A novel bifunctional maleimido CHX-A″ chelator for conjugation to thiol-containing biomolecules

Article abstract of DOI:10.1016/j.bmcl.2008.03.022

A novel bifunctional maleimido CHX-A″ DTPA chelator 5 was developed and conjugated to the monoclonal antibody trastuzumab (Herceptin) and subsequently radiolabeled with ¹¹¹In. The resulting ¹¹¹In labeled immunoconjugate 2 was demonstrated to bind to SKOV-3 ovarian cancer cells comparably to an isothiocyanato CHX-A″ DTPA modified native trastuzumab, 1. Through efficient thiol-maleimide chemistry, antibodies, peptides or other targeting vectors can now be modified with an established radioactive metal chelating agent CHX-A″ DTPA for imaging and/or therapies of cancer.

Full text of DOI:10.1016/j.bmcl.2008.03.022

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 2679–2683
A novel bifunctional maleimido CHX-A00 chelator for conjugation
to thiol-containing biomolecules
Heng Xu,a Kwamena E. Baidoo,a Karen J. Wongb and Martin W. Brechbiela,*
aRadioimmune & Inorganic Chemistry Section, Radiation Oncology Branch, National Cancer Institute,
National Institutes of Health, Building 10, Room 1B40, 10 Center Drive, Bethesda, MD 20892-1088, USA
bMolecular Imaging Program, Center for Cancer Research, National Cancer Institute, National Institutes of Health,
10 Center Drive, Bethesda, MD 20892-1088, USA
Received 23 January 2008; revised 5 March 2008; accepted 6 March 2008
Available online 10 March 2008
Abstract—A novel bifunctional maleimido CHX-A00 DTPA chelator 5 was developed and conjugated to the monoclonal antibody
trastuzumab (Herceptin) and subsequently radiolabeled with 111In. The resulting 111In labeled immunoconjugate 2 was demon-
strated to bind to SKOV-3 ovarian cancer cells comparably to an isothiocyanato CHX-A00 DTPA modified native trastuzumab,
1. Through efficient thiol-maleimide chemistry, antibodies, peptides or other targeting vectors can now be modified with an estab-
lished radioactive metal chelating agent CHX-A00 DTPA for imaging and/or therapies of cancer.
Published by Elsevier Ltd.
The use of monoclonal antibodies (mAbs) as targeting
vectors to selectively deliver a lethal dose of radiation
to cancer cells through either a bꢀ or a-emitting radionu-
clide, radioimmunotherapy (RIT), has been extensively
investigated for cancer therapies.1–3 The targeted nature
of such therapies as well as imaging with c- or b+-emit-
ters (SPECT/PET), radioimmunoimaging (RII), offers
the promise of greater efficacy and less toxicity.4 This
field has attracted greater attention particularly after
the recent Food and Drug Administration approval of
two radionuclide-bearing monoclonal antibody thera-
pies (90Y-ibritumomab and 131I-tositumomab) for the
treatment of lymphohematopoietic malignancies.5,6
purpose and some have been carried forward into anti-
body-targeted radiation therapy clinical trials (Fig. 1).4
The acyclic bifunctional ligand, CHX-A00 DTPA (N-
NCS
NCS
O
O
OH
OH
OH
OH
N
N
OH
N
O
O
OH
N
O
O
N
N
O
O
OH
OH
HO
O
HO
O
The development of suitable bifunctional chelating
agents for the modification of proteins for RII and
RIT continues to receive great attention. To achieve use-
ful radiolabeled mAbs in the clinical setting, chelating
agents must form thermodynamically and kinetically
stable complexes to prevent loss of radionuclides
CHX-A"-DTPA
1B4M-DTPA
COOH
COOH
HOOC
HOOC
NCS
HOOC
HOOC
N
N
N
N
COOH
N
N
in vivo, while retaining mAb immunoreactivity.7,8
A
N
N
NCS
variety of DTPA and DOTA derivatives bearing isothi-
ocyanate reactive groups are well documented for this
COOH
Keywords: Monoclonal antibody; Thiol-maleimide chemistry; Imaging
and/or therapies of cancer.
c-DOTA
PA-DOTA
Figure 1. Structures of CHX-A00 DTPA, 1B4M-DTPA, c-DOTA, and
PA-DOTA.
