Homogeneous bispecifics by disulfide bridging

Article abstract of DOI:10.1021/bc5002467

We report on a chemical platform to generate site-specific, homogeneous, antibody-antibody conjugates by targeting and bridging disulfide bonds. A bispecific antibody construct was produced in good yield through simple reduction and bridging of antibody fragment disulfide bonds, using a readily synthesized bis-dibromomaleimide cross-linker. Binding activity of antibodies was maintained, and in vitro binding of target antigens was observed. This technology is demonstrated through linking scFv and Fab antibody fragments, showing its potential for the construction of a diverse range of bispecifics.

Full text of DOI:10.1021/bc5002467

Communication
pubs.acs.org/bc
Homogeneous Bispecifics by Disulfide Bridging
Elizabeth A. Hull,† Maria Livanos,‡ Enrique Miranda,‡ Mark E. B. Smith,† Kerry A. Chester,*,‡
and James R. Baker*,†
†Department of Chemistry, University College London, 20 Gordon Street, London, WC1H OAJ, United Kingdom
‡UCL Cancer Institute, 72 Huntley Street, London, WC1E 6BT, United Kingdom
S
* Supporting Information
ABSTRACT: We report on a chemical platform to generate site-specific, homogeneous, antibody−antibody conjugates by
targeting and bridging disulfide bonds. A bispecific antibody construct was produced in good yield through simple reduction and
bridging of antibody fragment disulfide bonds, using a readily synthesized bis-dibromomaleimide cross-linker. Binding activity of
antibodies was maintained, and in vitro binding of target antigens was observed. This technology is demonstrated through linking
scFv and Fab antibody fragments, showing its potential for the construction of a diverse range of bispecifics.
he limited ability of conventional monoclonal antibody
therapies to induce significant antitumor activity has led to
Using this approach, Schultz et al. described the synthesis of a
homogeneous anti-HER2/anti-CD3 bispecific in good yield.10
This technology, while elegant, is not readily transferred; each
antibody to be conjugated must undergo prior investigation
to determine appropriate mutation sites, substitution for the
unnatural amino acid is often incomplete, and expression yields
are generally low due to the cellular toxicity of artificial amino
acids at the high concentrations necessary.11,12 To avoid these
difficulties, an ideal site-directed conjugation technique would
use residues natural to the protein that are revealed for modifi-
cation only under defined conditions.
Cysteine residues have a low natural abundance in proteins,
and are often found tied up in disulfide bonds.13 In the case of
antibodies and antibody fragments there are no free cysteine
residues, and site-directed conjugation has been attempted via
interchain disulfide bond reduction and subsequent conjugation
of the free cysteines. However, conjugation of chemical entities
to the generated cysteine residues results in significant physical
instability of conjugates, particularly under times of stress.14
Furthermore, targeting the cysteine residues responsible for
interchain disulfides using chemical cross-linking reagents results
in poor yields of bispecific due to the formation of homodimers
and intrachain coupling.15 Therefore, the ideal solution would be
to use reagents that bridge disulfide bonds, maintaining this key
stabilizing feature, and preventing the opportunity for product
heterogeneity.16−23 Herein we propose a conjugation strategy
using simple chemical reagents that selectively bridge disulfide
bonds. Through rapid reduction and bridging of disulfides,
T
the development of bispecifics: antibodies that can simulta-
neously bind two different antigens. In 2009, catumaxomab
became the first bispecific therapeutic to be clinically approved,
combining EpCAM targeting with T-cell recruitment for the
treatment of malignant ascites.1 Recombinant technologies have
produced a diverse range of bispecific antibodies, generating
45 formats in the past two decades.2 Despite this variety of topo-
logies, the approach is not suited to every protein combination.
The fusion of proteins via their N or C termini can result in a
reduction or loss of bioactivity and variable expression yields can
be observed due to complications in folding and processing.3−5
An alternative and potentially more versatile approach to
generating bispecific therapeutics is chemical conjugation. Until
now, this has been a less successful method of producing such
conjugates. A fundamental flaw in the chemical techniques
employed in this area has been their dependence on modifying
lysine residues. There is an average of 100 lysine residues per
antibody, and their distribution is uniform throughout the
surface topology of the Fab and Fc regions. As such, conjugation
techniques using lysine residues will randomly cross-link to
virtually all areas of the antibody molecule, resulting in a highly
heterogeneous mixture of products with unpredictable proper-
ties. One strategy to overcome this issue is provided by site-
directed mutagenesis, which enables a single nucleophilic
cysteine residue to be introduced at a desired site in an antibody.
