Extract
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mild reduction into free thiol-containing, univalent FabĄ¯
fragments. An advantage of having such FabĄ¯ fragments with
free thiol groups in the hinge region is that they can be
conjugated directly with other thiol-specific molecules for
site-specific conjugation[3,4]. Another desirable feature is that
the thiol groups in the hinge of FabĄ¯ are remote from the
antigen-binding site and therefore antigen-binding is not
sterically impaired by the conjugation. This method of
site-specific conjugation has been widely used for conjugating
antibodies with enzymes such as peroxidase and
glucosidase with minimal polymerization and without impairing the
activity of FabĄ¯[5,6]. The labeling of
antibodies with thiol-specific bifunctional chelating agents for metal ions has a
number of potential new applications, including in vivo
diagnosis and therapy and in vitro
immunoassays[7_9]. In the past linking agents such as 2-iminothiolane(2-IT), which
generates a linkage containing a disulfide bond and an amidinium
bond, were always used as "bridges" to connect
bifunctional chelating agents, such as
6-[p-(bromoacetamido)benzyl]-1,4,8,11tetraazacyclotetradecane-
N,NĄ¯,NĄ¯Ą¯,NĄ¯Ą¯Ą¯-tetraacetic acid (BAT) and antibodies containing no free thiol
groups[10,11]. This kind of linking agents was able to offer
antibody the free thiol groups, which may be used for
further conjugation, whereas free thiol-containing FabĄ¯
fragments do not need this step. We are not aware of any
previous reports in the literature on the site-specific conjugation
of bifunctional chelating agents with FabĄ¯ fragments directly
using their thiol groups in the hinge region.
In the present study, we prepared
F(abĄ¯)2 from mouse MoAb B43 by using a modified pepsin digestion method,
and reduced the divalent fragment with cysteine to generate
free thiol-containing FabĄ¯ in the hinge region. The FabĄ¯ was
then directly conjugated with BAT, a thiol-specific
bifunctional chelator for Cu, Co and similar metals without a linking
agent. The emphasis of the present report is on the detailed
description of the preparation of the conjugate molecule and
its antigen-binding properties compared with those of the
parent B43 molecule.
Materials and methods
Preparation of B43 F(abĄ¯)2 and FabĄ¯ fragments
Purified B43 directed against ovarian carcinoma-associated antigen
CA125 was from AltaRex (Edmonton, Canada). Stock solu
tions of the B43 were at a concentration of 4.7_5.0 mg/mL in
pH 7.5 phosphate buffered saline (PBS). The pH value of an
aliquot of the stock solution was adjusted by 1 mol/L pH 3.5
ammonium citrate buffer (Sigma, St Louis, USA) to pH 3.6.
The aliquot was then mixed with bovine pepsin (2660 U/mg,
Sigma) at a molar enzyme:B43 ratio of 1:5 . The mixture was
incubated at 37 oC for 6 h with continuous shaking. The
reaction was terminated by increasing the pH of the mixture
to 7.5 with the addition of 3 mol/L pH 8.3 Tris buffer. The
products were diluted 200 times with pH 7.5 PBS and spun in
30 kDa concentrator tubes (Amicon, Beverly, USA). The
final concentration of protein as measured by UV absorbance
at 280 nm was approximately 5 mg/mL on a model SP8-400
UV/VIS Spectrophotometer (Pye Unicam, Cambridge, UK).
The purity of F(abĄ¯)2 was analyzed by sodium dodecyl
sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under
non-reducing conditions. FabĄ¯ fragments were obtained by
the reduction of F(abĄ¯)2 with 10 mmol/L fresh cysteine (Sigma,
St Louis, USA) solution for 30 min at room temperature at pH
7.5. The reaction mixture was diluted 1000 times with pH 5.0
citrate buffer containing 1.0 mmol/L ethylenediamine
tetraacetic acid (EDTA) that had been purged with nitrogen,
loaded on a 30 kDa concentrator tube and spun in a
centrifuge to remove residual cysteine and small fragments of the
Fc portion. The concentration of the final sample was
approximately 5.0 mg/mL according to UV absorbance at 280
nm. The purity of FabĄ¯ was analyzed by size exclusion
high-performance liquid chromatography (SEC-HPLC) (Waters,
Milford, USA) and SDS-PAGE.
Determination of concentration of thiol groups in FabĄ¯
fragment EllmanĄ¯s reagent (Boehringer-Mannheim,
Mannheim, Germany) was employed to determine the
concentration of thiol groups[12]. Five microliters of EllmanĄ¯s
solution (4.0 mg/mL in distilled water) and 25 mL each of 6
cysteine standard solutions ( 0, 31.5, 62.5, 125, 250, 500
µmol/L) were added to each well of a 96-well plate. Reaction buffer
(200 µL; 0.1 mol/L sodium phosphate; pH 8.0) was then added
to each well. The reaction was allowed to proceed at room
temperature for 15 min. Absorbance of the wells at 405 nm
was measured on an OTC 400 96-well plate reader (Organon
Teknika, Durham, USA). The values obtained for the
standards were plotted to give a standard curve. The
concentration of the unknown sample was inferred from the curve.