*
Corresponding author. Fax: +1 301 402 1923; e-mail: martinwb@
mail.nih.gov
0960-894X/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2008.03.022

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H. Xu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2679–2683
[(R)-2-amino-3-(p-aminophenyl)propyl]-trans-(S,S)-cyclo-
hexane-1,2-diamine-N,N,N0,N00,N00-pentaacetic acid) re-
ported in 1997, has been found to be excellent at
sequestering radionuclides such as 86/90Y, 212/213Bi,
so if there are lysine residues in the antigen binding
region.
An alternate conjugation strategy is available via the
selectivity of the Michael addition between a thiol and
a maleimide.13 The maleimide group reacts efficiently
and specifically with thiol-terminal biomolecules and
has been widely used to form stable protein conjugates
through thioether linkages. This conjugation method is
widely considered to give greater control over the posi-
tion of the chelating agent on a protein when the thiol
group(s), for example, engineered cysteine residues,
can be introduced at the genetic level. On the other
hand, thiol groups are less likely to be currently used
than charged groups (e.g., amino groups) for conjuga-
tion to be found at mAb’s binding sites, thereby offering
better chemistry to retain immunoreactivity of mAbs. A
small number of chelating agents that exploit this strat-
egy have been reported,14–17 but few have seen extensive
use for in vivo studies of radiolabeled antibodies or pep-
tides for either imaging or therapy. In part, this may be
due to a lack of familiarity with this chemistry and/or
177
111In, and Lu with great in vivo stability.9–12 In par-
ticular, radiolabeling of CHX-A00 DTPA modified
mAb with the therapeutic a-emitter 212Bi was found to
be statistically comparable to protein conjugates formed
with a bifunctional DOTA derivative (2-(p-isothiocy-
anatobenzyl)-l,4,7,10-tetraazacyclododecane tetraacetic
acid) in stability, without the detraction of the slow for-
mation kinetics inherent to the DOTA macrocycle.11
In this laboratory, the isothiocyanate form of CHX-A00
DTPA reacts with primary e-amine groups of lysine or
terminal a-amine residues of proteins and has routinely
been used for conjugation to mAb (Fig. 2). This conju-
gation method can be problematic in controlling the pre-
cise number and exact position of chelates that have
been attached. This can lead to concerns regarding po-
tential interference with antigen binding, particularly
OH
OH
OH
OH
O
O
O
O
O
O
N
NH
OH
O
N
OH
O
S
O
O
S
N
N
HN
N
H
N
H
N
N
H
OH
OH
N
N
O
HO
HO
O
O
1
2
Figure 2. A schematic presentation of CHX-A00 DTPA conjugated trastuzumab 1, conjugation through an isothiocyanato CHX-A00 DTPA chelator,
and 2 conjugation through the maleimido CHX-A00 DTPA chelator, 5, to prepare 2.
O
O
O
O
O
O
O
O
N
O
O
O
O
O
O
N
O
N
EDCI, HOBt, Et3N
EMAC, 63%
O
N
N
O
N
N
O
H
O
H2N
N
O
O
O
O
4
3
TFA, 92%
OH
OH
O
O
O
OH
O
N
O
O
N
N
OH
N
N
H
O
HO
O
5
Figure 3. Synthesis of the bifunctional maleimido CHX-A00 DTPA chelator, 5.

H. Xu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2679–2683
2681
more problematically, due to the lack of appropriate
derivatives of previously established chelating agents.
was recently developed in this laboratory (Fig. 3).18,19
This aniline derivative was reacted with N-e-maleimido-
caproic acid (EMCA) using standard peptide coupling
conditions to provide maleimide 4 (63%).20 The tert-bu-
tyl esters in 4 were quantitatively cleaved by TFA as
monitored by TLC to generate 5.21 Thereafter, the liber-
ated five carboxyl groups, along with the three tertiary
amines, became available for sequestering radioactive
metals, such as 111In, 86/90Y, 212/213Bi or 177Lu. The
maleimide moiety in 5 provides a highly reactive group
toward thiol groups either extant or introduced into
proteins or peptides. To conjugate 5 to trastuzumab,
the mAb was first thiolated with 15 equiv of Traut’s
agent using standard procedures with minor modifica-
tions.22 Routinely, ꢁ2–4 thiol groups per trastuzumab
were introduced as quantified by Ellman’s reagent. Thi-
Herein, the synthesis of a novel maleimido CHX-A00
DTPA chelator, 5, is described as well as its successful
conjugation to the monoclonal antibody trastuzumab.
