1Crump Institute for Molecular Imaging, Department of Molecular and Medical Pharmacology, David Geffen School of Medicine at UCLA, 700 Westwood Plaza, Los Angeles, CA 90095, 3Division of Molecular Biology, 4Division of Immunology and 6Division of Biology, Beckman Research Institute of the City of Hope, 1450 East Duarte Road, Duarte, CA 91010, 5Department of Radiology and Bio-X Program, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, CA 94305, 7Division of Radiology and 8Department of Radioimmunotherapy, City of Hope National Medical Center, 1500 East Duarte Road, Duarte, CA 91010, USA
2 To whom correspondence should be addressed. e-mail: tolafsen{at}mednet.ucla.edu
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Abstract |
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Keywords: reactive thiol groups/scFv fragment/site-specific labeling
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Introduction |
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Recombinant fragments such as diabodies [dimers of single-chain Fvs, 55 kDa (Holliger et al., 1993)] or minibodies [scFv-CH3 fusion proteins, 80 kDa (Hu et al., 1996
)] have shown promise as in vivo imaging agents in preclinical studies when radiolabeled with single-photon emitting radionuclides such as In-111 or I-123 or positron emitters such as Cu-64 or I-124 for positron emission tomography (PET) in preclinical studies (Wu et al., 2000
; Sundaresan et al., 2003
). Targeting and imaging of I-123 radiolabeled single-chain Fv (scFv, 27 kDa) fragments has been demonstrated clinically, although the size and monovalency of scFv may limit their utility (Begent et al., 1996
). Recent clinical imaging studies using I-123 radiolabeled diabodies appear promising (Santimaria et al., 2003
).
Most current antibody radiolabeling approaches involve conjugation to random sites on the surface of the protein. For example, standard radioiodination methods using Chloramine T or Iodogen result in modification of random surface tyrosine residues. As the size of the antibody decreases, the protein may become inactive following iodination due to modification of key tyrosines in or near the binding site (Nikula et al., 1995; Olafsen et al., 1996
). Alternative iodination approaches or radiometal labeling through conjugation of bifunctional chelates direct modifications to
-amino groups of lysine residues, again randomly located on the surface of antibodies. Chemical modification of lysines located in or near the antigen-binding site could also potentially interfere with binding through steric hindrance if a bulky group is added (Benhar et al., 1994
; Olafsen et al., 1995
). Hence the issue of inactivation following radiolabeling becomes more pressing with smaller antibody fragments, if equal reactivity is assumed, because the binding site(s) represent a larger proportion of the protein surface and fewer safe sites for conjugation are available.
Site-specific radiolabeling approaches provide a means for both directing chemical modification to specific sites on a protein and for controlling the stoichiometry of the reaction. Several strategies capitalize on naturally occurring moieties or structures on antibodies that can be targeted chemically. For example, the carbohydrate found on constant domains of immunoglobulins can be oxidized and conjugated with bifunctional chelates for radiometal labeling (Rodwell et al., 1986). In one instance, an unusual carbohydrate moiety occurring on a hypervariable loop of a kappa light chain was modified for site-specific chelation and radiometal labeling (Leung et al., 1995
). Others have exploited selective reduction of interchain disulfide bridges to enable modification using thiol-specific reagents. C-terminal cysteine residues on antibody Fab or Fab' fragments have been used for direct labeling using 99mTc (Behr et al., 1995
; Verhaar et al., 1996
). Novel approaches include the identification of a purine binding site in antibody Fv fragments, allowing specific photoaffinity labeling (Rajagopalan et al., 1996
).
More recently, genetic engineering approaches have been used to introduce specific sites for modification or radiolabeling of proteins and antibodies. Building on the above-mentioned work, glycosylation sites have been engineered into proteins to provide novel carbohydrate targets for chemical modification. (Leung et al., 1995; Qu et al., 1998
). The six-histidine tail commonly appended to recombinant proteins to provide a purification tag has been used in a novel 99mTc labeling method (Waibel et al., 1999
). Alternatively, a popular strategy has been to use site-directed mutagenesis to place cysteine residues on the surface of proteins to provide reactive sulfhydryl groups. This approach has been implemented by numerous groups to allow site-specific labeling of antibodies (Lyons et al., 1990
; Stimmel et al., 2000
) and other proteins (Haran et al., 1992
; Kreitman et al., 1994
).
Introduction of cysteine residues into engineered antibody fragments has also been used for stabilization or multimerization purposes. For example, introduction of strategically placed cysteine residues in the interface between the VH and VL domains of antibody Fv fragments has allowed covalent linkage and stabilization of these fragments (disulfide-stabilized Fv or dsFv) (Glockshuber et al., 1990; Webber et al., 1995
). FitzGerald et al. described a disulfide-bonded diabody in which cysteine residues were introduced into the VL/VH interface for stability and demonstrated its utility for fluorescent imaging of tumors (FitzGerald et al., 1997
). Others have appended cysteine residues to the C-termini of single-chain Fv fragments (scFv, formed by fusing VH and VL domains with a synthetic peptide linker) to allow multimerization into scFv'2 fragments (Adams et al., 1993
; Kipriyanov et al., 1995
).