Preparation of B43 FabĄ¯-BAT The production of
milligram quantities of thiolated FabĄ¯fragments of B43 prepared
as described earlier has allowed the design and
implementation of a new conjugation methodology for labeling FabĄ¯
with radiometals. The central strategy here was to exploit the
high chemical reactivity of the hinge thiol groups on the FabĄ¯
fragment as attachment sites for BAT and the thiol reactive
bromoacetamido linking chemistry. To a solution of 150
µmol/L FabĄ¯ in sodium citrate buffer (pH 5.0) containing 1 mmol/L
EDTA was added a freshly diluted solution of BAT in 0.2
mol/L Na2HPO4 (pH 8.5) buffer to afford final concentrations
of FabĄ¯ and BAT of 72 µmol/L and 11 mmol/L, respectively, at
pH 7.4. After various time periods at 37
oC, the reaction was fractionated according to molecular weight by SEC-HPLC.
Thirty minutes reaction time was found to be optimal. The
monovalent conjugate fraction was collected and
concentrated using a 30 kDa concentrator. The conjugate was stored
at -70 oC until immediately before use.
Preparation of 67Cu- BAT-FabĄ¯
The specific activity of the
67CuCl2 in 2 mol/L HCl (Nordion International,
Kanata, Canada) varied from 0.8 to 6 mCi/µg copper.
67Cu in dilute HCl was dried on a 70
oC heat block under a gentle stream of nitrogen gas. B43 FabĄ¯-BAT in 0.1 mol/L ammonium citrate
(pH 5) was added to the dried 67Cu and incubated for 30 min
at room temperature. Free 67Cu was scavenged by the
addition of 100 mmol/L EDTA to a final concentration of 10
mmol/L. The immunoconjugate was separated from
67Cu-EDTA by SEC-HPLC. The specific activity of
67Cu-BAT-FabĄ¯ was 4.5_6 mCi/µg. The purified
67Cu-BAT-FabĄ¯ was formulated at 1 mCi/mL in 4% human serum Alb/saline for future
uses[13].
Comparison of the immunoreactivities of intact B43 with
its FabĄ¯ fragment and FabĄ¯-BAT Enzyme-linked immunosorbent assay (ELISA) was used. OVCAR, a human
ovarian carcinoma cell line positive for the CA125 antigen
was obtained from AltaRex. DulbeccoĄ¯s modified EagleĄ¯s
medium (DMEM) with 10% (v/v) heat-inactivated fetal
bovine serum (FBS) supplemented with 2 mmol/L L-glutamine
and 100 U/mL penicillin was used to maintain the cells. One
day before each experiment, 1×105 OVCAR cells were added
to each well of a 96-well flat-bottom tissue culture plate and
incubated at 37 oC overnight in a sterile 5%
CO2 humidified incubator. The attached cells were washed with 37
oC DMEM medium without FBS 3 times and were incubated with DMEM
medium containing various concentrations of the antibody
preparations in a 37 oC incubator for 2 h. MOPC21 (Sigma), a
mouse IgG1 MoAb, was used as a negative control. The
cells were then washed with 37 oC DMEM medium without
FBS 3 times. Peroxidase-conjugated rabbit anti-mouse IgG
(1/4000 diluted; Dako, Carpenteria, USA) was added to the
wells for 1.0 h incubation at 37 oC. After 3 washings with 37
oC DMEM medium without FBS, 3,3Ą¯,5,5Ą¯-tetramethylbenzidine
(TMB) peroxidase substrate (Bio-Rad, Hercules, CA, USA)
was added to the wells for approximately 20 min incubation
at room temperature with continuous shaking. The reaction
was quenched with 1.0 mol/L HCl. Optical intensity at 450
nm in each well was determined using a 96-well microplate
reader.
Comparison of the immunoreactivities of FabĄ¯-BAT with
67Cu- BAT-FabĄ¯ As described earlier, the serial-diluted
FabĄ¯-BAT and 67Cu-BAT-FabĄ¯ were used to determine the loss of
immunoreactivity.
Statistical analysis Data are presented as
mean±SD. Statistical differences between groups were determined by
using StudentĄ¯s t-test. For all
analyses, P<0.05 was considered statistically significant.