However, this approach is limited, as cysteine mutagenesis
commonly leads to reduced expression yields and undesirable
properties such as susceptibility to dimerization, mixed disulfide
formation, or disulfide scrambling.6−8
Received: June 5, 2014
Revised: July 11, 2014
Published: July 17, 2014
Recently the site-specific introduction of chemical linkers
has been reported through unnatural amino acid insertion.9,10
© 2014 American Chemical Society
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Scheme 1. Strategy for the Production of a Homogeneous Bispecific through Disulfide Bridging of Two Antibody Fragments
a
Scheme 2. Synthetic Route to Linkers
a
(a) ClCO2Me, NMM, THF, 97%; (b) For BDBM(PEG)2: NH2CH2CH2(OCH2CH2)2NH2, DCM, 70%. For BDBM(PEG)19:
NH2CH2CH2(OCH2CH2)19NH2, DCM, 65%.
homogeneous bispecific antibodies could be readily generated
with no effect on stability or activity. To demonstrate the versati-
lity of this chemical conjugation approach to varying antibody
fragment formats, we aimed to generate a homogeneous scFv-
Fab conjugate (Scheme 1).
∼50 kDa was observed on incubation of BDBM(PEG)19 with
reduced scFv after only 1 h at room temperature (Supporting
Information Figure 1). This corresponds to the molecular weight
of scFv dimer. In contrast, the reduced scFv incubated with linker
BDBM(PEG)2, although rebridged, reveals only a faint band at
∼50 kDa, suggesting poor dimer formation.
In prior work we have demonstrated that next generation
maleimides can be used for the extremely efficient rebridging
of disulfide bonds in Fab and disulfide-stabilized scFv antibody
fragments, to produce fully active, homogeneous protein
conjugates in near-quantitative yields.20,21 Antibody fragments
including Fabs and scFvs are commonly used in a range of
bispecific topologies. Thus, we envisaged that next generation
maleimide based cross-linking reagents could be used to produce
homogeneous bispecific constructs. To this end homobifunc-
tional linkers were designed, incorporating two dibromomalei-
mide moieties linked by a PEG chain, conferring some flexibility
to the molecule (Scheme 2). Using commercially available
dibromomaleimide and diamine PEG, two linkers of distinct
length were readily synthesized. The reaction proceeds under mild
conditions in good yield, requiring only a single purification step.24
To examine the feasibility of this approach to producing a
homogeneous protein−protein conjugate, we decided to first
work with the anti-CEA disulfide-stabilized scFv fragment.
Carcinoembryonic antigen (CEA) is a cell surface glycoprotein
overexpressed in a wide range of cancers, particularly in
colorectal carcinoma.25 A scFv is the smallest antibody fragment
which retains full binding activity, and is a common component
of many bispecific antibody formats, e.g., BiTEs (Bispecific T-cell
Engager) and DARTs (Dual-Affinity Re-Targetting). Initially,
the ability of the linkers to generate scFv homodimer was
investigated (Scheme 3).
The longer length of linker BDBM(PEG)19 (19 PEG units,
∼1 kDa), in combination with its flexibility, is likely to signifi-
cantly reduce steric hindrance which creates difficulty when
attempting to link two large proteins. This is observed in the
far greater conversion to homodimer obtained compared to
linker BDBM(PEG)2 (Supporting Information Figure 1). It was
therefore decided that conditions for homogeneous protein−
protein conjugation should be optimized using linker BDBM-
(PEG)19. As a model system, generation of homogeneous scFv
dimer was further pursued in order to determine ideal conditions
for maintaining antibody fragments in their reduced form while
promoting bridging.
Following reduction, the scFv was eluted over a Sephadex
column to remove DTT and to buffer exchange the protein
into conditions suitable for maintaining the reduced antibody
(20 mM phosphate buffer, 5 mM EDTA, pH 7.4). The antibody
fragment was then concentrated to no greater than 1 mg/mL,
since higher concentrations promoted disulfide reoxidation.
