scFv-K protein for 20 min, respectively. Cells were intensively washed with FACS solution and analyzed by
the BD LSR II flow cytometer. The fluorescence intensity of FITC or Alexa Fluor 647 on live (Hoechst33258-negative) cells was analyzed.
enabling cleavage of the protected peptide by reduction with
Cu(II) acetate in the presence of pyridine. Although this
method was much faster and easier than the original method,
the benefits have a trade-off of a relatively low yield of 28%
(based on the resin substitution) that may be caused by poor
solubility of the reagents and the product in the water-dioxane
mixture. There are a number of publications describing the
successful preparation of peptide amides, esters and thioesters
using the hydrazinobenzoyl resin and corresponding amines
and alcohols, respectively.16−18 Unfortunately, we have not
found any examples of the preparation of peptide acids with
this resin.
Polymerizations. The majority of the HPMA copolymers
used for the preparation of polymer drug carriers and their
conjugates with drugs have been prepared by free radical
solution polymerization. Although very encouraging biological
activity data were obtained with the polymer−drug conjugates
prepared this way, we are aware that broad distribution of
molecular weights may have undesired consequences, namely,
in terms of the pharmacokinetics of such polymer therapeutics
(or diagnostics).
Recently, several papers have reported the preparation of
HPMA copolymers by RAFT polymerization.19−22 The
resulting copolymers generally have a much lower polydisper-
sity index compared to those prepared by free radical
polymerization. It is obvious that the molecular weight
distribution has a significant impact on the biological behavior
of the copolymers. Therefore, we prepared the polymer−Dox
conjugates described in this paper using both types of
polymerization. In the case of polymer 4 prepared by RAFT
copolymerization, the terminal DTB group was removed by
reaction with excess AIBN prior to the reaction of the resulting
polymer 5 with peptide 1. Removal of the DTB groups is quite
important due to the reactivity of these groups with
nucleophiles; DTB might react in the subsequent step, e.g.,
with lysine amino groups of peptide 1. This would lead to
undesired branching of the polymer−peptide conjugate.
Moreover, the presence of DTB functionality in the copolymer
containing TT does not allow for spectrophotometric
determination of the content of reactive TT groups due to
the overlapping UV spectra of the two chromophores.
We successfully prepared copolymer 4 containing HPMA
and Ma-GG-TT repeating units using RAFT polymerization.
Unfortunately, our attempts to prepare an analogous copolymer
of HPMA with Ma-GFLG-TT led to products with relatively
high polydispersity and low yields. Therefore, we used
copolymer 4 for the attachment of Dox derivative 2 containing
an enzymatically degradable GFLG spacer.
Polymer Analogous Reactions. Reactive polymer pre-
cursors 3 and 5 were used as starting materials for all polymer
analogous reactions. Copolymer 3 was reacted with propargyl
amine to yield copolymer 6. Copolymer 7 was obtained by
reaction of polymer 3 with Dox followed by the addition of
propargyl amine. Reaction of copolymer 5 with tetrapeptide-
Dox derivative 2 yielded polymer−Dox conjugate 8 with a
relatively narrow molecular weight distribution. Biotinylated
polymer peptide conjugate 9 was prepared by reaction of the
reactive copolymer p(HPMA-co-Ma-GG-TT) with N-(2-
aminoethyl)biotinamide and propargyl amine followed by
copper-catalyzed azide−alkyne cycloaddition (“click” reaction)
of peptide 1 as described previously.2 The “click” reaction
between peptide azide 1 and the propargyl groups of polymers
6 and 7 resulted in polymer−peptide conjugates 10 and 11,
respectively. Analogously, the “click” reaction between peptide
1 and polymer 8 afforded polymer peptide conjugate 12 with
low polydispersity. Because our initial attempts to use copper-
catalyzed “click” chemistry for attachment of peptide 1 to
doxorubicin-containing polymers 7 and 8 were unsuccessful
due to the formation of copper−doxorubicin complexes,23,24
t h e r u t h e n i u m - b a s e d c a t a l y s t , 2 5 n a m e l y ,
pentamethylcyclopentadienyl(cyclooctadiene) ruthenium(II)
chloride, was used in the synthesis. This catalyst enabled
successful attachment of the azide peptide 1 to the polymer−
Dox conjugates in quantitative yields.
Binding Activity of the scFv-K Protein to BCL1 Cells.