Results
Preparation of B43 F(abĄ¯)2 by pepsin digestion The products of digestion were analyzed by SDS-PAGE under
non-reducing conditions. Intact B43 and
F(abĄ¯)2 fragments yielded single bands of apparent molecular weights of 150 kD and
100 kDa, respectively (Figure 1). Most of the intact B43
(>90%) was converted to F(abĄ¯)2 after 6 h digestion,
according to densitometer readings (Bio-Rad). There was no
indication of further degradation of
F(abĄ¯)2 after longer digestion times, and no further purification of
F(abĄ¯)2 was deemed necessary.
Analysis of B43 FabĄ¯ fragments SEC-HPLC analysis
indicated that approximately 96% of the
F(abĄ¯)2 fragments of B43 were cleaved to form FabĄ¯ after 30 min reduction with 10
mmol/L cysteine. The number of thiol groups in each FabĄ¯
was determined to be 1.8 by the use of EllmanĄ¯s reagent. The
products of reduction were analyzed by SDS-PAGE under
reducing and non-reducing conditions. The FabĄ¯ fragments
yielded single bands of apparent molecular weights of 25
kDa under reducing conditions and 50 kDa under
non-reducing conditions, respectively (Figure 1). The free thiol
groups in the hinge-region of FabĄ¯ can be used for
conjugation with other molecules.
Determination of immunoreactivities of
FabĄ¯-BAT The ELISA results (Figure 2) showed that the binding of the FabĄ¯
fragment to antigen CA125 expressed on OVCAR cells was
similar to that of intact B43 (P>0.05), indicating that the
immunoreactivity of the antibody had not been destroyed
during pepsin digestion and cysteine reduction. The
immunoreactivity of the conjugate FabĄ¯-BAT was reduced by less than
2 orders of magnitude relative to that of the intact B4
(P<0.05).
Determination of immunoreactivities of
67Cu-BAT-FabĄ¯ The ELISA results (Figure 3) showed that the binding of the
67Cu-BAT-FabĄ¯ fragment to antigen CA125 expressed on
OVCAR cells was similar to that of BAT-FabĄ¯
(P>0.05), indicating that the immunoreactivity of the antibody had not
been destroyed during 67Cu labeling.
Discussion
Antibodies labeled with longer-lived, positron-emitting
isotopes such as 67Cu have advantages over traditionally
labeled antibodies because of their improved sensitivity for
detecting tumors and small metastases. MoAb FabĄ¯
fragments have a shorter biological half-life than
F(abĄ¯)2 fragments or whole IgG molecules, and are rapidly cleared through
both the liver and kidneys. The use of FabĄ¯ fragments yields
a lower blood-pool activity and improves image contrast
compared with intact MoAb and F(abĄ¯)2
fragments. 67Cu-labeled FabĄ¯ fragments may be more sensitive agents for
detecting tumors, and cause the formation of fewer human
anti-mouse antibodies
(HAMA)[9,14,15].
Current techniques for preparing FabĄ¯ fragments
necessitate purification steps that may cause irreversible damage
to the antigen-binding sites due to denaturation of the
antibody[16,17]. For several conjugation methods of antibody
fragments and other molecules, such as glutaraldehyde and
periodate methods, some active groups or linking agents
have to be introduced to the FabĄ¯ fragments before they are
conjugated to the other
molecules[9,18_20]. These steps may further destroy the immunoreactivities of the FabĄ¯
fragments. However, some bifunctional chelators, such as BAT, can
also be attached to amine, sulfhydryl, imidazole, or thiol
groups on the amino acid side chains of the FabĄ¯. The thiol
group has been found to be the most reactive among
these[21]. The direct use of the thiol group for conjugation may be
beneficial for preserving the bioactivities of conjugates
during the purification process.
In the present study we introduced a simple, 2-step
approach for site-specific conjugation of B43 FabĄ¯ fragments
with BAT directly using the thiol groups in the hinge-region.
This method in principle ensures that the coupling sites
between BAT and FabĄ¯ are distal to the hypervariable region.
The method should dramatically reduce the risk of
inactivation of FabĄ¯ due to modification of amino acid residues that
are essential to the spatial organization of the
antigen-binding sites. In practice, site-specific conjugation has shown a
high yield and an acceptable loss of immunoreactivity of the
product.
References
References
1 Sundaresan G, Yazaki PJ, Shively JE, Finn RD, Larson SM.
124I-labeled engineered anti-CEA minibodies and diabodies allow
high-contrast, antigen-specific small-animal PET imaging of
xenografts in athymic mice. J Nucl Med 2003; 44: 1962_9.
2 Neumaier M, Gaida FJ, Lewis MR, Hefta LJ, Shively LE. A
chimeric anti-CEA antibody with heavy interchain disulfide bonds
deleted: molecular characterization and biodistributions in
normal and tumor bearing mice. Anticancer Res 1999; 19:
13_21.
3 Lu ZR, Shiah JG, Sakuma S, Kopeckova P, Kopecek J. Design of
novel bioconjugates for targeted drug delivery. J Control Release
2002; 78: 165_73.