A series of optimization experiments revealed incubation with
0.42 equiv of bis-dibromomaleimide cross-linker BDBM(PEG)19
for 1 h at room temperature, or 4 °C overnight, yielded the
highest levels of homogeneous dimer (Supporting Information
Figure 2). In fact, after 1 h approximately 80% of starting material
was consumed (Figure 1[i], lane 3, measured using ImageJ). After
purification of the homodimer by size exclusion chromatography,
an excellent 64% yield of pure, homogeneous scFv−scFv
conjugate was obtained from 1 mg of scFv (Figure 1[iii], 75%
yield with respect to the limiting reagent BDBM(PEG)19). This
yield represents a significant improvement on previous reports
of direct chemical cross-linking of antibodies using natural amino
acids, which have achieved yields in the 10−40% range.26−29
Notably the conjugate is homogeneous due to the site-selectivity
of the methodology.
Scheme 3. Generation of scFv Homodimer
The scFv was reduced (DTT, 20 equiv with respect to
antibody) and, following buffer exchange, incubated with 0.5 equiv
of linker BDBM(PEG)2 or BDBM(PEG)19. The conjugation
reaction was monitored by SDS-PAGE, and a distinct band at
Given the success of our technology in generating homo-
geneous scFv homodimer in high yield, we targeted the
generation of a bispecific antibody conjugate. The monoclonal
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Figure 1. Application of optimized conditions to dimerization of scFv: [i] SDS-PAGE analysis (1) scFv; (2) scFv + 20 equiv DTT to afford reduced scFv
(note - scFv with intact disulfide runs faster through gel due to more compact structure); (3) reduced scFv + 0.42 equiv BDBM(PEG)19, 1 h rt to afford
scFv homodimer (∼50 kDa) and disulfide bridged scFv monomer (∼25 kDa). [ii] Size exclusion chromatography purification of pure scFv homodimer
(peak a, ∼50 kDa) from monomer starting materials (peak b) and unreacted linker (peak c) (Superdex 75, GE Healthcare).
IgG1 antibody Traztuzumab (Herceptin) targets the HER2/neu
receptor and has successfully been used to treat HER2+ breast
cancer patients.30 The Fab fragment of this clinically relevant
antibody can be readily obtained by enzymatic digest, and its
incorporation into bispecific formats has therapeutic potential.10
A Fab fragment contains one variable and one constant region of
each of the heavy and light antibody chains, and so is twice the
size of a scFv fragment. The heavy and light chains are covalently
linked via a single interchain disulfide bond. Thus, we envisaged
linking the disulfide-stabilized scFv to the Fab fragment using
BDBM(PEG)19, generating a conjugate that could simulta-
neously bind two different antigens (CEA and HER2) as a proof
of concept (Scheme 4).
a strong band at ∼80 kDa could be observed, corresponding
to the scFv-Fab conjugate. After purification by size exclusion
chromatography, a pleasing 52% yield of homogeneous scFv-Fab
bispecific was achieved.
Next, we used enzyme-linked immunosorbent assay (ELISA)
to assess the immuno-reactivity of the purified conjugates, in
comparison to the unmodified scFv and Fab fragments that
bound either CEA or HER2, respectively (Figure 3[i] and [ii]).
Against CEA, the activity of the scFv homodimer was not only
maintained, but remained high at low dilutions, suggesting
dimerization has successfully increased the avidity of the
antibody. Most importantly, the scFv-Fab conjugate showed
comparable antigen binding activity against both CEA and
HER2. Given this success, we wanted to demonstrate that our
heterodimeric conjugate could simultaneously bind its two
target antigens. To achieve this we developed a sandwich ELISA.
A 96-well plate was coated with CEA and the sample to be tested,
e.g., scFv-Fab conjugate, was applied. Subsequent incubation
with HER2-Biotin and Extravadin-Peroxidase would thus only
lead to a signal if the sample successfully bound both CEA and
HER2 antigens. Pleasingly, simultaneous binding activity was
confirmed (Figure 3[iii]). Hence we have successfully produced
a homogeneous antibody conjugate with bispecific ability using
our disulfide-bridging technology.
Scheme 4. Generation of anti-CEA/anti-HER2 scFv-Fab
Bispecific
In a final investigation, we assessed the binding of our anti-
body conjugates to a CEA-positive cell line, A375 CEA, and a
HER2-positive cell line, BT-474 (controls shown in Supporting
Information Figure 3). The monomer controls demonstrate
that anti-CEA scFv binds only to A375CEA cells and anti-HER2
Fab only to BT474 cells (Figure 4, monomer). Application of
our scFv homodimer conjugate reveals that CEA binding activity
and selectivity is maintained after conjugation, with a shift in
fluorescence being observed only on the A375CEA cell line
(Figure 4, homodimer). Following this success, we tested our
bispecific construct (Figure 4, heterodimer). In this case, scFv
and Fab were detected in both the CEA-positive cell line
A375CEA and the HER2-positive BT474. This demonstrates
that the antibody fragments have maintained their distinct
selectivity and binding activity in vitro, while being successfully
chemically linked.