First, we checked the receptor-specific binding activity of the
B1 mAb and the recombinant protein scFv-K to BCL1
leukemia cells using flow cytometry (Figure 1). Formation of
the biotin/streptavidin complex was used for detection of cell
surface-bound mAb or polymer conjugates. The recombinant
protein scFv-K bound very well to target BCL1 cells, to a
similar extent as intact B1 mAb. Unfortunately, a quantitative
comparison of the binding efficacy of the B1 mAb, the scFv-K
and the scFv-K/polymer complexes was very difficult due to
different amounts of biotin on B1 mAb and the polymer and
due to the requirement of a different method of detection of
the free scFv-K protein bound to the cells (detected with
antimyc-Alexa Fluor 647 antibody).
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Noncovalent Polymer−scFv Complex Formation and
Its Binding Activity to BCL1 Cells. The polymer−protein
complexes were prepared by simple mixing of the polymer−
peptide conjugates 9−13 with the corresponding amounts of
the recombinant protein scFv-K. The formation of the
complexes due to the coiled coil interactions between peptides
E and K was confirmed by SEC (Figure 2) as we have already
described.2
Table 3. Cytostatic Activity of the scFv-Targeted and
Nontargeted Polymer−Dox Conjugates 11 and 12, PK1 and
Free Dox
a
IC50 (μg/L)
substance
Polymer 11
BCL1
38C13
146 42
197 23
826 35
621 49
1065 135
899 18
Polymer 12
Polymer 11 + scFv-K
Polymer 12 + scFv-K
Dox
2
3
1
1
1.2 0.3
8
5
PK1
1279 273
4074 2296
a
IC50 values in μg/L, concentration of Dox in copolymer.
B1 mAb, were used as a control to confirm high specificity of
the scFv-K binding. Binding to these cells was not observed for
either scFv-K or for the whole targeted complex scFv-K/
polymer 13 (Figure 4). The binding activity of scFv-K/polymer
13 complexes was measured directly using the fluorescence of
doxorubicin conjugated to polymer carrier (data not shown).
Although Dox is a relatively poor fluorophore, we have
observed the same dependence of fluorescence intensity on
increasing concentration of scFv fragment of B1 antibody using
BCL1 cells. Almost no increase in the fluorescence intensity
with increasing ligand concentration was observed in the case of
the control 38C13 cells.
The results presented in Figure 4 show that the binding
activity of the scFv-targeted conjugate to BCL1 cells is
concentration dependent. The binding affinity is 50 − 100
times higher than that of the control 38C13 cells (at the same
scFv concentration), thus demonstrating high specificity of the
targeted polymer for BCL1 cells.
Cytostatic Activity of the scFv-Targeted Polymer−
Dox Complexes. Dox-containing polymer−peptide conju-
gates 11 and 12, as well as the corresponding scFv-K/polymer
complexes, were tested in proliferation assay using the receptor-
positive murine B cell leukemia line BCL1 and murine B cell
lymphoma 38C13 as a negative control. As a nontargeted
control, the polymer−Dox conjugate called PK1 (prepared as
described previously)13 and consisting of HPMA and Ma-
GFLG-Dox (2 mol %) repeating units was used. This conjugate
successfully passed phases I and II of clinical trials.26 We
considered the polymer PK1 to be the “gold standard” of
nontargeted polymer cytostatic drugs. The results of the
cytostatic activities are summarized in Table 3 and Figure 5.
Figure 2. SEC chromatograms (UV detector, 220 nm, MicroSuperose
12, 0.05 M phosphate buffer with 0.15 M NaCl, pH 6.5, 0.1 mL/min)
of the product of the mixture of polymer 9 (red circles) with the K-
scFv fragment (blue crosses) in two different molar ratios of peptide
E/peptide K, 1:1 (green squares) and 1:4 (black line).
The binding activity of the whole scFv-K/polymer 9
complexes to target cells was evaluated for conjugates differing
in ratios of scFv-K/polymer peptide E (Figure 3). Figure 3
shows that an excess of the targeting ligand scFv-K relative to
the concentration of peptide E in the mixture led to a decrease
in the fluorescence associated with the cells. It is very likely that
the free protein competes with the scFv-K/polymer complexes
in binding to the cell receptors; this may explain the lower level
of cell binding of the complexes in presence of excess of the
free scFv-K.
Selectivity of Binding Activity of the Conjugates to
BCL1 Cells in Vitro. The in vitro binding activity of scFv-K/
polymer complexes of polymer 13 and scFv-K to BCL1
leukemia was confirmed by flow cytometry. Polymer 13 carried
biotin in addition to the cytostatic drug doxorubicin. The biotin
bound to the polymer was detected with streptavidin-APC.