This novel maleimide CHX-A00 DTPA derivative thus
offers an alternative route to modify antibodies, pep-
tides, and other targeting vectors with an established
radiometal chelate. This conjugation strategy might be
particularly useful considering the availability of engi-
neered Fab or Fab0 antibody fragments containing free
sulfhydryl groups.
For the synthesis of 5, we used the tert-butyl ester pro-
tected p-amino functionalized CHX-A00 DTPA, 3, which
Figure 4. SE-HPLC chromatograph of (a) 1; (b) 2; (c) 111In-1; (d) 111In-2; (e) trastuzumab. (a), (b), and (e) were monitored by UV at 280 nm, while
(c) and (d) were recorded by an in-line HPLC radiodetector.

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H. Xu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2679–2683
6. Borghaei, H.; Schilder, R. J. Semin. Nucl. Med. 2004, 34,
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7. Milenic, D. E.; Garmestani, K.; Brady, E. D.; Albert, P.
S.; Ma, D.; Abdulla, A.; Brechbiel, M. W. Clin. Cancer
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8. Milenic, D. E.; Garmestani, K.; Brady, E. D.; Albert, P.;
Abdulla, A.; Flynn, J.; Brechbiel, M. W. Clin. Cancer Res.
2007, 13, 1926.
9. Wu, C.; Kobayashi, H.; Sun, B.; Yoo, M.; Paik, C. H.;
Gansow, O. A.; Carrasquillo, J. A.; Pastan, I.; Brechbiel,
M. W. Bioorg. Med. Chem. 1997, 5, 1925.
10. Kobayashi, H.; Wu, C.; Yoo, T. M.; Sun, B.; Pastan, I.;
Paik, C. H.; Gansow, O. A.; Carrasquillo, J. A.; Brechbiel,
M. W. J. Nucl. Med. 1998, 39, 829.
11. Nikula, T. K.; McDevitt, M. R.; Finn, R. D.; Wu, C.;
Kozak, R. W.; Garmestani, K.; Brechbiel, M. W.; Curcio,
M. J.; Pippin, C. G.; Tiffany-Jones, L.; Geerlings, M. W.,
Sr.; Apostolidos, C.; Molinet, R.; Geerlings, M. W., Jr.;
Gansow, O. A.; Scheinberg, D. A. J. Nucl. Med. 1999, 40,
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12. Milenic, D. E.; Garmestani, K.; Chappell, L. L.; Dadach-
ova, E.; Yordanov, A.; Ma, D.; Schlom, J.; Brechbiel, M.
W. Nucl. Med. Biol. 2002, 29, 431.
13. Hermanson, G. T. Bioconjugate Techniques; Academic
Press: London, 1996, pp 419–455.
olated trastuzumab was then reacted with 10 equiv of 5
to produce conjugate 2.23 Unreacted thiolated conjugate
was capped with iodoacetamide to minimize cross-link-
ing of the mAb conjugate product and to promote a
longer shelf life of the immunoconjugate. Finally, the
reaction mixture was dialyzed into PBS buffer at 4 ꢀC
to remove all small molecules from the protein solution.
The final average ratio of CHX-A00 DTPA moieties per
trastuzumab achieved by this process was calculated to
be ꢁ2 based on an Arsenazo(III) assay.24 The immuno-
conjugate 2 was radiolabeled efficiently (>95%) with the
SPECT radionuclide 111In within 30 min at room tem-
perature.25 Figure 4 shows the size-exclusion HPLC pro-
file of the isothiocyanate and maleimide-functionalized
trastuzumab as compared to their respective 111In la-
beled analogs as well as to the unmodified mAb. The
profiles of the maleimide-modified analogs were similar
to those of the previously reported isothiocyanate-mod-
ified analogs, both radiolabeled and unlabeled, as well as
to unmodified trastuzumab. Immunoreactivity of the
resulting 111In labeled immunoconjugate 2 was demon-
strated by binding to SKOV-3, a human ovarian carci-
noma cell line. There was comparable binding between
111In labeled immunoconjugate 2 (44.9%) and the 111In
labeled isothiocyanato CHX-A00 modified trastuzumab,
1 (38.6%).19,26
14. Arano, Y.; Uezono, T.; Akizawa, H.; Ono, M.; Wakisaka,
K.; Nakayama, M.; Sakahara, H.; Konishi, J.; Yokoy-
ama, A. J. Med. Chem. 1996, 39, 3451.