To efficiently link these two different antibody fragments,
we decided to first functionalize the scFv with BDBM(PEG)19.
This was cleanly achieved by reducing the scFv as previously
described, purifying by Sephadex column and subsequently
adding 30 equiv of linker to scFv. This reaction was complete in
less than 5 min at room temperature, yielding bromomaleimide
functionalized scFv in quantitative yield (Figure 2[i], lane 3).
Excess linker was removed by buffer exchange. In tandem with
this, the Fab fragment was reduced (TCEP, 10 equiv relative to
antibody) and purified from reducing agent into pH 7.4
phosphate buffer containing 5 mM EDTA. The concentrations
of bridged scFv and reduced Fab were adjusted to 37.2 μM
(1 mg/mL for the scFv), and the antibodies mixed in a 2:1 ratio
by volume (2 equiv bridged scFv to 1 equiv reduced Fab).
This slight excess of functionalized scFv was found to be
sufficient to promote bridging of the reduced Fab. The reaction
was monitored by SDS-PAGE, and after 1 h at room temperature
CONCLUSION
■
To date, the generation of bispecifics for the clinic by chemical
cross-linking has been unsuccessful, due to low yields and
product heterogeneity. Here we have presented the rapid
production of two homogeneous conjugates in high yield: an
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Figure 2. Production of homogeneous scFv-Fab: [i] SDS-PAGE analysis (1) scFv; (2) scFv + 20 equiv DTT to afford reduced scFv (note - scFv with
intact disulfide runs faster through gel due to more compact structure); (3) Reduced scFv + 30 equiv BDBM(PEG)19 to afford disulfide bridged scFv
monomer (note - dibromomaleimide functionalized scFv runs faster than reduced through gel as compact structure is recovered through disulfide
bridging); (4) Fab; (5) Fab +10 equiv TCEP to afford component heavy and light Fab chains; (6) Bridged scFv + Reduced Fab (2:1), 1 h rt to afford
scFv-Fab conjugate (∼80 kDa), reoxidized Fab (∼50 kDa) and scFv starting material (∼25 kDa) . [ii] Size exclusion chromatography purification of
pure scFv-Fab heterodimer (peak a) from starting materials (peak b and c) (Superdex 75, GE Healthcare).
Figure 3. ELISA analysis of conjugates and unmodified antibody fragments: [i] ELISA against full length CEA. [ii] ELISA against HER2. [iii] Sandwich
ELISA using full length CEA coated plates and HER2 conjugated to biotin, followed by Extravadin-Peroxidase.
anti-CEA scFv homodimer and an anti-CEA/anti-HER2 scFv-
Fab heterodimer. Our chemical conjugation approach uses
bis-dibromomaleimide PEG linker readily synthesized over two
steps from the commercially available dibromomaleimide.
Through targeting and bridging the disulfide bond of antibody
fragments, conjugates were produced which exhibit retention
of activity by ELISA and cell binding assays. This platform has
the potential to enable the facile generation of bispecifics from
a range of antibody fragment formats, and could be readily
translated to other protein conjugates of choice, exploiting the
versatility of the chemical conjugation approach.
EXPERIMENTAL PROCEDURES
■
N,N-PEG2-bis-3,4-dibromomaleimide (BDBM(PEG)2).24
2,2′-(Ethylenedioxy)bis(ethylamine) (0.2 mL, 1.38 mmol)
was added to a stirred solution of N-(methoxycarbonyl)-3,4-
dibromomaleimide24 (1.08 g, 3.45 mmol) in DCM (8 mL).
After 20 min EtOAc (80 mL) was added and the organic layer
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Figure 4. Flow cytometry based binding assay of unmodified scFv and Fab (monomer), scFv dimer (homodimer), and scFv-Fab conjugate
(heterodimer) to a CEA-positive cell line (A375CEA) and HER2-positive cell line (BT474).
extracted with saturated NH4Cl solution (2 × 40 mL) and
H2O (3 × 40 mL). The organic layer was dried with MgSO4 and
concentrated in vacuo. Purification by column chromatography
(gradient elution from 1:9 to 3:7 EtOAc/Petrol) yielded
BDBM(PEG)2 as a pale yellow solid (600 mg, 70%): mp
overnight at 4 °C, the reaction was purified by size exclusion (500
mL Superdex 75 column, GE Healthcare).