4 Dhawan S. Design and construction of novel molecular
conjugates for signal amplification (I): conjugation of multiple
horseradish peroxidase molecules to immunoglobulin via primary amines
on lysine peptide chains. Peptides 2002; 23:
2091_8.
5 Hashida S, Imagawa M, Inoue S, Ruan KH, Ishikawa E. More
useful maleimide compounds for the conjugation of Fab' to
horseradish peroxidase through thiol groups in the hinge. J Appl
Biochem 1984; 6: 56_63.
6 Govindon SV, Goldenberg DM, Griffiths GL, Leung S, Losman
MJ, Hansen HJ. Site-specific modification of light chain
glycosylated antilymphoma(LL2) and anti-carcinoembryonic
antigen(hImmu-14-N) antibody divalent fragments. Cancer Res
1995; 55: 5721s_5725s.
7 Griffiths GL, Goldenberg DM, Roesch F, Hansen HJ.
Radiolabeling of an anti-carcinoembryonic antigen antibody Fab' fragment
(CEA-Scan) with the positron-emitting radionuclide Tc-94m. Clin
Cancer Res 1999; 5: 3001s-3003s.
8 Smith A, Alberto R, Blaeuenstein P, Novak-Hofe I, Maecke H,
Schubiger PA. Pre-clinical evaluation of
67Cu-labeled intact and fragmented anti-colon-carcinoma monoclonal antibody MAb35.
Cancer Res 1993; 66: 5727_33.
9 Stimmel JB, Merrill BM, Kuyper LF, Moxham CP, Hutchins JT.
Site-specific conjugation of serine ® cysteine variant
monoclonal antibodies. J Biol Chem 2000; 275:
30445_50.
10 Lewis MR, Boswell CA, Laforest R, Buettner TL, Ye D.
Conjugation of monoclonal antibodies with TETA using activated esters:
biological comparison of 64Cu-TETA-1A3 with
64Cu-BAT-2IT-1A3. Cancer Biother Radiopharm 2001; 16:
483_94.
11 O'Donnell RT, Shen S, Denardo SJ, Wun T, Kukis DL. A phase I
study of 90Y-2IT-BAD-Lym-1 in patients with non-Hodgkin's
lymphoma. Anticancer Res 2000; 20: 3647_55.
12 Riddles PW, Blakeley RL, Zerner B. Reassessment of Ellman's
reagent. Meth Enzymol 1983; 91: 49_60.
13 Anderson CJ, Connett JM, Schwarz SW. Copper-64-labeled
antibodies for PET imaging. J Nucl Med 1992; 33: 1685_91.
14 Mirick GR, O'Donnell RT, DeNardo SJ, Shen S, Meares CF.
Transfer of copper from a chelated 67Cu-antibody conjugate to
ceruloplasmin in lymphoma patients. Nucl Med Biol 1999; 26:
841_5.
15 Kukis DL, DeNardo GL, DeNardo SJ, Mirick GR, Miers LA.
Effect of the extent of chelate substitution on the
immunoreactivity and biodistribution of 2IT-BAT-Lym-1 immunoconjugates.
Cancer Res 1995; 55: 878_84.
16 Weston PD, Devries JA, Wrigglesworth R. Conjugation of
enzymes to immunoglobulins using dimaleimides. Biochem Biophys
Acta 1980; 612: 40_9.
17 Imagawa M, Hashida S, Ishikawa E, Sumiyoshi A. Evaluation of
Fab'-horseradish peroxidase conjugates prepared using
pyridyl disulfide compounds. J Appl Biochem 1982; 4:
400_10.
18 Wilbur DS, Stray JE, Hamlin DK, Curtis DK, Vessella RL.
Monoclonal antibody Fab' fragment cross-linking using equilibrium
transfer alkylation reagents. A strategy for site-specific
conjugation of diagnostic and therapeutic agents with
F(ab')2 fragments.
Bioconjug Chem 1994; 5: 220_35.
19 McCall MJ, Diril H, Meares CF. Simplified method for
conjugating macrocyclic bifunctional chelating agents to antibodies via
2-iminothiolane. Bioconjugate Chem 1990; 1:
222_6.
20 Smith-Jones PM, Fredrich R, Kaden TA, Novak-Hofer I, Siebold
K. Antibody labeling with copper-67 using the bifunctional
macrocycle 4-[(1,4,8,11-tetraazacyclotetradec-l-yl)methy]
benzoic acid. Bioconjugate Chem 1991; 2:
415_21.
21 Albrecht H, Burke PA, Natarajan A, Xiong CY, Kalicinsky M.
Production of soluble ScFvs with C-terminal-free thiol for
site-specific conjugation or stable dimeric ScFvs on demand. Bioconjug
Chem 2004; 15: 16_26.
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