15. Ali, M. S.; Quadri, S. M. Bioconjugate Chem. 1996, 7, 576.
16. Lewis, M. R.; Shively, J. E. Bioconjugate Chem. 1998, 9,
72.
In conclusion, a novel bifunctional maleimido DTPA
derivative 5 was designed, synthesized and character-
ized for conjugation to thiol-containing biomolecules
such as antibodies, peptides or other targeting vectors,
which was based on a previously established chelating
agent, CHX-A00. Successful conjugation of 5 to the
monoclonal antibody trastuzumab (Herceptin) was
achieved by efficient thiol-maleimide chemistry. One
should note that the direct use of 4 would also be
possible for solution or solid phase conjugation to
peptides and other vectors. Subsequent cleavage of
the esters would provide a conjugate product suitable
for radiolabeling. Therefore, this newly developed
maleimido CHX-A00 DTPA allows a new route to pro-
viding an established radioactive metal chelating agent
on biomolecules for imaging and/or therapies of
cancers.
17. Lattuada, L.; Gabellini, M. Synth. Commun. 2005, 35,
2409.
18. Clifford, T.; Boswell, A. C.; Biddlecombe, G. B.; Lewis, J.
S.; Brechbiel, M. W. J. Med. Chem. 2006, 49, 4297.
19. Xu, H.; Baidoo, K.; Gunn, A. J.; Boswell, C. A.; Milenic,
D. E.; Choyke, P. L.; Brechbiel, M. W. J. Med. Chem.
2007, 50, 4759.
20. Synthesis of maleimido CHX-A00 penta tert-butyl ester 4: N-
e-Maleimidocaproic acid (0.28 g, 1.32 mmol), 1-[3-
(dimethylamino)propyl]-3-ethylcarbodiimide hydrochlo-
ride (EDCI) (0.50 g, 2.62 mmol), 1-hydroxybenzotriazole
(HOBt) (0.35 g, 2.62 mmol), and Et3N (0.18 mL,
1.32 mmol) were added to a stirred solution of 3 (1.00 g,
1.20 mmol) in DMF (50 mL). The mixture was stirred for
24 h at room temperature (rt) and then concentrated under
reduced pressure, diluted with CH2Cl2 (100 mL), and
washed successively with water (2· 100 mL), 5% NaHCO3
(2· 100 mL), and water (1· 100 mL). The organic layer
was concentrated and the residue was chromatographed
on silica gel eluted with EtOAc/MeOH (9:1) to afford 4
(0.78 g, 63%). 1H NMR (DMSO-d6) d 9.70 (s, 1H), 7.45 (d,
J = 8.1 Hz, 2H), 7.15 (d, J = 8.1 Hz, 2H), 7.00 (s, 2H), 3.5–
3.20 (m, 12H), 3.15 (br d, J = 15.6 Hz, 1H), 2.8–3.0 (m,
2H), 2.75 (m, 1H), 2.55 (m, 3H), 2.39 (m, 1H), 2.24 (t,
J = 7.2 Hz, 2H), 1.90 (br s, 2H), 1.70–1.10 (m, 10H), 1.37
(s, 45H), 1.03 (m, 4H). 13C NMR (CDCl3) d 171.7, 171.3,
170.8, 136.5, 134.1, 129.6, 119.5, 80.4, 63.9, 63.2, 62.6,
53.5, 53.0, 52.5, 39.0, 37.2, 36.1, 28.1, 27.2, 26.2, 25.8, 25.0;
ES-MS: Calcd for C55H88N5O13 [M+H+]: 1026.63786.
Found 1026.63822.
Acknowledgment
This research was supported by the Intramural Research
Program of the NIH, National Cancer Institute, Center
for Cancer Research.
References and notes
1. Goldenberg, D. M. Cancer Immunol. Immunother. 2003,
52, 281.
2. Milenic, D. E.; Brechbiel, M. W. Cancer Biol. Ther. 2004,
3, 361.
3. Wu, A. M.; Senter, P. D. Nat. Biotechnol. 2005, 1137.
4. Milenic, D. E.; Brady, E. D.; Brechbiel, M. W. Nat. Rev.
Drug Disc. 2004, 3, 488.