Disulfide Bridging of scFv with Linker BDBM(PEG)19 to
Generate scFv−scFv Homodimer. Anti-CEA ds-scFv in PBS
(1 mg/mL, 37.2 μM) was reduced with DTT (20 equiv relative
to scFv, 37.2 mM stock in PBS) for 1 h at room temperature.
DTT was then removed using a desalting column (PD-10, GE
Healthcare) and the reduced scFv buffer exchanged into
conjugation buffer (20 mM phosphate buffer, 5 mM EDTA,
pH 7.4). The scFv was concentrated to approximately 1 mg/mL
and linker BDBM(PEG)19 added (0.42 equiv relative to scFv,
3.72 mM stock in water). After 1 h at room temperature or
overnight at 4 °C, the reaction was purified by size exclusion
(500 mL Superdex 75 column, GE Healthcare).
Disulfide Bridging of scFv and Fab to Generate scFv-
Fab Heterodimer. Anti-CEA ds-scFv in PBS (1 mg/mL,
37.2 μM) was reduced with DTT (20 equiv relative to scFv,
37.2 mM in PBS) for 1 h at room temperature. DTT was then
removed using a desalting column (PD-10, GE Healthcare) and the
reduced scFv buffer exchanged into conjugation buffer (20 mM
phosphate buffer, 5 mM EDTA, pH 7.4). The scFv was con-
centrated to approximately 1 mg/mL and linker BDBM(PEG)19
added (30 equiv relative to scFv, 37.2 mM in water). After 10 min
excess linker was removed by buffer exchange (repeat 3 times,
Amicon Ultra-4 Centrifugal Filter Units, 10 kDa cutoff) into
conjugation buffer, and the bridged scFv concentrated to
approximately 1 mg/mL. Meanwhile, Herceptin Fab in Borate
buffer (1 mg/mL, 25 mM sodium borate, 25 mM NaCl, 1 mM
EDTA, pH 8.0) was reduced with TCEP (10 equiv relative to Fab,
37.2 mM in borate buffer) for 1 h at room temperature. TCEP was
then removed using a desalting column (PD-10, GE Healthcare)
and the reduced Fab buffer exchanged into conjugation buffer
(20 mM phosphate buffer, 5 mM EDTA, pH 7.4). The reduced
Fab concentration was adjusted to 37.2 μM, and bridged scFv
(37.2 μM) mixed with the reduced Fab in a 2:1 ratio by volume,
respectively. After 1 h at room temperature or overnight at 4 °C,
the reaction was purified by size exclusion (500 mL Superdex
75 column, GE Healthcare).
1
116−117 °C; H NMR (500 MHz, CDCl3) δ 3.82 (4H, t, J =
5.6 Hz), 3.65 (4H, t, J = 5.6 Hz), 3.58 (4H, s); 13C NMR
(125 MHz, CDCl3) δ 164.2 (C), 129.8 (C), 70.4 (CH2), 68.0
(CH2), 39.3 (CH2); IR (solid) 3489, 2913, 1784, 1720, 1597;
LRMS (ES+) 651 ([81,81,81,81M+Na], 20), 649 ([81,81,81,79M+Na],
70), 647 ([81,81,79,79M+Na], 100), 645 ([81,79,79,79M+Na], 70),
643 ([79,79,79,79M+Na], 20); HRMS (ES+) calculated for
C14H12N2O6Na79Br4 642.7327, observed 642.7355.
N,N-PEG20-bis-3,4-dibromomaleimide (BDBM(PEG)19).
O,O′-Bis(2-aminoethyl)octadecaethylene glycol (50 mg, 0.06 mmol)
was added to a stirred solution of N-(methoxycarbonyl)-3,4-
dibromomaleimide24 (44 mg, 0.14 mmol) in DCM (2 mL).