Whole complex scFv-K/polymer 13 bound to BCL1 cells very
well and in a concentration-dependent manner. 38C13 B
lymphoma cells, the cell line without the epitope recognized by
Figure 3. Binding of the scFv-K/polymer 9 bearing biotin and targeted to BCL1 cells at various molar ratios of the peptide K/peptide E was
evaluated by flow cytometry. BCL1 cells were incubated with scFv-K/polymer 9 complexes for 20 min and afterward cells were incubated with
streptavidin-APC for the next 20 min. The cells were intensively washed with FACS solution and analyzed by the BD LSR II flow cytometer. The
fluorescence intensity of APC bound to scFv-K/polymer 9 complexes in live (Hoechst33258-negative) BCL1 cells was analyzed.
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Figure 4. Binding activity of the scFv-K/polymer 13 complexes to BCL1 and 38C13 (inset) cells was evaluated by flow cytometry based on
fluorescence of APC (662 nm). BCL1 cells were incubated with scFv-K/polymer 13 complexes for 20 min. The molar ratio of the peptides K: E was
1:2. Next, the cells were incubated with streptavidin-APC for 20 min to detect biotin bound on polymer 13. The cells were extensively washed and
analyzed on a BD LSR II flow cytometer. The fluorescence intensity of APC bound to scFv-K/polymer 13 complexes in live (Hoechst33258-
negative) BCL1 and 38C13 cells was analyzed.
Figure 5. Proliferation of BCL1 and 38C13 cells in the presence of scFv-targeted and nontargeted polymer 11 and free Dox. The cells were
incubated with scFv-K/polymer complexes, polymer without targeting structure, PK1 or free doxorubicin for 72 h. Proliferation of the cells was
determined using the [3H] thymidine, which was added to the cells for last 6 h of incubation. The radioactivity of samples after harvesting the cells
was determined using a scintillation counter.
Polymers 11 and 12 without the targeting ligand scFv-K
exhibited low cytostatic activity that was comparable to that of
the control polymer-Dox conjugate PK1. A significant increase
of approximately 2 orders of magnitude in cytostatic activity of
the polymer−Dox conjugates 11 and 12 against BCL1 cells was
observed upon addition of the targeting scFv-K ligand. The
IC50 values of polymers 11 and 12 decreased from 146 to 2 μg/
L and from 197 to 3 μg/L, respectively (see Table 3 and Figure
5), which is similar to the value obtained for free Dox. The
lowest cytotoxic effect was found for the PK1 control polymer.
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No significant difference in cytotoxicity of the targeted and
nontargeted polymer drugs was observed in the case of
treatment of the 38C13 cells used as a negative control, which
lack the appropriate antigen recognized by scFv-K. Here, a
slight decrease in the cytotoxic activity (increase in IC50) of the
scFv-modified polymer conjugate can be ascribed to slower
uptake of the more bulky conjugate by 38C13 cells that do not
express the cognate antigen. The results clearly demonstrate
that antigen-specific delivery of the polymer-drug conjugate to
the target cells significantly increases the cytostatic activity of
these conjugates in vitro.
As expected, a very small difference in cytostatic activity of
the polymers 11 (prepared by classical radical polymerization)
and 12 (prepared by RAFT) was found in vitro. In this case, all
molecules of the conjugates are in contact with cancer cells and
can exhibit cytostatic effects. Conversely, under in vivo
conditions, polymer 12 with narrower molecular weight
distribution might exhibit better biological activity than the
“classical,” more polydisperse polymer 11. Lower-molecular-
weight fractions of polymer 11 (necessarily present in the
conjugate with higher polydispersity) can be rapidly excreted by
glomerular filtration,27 thus decreasing the effective concen-
tration of the polymer drug in the organism. In addition, we can
expect that the more uniform polymer 12 prepared by the
RAFT technique will exhibit better and more specific
pharmacokinetics, allowing for a more precise study of the
mechanism of action of the conjugate. We believe that the
forthcoming in vivo experiments will confirm our expectations.
IAAX00500803 grant by the Grant Agency of the Academy of
Sciences of the Czech Republic, and in part by research project
no. AV0Z50520514 awarded by the Academy of Sciences of the
Czech Republic. We also acknowledge Anna Vankova for
̌ ́
excellent technical assistance in peptide synthesis.
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CONCLUSION
■
The water-soluble polymer conjugates bearing cancerostatic
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ruthenium(II) chloride. Peptide E can form a coiled coil
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protein consisting of the scFv fragment of B1 mAb. Both B1
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affinity. The specific binding of the scFv-K/polymer complexes
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activity of the targeted scFv-K/polymer-Dox complexes against
BCL1 leukemia cells was approximately 100-times higher than
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AUTHOR INFORMATION
Corresponding Author
*E-mail: pola@imc.cas.cz.
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■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors gratefully acknowledge the financial support from
P301/11/0325 grant by Czech Science Foundation, from
Institutional Research Concept RVO 61388971 and from
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