21. Synthesis of maleimido CHX-A00 DTPA 5: CHX-A00
derivative 4 (0.30 g, 0.29 mmol) was stirred with 15 mL
of TFA for 4 h. The reaction mixture was concentrated
under reduced pressure, treated with Et2O (50 mL), and
1
filtered to afford 5 (0.20 g, 92%). H NMR (DMSO-d6) d
9.78 (s, 1H), 7.45 (d, J = 8.1 Hz, 2H), 7.18 (d, J = 8.1 Hz,
2H), 7.00 (s, 2H), 3.60–2.40 (m, 17H), 2.24 (t, J = 7.2 Hz,
2H), 2.00 (m, 2H), 1.50 (m, 8H), 1.20 (m, 6H). 13C NMR
5. Wahl, R. L. J. Nucl. Med. 2005, 46, 128s.

H. Xu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2679–2683
2683
(DMSO-d6) d 173.1, 172.7, 171.3, 171.2, 168.8, 137.8,
134.6, 133.4, 129.6, 119.3, 61.5, 53.2, 52.4, 37.2, 36.4, 28.0,
26.1, 24.8, 24.5, 24.5, 24.1; ES-MS: Calcd for
C35H48N5O13 [M+H+]: 744.30922. Found 744.30692.
22. Jue, R.; Lambert, J. M.; Pierce, L. R.; Traut, R. R.
Biochemistry 1978, 17, 5399.
25. 111In Radiolabeling of immunoconjugate 2: Caution: 111In
(t1/2 = 2.8 d) is a c-emitting radionuclide. Appropriate
shielding and handling protocols should be in place when
using this radionuclide. NH4OAc buffer (0.15 M, pH 7,
100 lL) was added to a solution of the maleimido CHX-A00
DTPA chelator, 5, conjugated to trastuzumab, 2, in PBS
(1 mg/mL, 50 lL). 111InCl3 in 0.05 M HCl (1 mC, 3 lL)
(Perkin-Elmer) was then added to the mixture and vortexed.
The reaction mixture was incubated at room temperature
for 30 min. The reaction was quenched by the addition of a
solution of EDTA (0.1 M EDTA, 4 lL). The 111In labeled
product was purified on a PD-10 column using PBS as the
eluent. The radiolabeling efficiency was 95.2%.
26. Radioimmunoassay: Immunoreactivities of the radiola-
beled preparations were assessed in a radioimmunoassay
using methanol-fixed cells. Briefly, SKOV-3 cells were
trypsinized, pelleted, re-suspended in PBS (pH 7.2) and
fixed with cold methanol at a final concentration of 80%
methanol. After 1 h at 4 ꢀC, the cells were washed with
PBS (3·) and re-suspended in PBS containing 1% BSA
(PBS/BSA). Serial dilutions of radiolabeled trastuzumab
(ꢁ140–8 nCi) were added in duplicate to cells (1 · 106/
50 lL) in 50 lL of PBS/BSA. The cells were washed with
3 mL of 1% BSA in PBS following an 18 h incubation at
room temperature, pelleted at 1000g for 5 min and the
supernatant decanted. The radioactivity was measured in
a c-counter (WizardOne, Perkin-Elmer, Shelton, CT) and
the percent bound calculated for each dilution. The value
presented is the average percent bound.
23. Thiolation of and conjugation with trastuzumab: Trast-
uzumab (ꢁ7.7 mg) was reacted with Traut’s reagent at a
1:15 molar ratio for 1 h at rt in 1 mL of PBS containing
5 mM EDTA buffer. Excess Traut’s reagent was removed
by passage of the reaction solution through a PD-10
column eluted with PBS containing 5 mM EDTA buffer.
Just prior to protein conjugation, 5 was dissolved in the
same buffer and then added drop wise to the mAb solution
to achieve a molar reaction ratio of 10:1 (5:trastuzumab)
and gently vortexed. The solution was then gently agitated
in the dark at 25 ꢀC for 1 h. Excess free unreacted SH
groups were capped by the addition of iodoacetamide
solution (2.0 mM). Finally, the reaction mixture was
dialyzed into PBS buffer at 4 ꢀC with 4 buffer changes
(4· 1 L) over 48 h to afford immunoconjugate 2. Protein
concentration and the number of the CHX-A00 chelate per
protein were determined by Lowry assay and Arse-
nazo(III) assay, respectively. The purity of immunocon-
jugate 2 was evaluated by both SE-HPLC and SDS–
PAGE (data not shown), and was found to be comparable
to native trastuzumab.
24. Pippin, C. G.; Parker, T. A.; McMurry, T. J.; Brechbiel,
M. W. Bioconjugate Chem. 1992, 3, 342.