The reaction mixture was left at room temperature overnight,
and then the solvent removed in vacuo. Purification by column
chromatography (gradient elution from DCM to 20:1 DCM/
MeOH) yielded BDBM(PEG)19 as a pale yellow oil (53 mg,
65%). 1H NMR (600 MHz, CDCl3) δ 3.81 (4H, t, J = 6.0), 3.67−
3.59 (76H, m); 13C NMR (150 MHz, CDCl3) δ 164.0 (C), 129.6
(C), 70.7 (CH2), 70.2 (CH2), 67.7 (CH2), 39.0 (CH2); IR (oil,
cm−1) 2865, 1722; LRMS (EI) 1394 ([81,81,81,81M+NH4], 20),
1392 ([81,81,81,79M+NH4], 70), 1390 ([81,81,79,79M+NH4], 100),
1388 ([81,79,79,79M+NH4], 70), 1386 ([79,79,79,79M+NH4], 20),
1346 (40), 1300 (10); HRMS (EI) calculated for
C48H8081,81,79,79Br4N2O3NH4 1390.2192, observed 1390.2181.
Disulfide Bridging of scFv with Linker BDBM(PEG)2 to
Generate scFv−scFv Homodimer. Anti-CEA ds-scFv in PBS
(1 mg/mL, 37.2 μM) was reduced with DTT (20 equiv relative
to scFv, 37.2 mM stock in PBS) for 1 h at room temperature.
DTT was then removed using a desalting column (PD-10,
GE Healthcare) and the reduced scFv buffer exchanged into
conjugation buffer (20 mM phosphate buffer, 5 mM EDTA,
pH 7.4). The scFv was concentrated to approximately 1 mg/mL
and linker BDBM(PEG)2 added (0.42 equiv relative to scFv,
3.72 mM stock in DMF). After 1 h at room temperature or
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(9) Hudak, J. E., Barfield, R. M., de Hart, G. W., Grob, P., Nogales, E.,
Bertozzi, C. R., and Rabuka, D. (2012) Synthesis of heterobifunctional
protein fusions using copper-free click chemistry and the aldehyde tag.
Angew. Chem., Int. Ed. 51, 4161−4165.
ASSOCIATED CONTENT
* Supporting Information
Full experimental procedures and data are provided. This material
is available free of charge via the Internet at http://pubs.acs.org.
■
S
(10) Kim, C. H., Axup, J. Y., Dubrovska, A., Kazane, S. A., Hutchins, B.
A., Wold, E. D., Smider, V. V., and Schultz, P. G. (2012) Synthesis of
bispecific antibodies using genetically encoded unnatural amino acids. J.
Am. Chem. Soc. 134, 9918−21.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: uccajrb@ucl.ac.uk.
*E-mail: j.r.baker@ucl.ac.uk.
(11) Loscha, K. V., Herlt, A. J., Qi, R., Huber, T., Ozawa, K., and Otting,
G. (2012) Multiple-site labeling of proteins with unnatural amino acids.
Angew. Chem., Int. Ed. 51, 2243−6.
Notes
(12) Liebscher, S., Schopfel, M., Aumuller, T., Sharkhuukhen, A., Pech,
̈
̈
The authors declare the following competing financial
interest(s): J.R.B. and M.E.B.S. are founders and directors of
Thiologics, a company developing homogeneous protein
modification technology.
A., Hoss, E., Parthier, C., Jahreis, G., Stubbs, M. T., and Bordusa, F.
̈
(2014) N-terminal protein modification by substrate-activated reverse
proteolysis. Angew. Chem., Int. Ed. 53, 3024−8.
(13) Fodje, M. N., and Al-Karadaghi, S. (2002) Occurrence,
conformational features and amino acid propensities for the α-helix.
Protein Eng., Des. Sel. 15, 353−358.
ACKNOWLEDGMENTS
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instability and the role of drug payload. Bioconjugate Chem. 25, 656−64.
(15) Ellerman, D., and Scheer, J. M. (2011) Generation of Bispecific
Antibodies by Chemical Conjugation. In Bispecific Antibodies
(Kontermann, R. E., Ed.) pp 47−63, Chapter 3, Springer.
(16) Smith, M. E. B., Schumacher, F. F., Ryan, C. P., Tedaldi, L. M.,
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We gratefully acknowledge UCL, the Wellcome Trust, BBSRC,
UCLB, the KCL and UCL Comprehensive Cancer Imaging
Centre (CCIC) Grant C1519/A10331, The Department of
Health and Cancer Research UK Experimental Cancer Medicine
Centre (ECMC) Grant C34/A7279 and The EU Seventh
Framework Programme (IMAGINT Project) Grant 259881 for
support of our programme. We also thank Dr Berend Tolner for
kindly providing the anti-CEA ds-scFv, Dr Kersti Karu for the
MALDI data and the EPSRC UK National Mass Spectrometry
Facility (NMSF), Swansea.
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