We have previously produced an anti-carcinoembryonic antigen (anti-CEA) diabody, assembled VLeight amino acid linkerVH. Following radiolabeling (at random sites on the protein) with radioiodine or radiometals, this fragment exhibits rapid tumor targeting in a nude mouse/LS174T human colon carcinoma xenograft model in biodistribution and imaging studies (Wu et al., 1999; Yazaki et al., 2001b
). In order to allow site-specific radiolabeling using thiol-specific reagents, this paper describes four mutant anti-CEA diabodies engineered by substitution or addition of unique cysteine residues. Two variants, with the C-terminal sequence -LGGC or -SGGC, were found to exist as a stable disulfide-linked dimer. The -LGGC Cys-diabody demonstrated equivalent antigen binding in vitro and tumor targeting in vivo and had the added advantage of allowing site-specific chemical modification following reduction of the interchain disulfide bridge.
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Materials and methods |
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Four variants of anti-CEA diabodies (Figure 1) were constructed by PCR-based mutagenesis (QuikChange site-directed mutagenesis kit, Stratagene, La Jolla, CA) of the pEE12 expression vector (Lonza Biologics, Slough, UK) containing the original anti-CEA diabody constructed with an eight amino acid glycineserine linker (GS8) (Wu et al., 1999). The first variant contained a cysteine instead of serine in the GS8 linker between the variable domains (Cys-linker). The other three contained a cysteine at the C-terminus of the VH. In one construct the terminal serine (Kabat residue 113) was replaced by a cysteine (VTVS-S changed to VTVS-C). The remaining two constructs contained two glycines inserted in front of the cysteine as a spacer. One retained the original C-terminal sequence with GGC appended (VTVS-SGGC) and in the other, serine 113 was exchanged to a leucine (VTVS-LGGC).
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A total of 1 x 107 NS0 cells (provided by Lonza Biologics) (Galfre and Milstein, 1981) were transfected with 40 µg of linearized vector DNA by electroporation and selected in glutamine-deficient media as described (Yazaki et al., 2001a
). Clones were screened for expression by ELISA, in which the desired protein was captured either by protein L or by a recombinant CEA fragment, N-A3 (You et al., 1998
), and detected using alkaline phosphatase-conjugated goat anti-mouse Fab antibodies (Sigma, St Louis, MO). Supernatants were also examined by western blot for size analysis, using the alkaline phosphatase-conjugated goat anti-mouse Fab antibodies. The best producing clones were expanded. Cys-diabodies were purified from cell culture supernatant, using a BioCad 700E chromatography system (Applied Biosystems, Foster City, CA) as described (Yazaki et al., 2001a
). Briefly, the supernatants were treated with 5% AG1-X8 (Bio-Rad Laboratories, Hercules, CA) overnight to remove phenol red and cell debris and then dialyzed versus 50 mM TrisHCl, pH 7.4. Treated supernatant was loaded on to an anion-exchange chromatographic column (Source15Q; Amersham Pharmacia Biotech, Uppsala, Sweden) and proteins were eluted with an NaCl gradient to 0.2 M in the presence of 50 mM HEPES, pH 7.4. Eluted fractions, containing the desired protein, were subsequently loaded on to a Ceramic Hydroxyapatite (Bio-Rad Laboratories) column and eluted with a KPi gradient to 0.15 M in the presence of 50 mM MES, pH 6.5. Fractions containing pure proteins were pooled and concentrated with a Centriprep 10 (Amicon, Beverly, MA). Elution was monitored by absorption at 280 nm. The concentration of purified protein per milliter was determined by measuring the OD280, but also by applying a small sample on protein L using known amounts of parental diabody and later Cys-diabody standards quantitated by amino acid composition analysis (Wu et al., 1996
).
Characterization of purified Cys-diabodies
Aliquots of purified proteins were analyzed by SDSPAGE pre-cast 420% polyacrylamide Ready Gels (Bio-Rad Laboratories) under non-reducing and reducing (1 mM DTT) conditions and stained using MicrowaveBlue (Protiga, Frederick, MD). Samples were also subjected to size-exclusion HPLC on Superdex 75 (Amersham Biosciences). Retention time was compared with a standard of parental diabody. Binding to CEA was initially assessed by ELISA as described above. Competition/Scatchard was also carried out in ELISA microtiter plates wells coated with N-A3, using a fixed concentration (1 nM) of biotinylated chimeric T84.66 antibody and increasing concentration of non-biotinylated competitors (0.01100 nM). Displacement was monitored with alkaline phosphatase-conjugated streptavidin (1:5000 dilution) (Jackson ImmunoResearch Laboratories, West Grove, PA) and color was developed with phosphatase substrate tablets (Sigma) dissolved in diethanolamine buffer, pH 9.8. All assays were carried out in triplicate.
Radioiodination
A 70 µg amount of purified Cys-diabody was radiolabeled with 140 µCi Na131I (Perkin-Elmer Life Sciences, Boston, MA) in 0.1 M phosphate buffer at pH 7.5, using 1.5 ml polypropylene tubes coated with 10 µg of Iodogen (Pierce, Rockford, IL). Following 57 min of incubation at room temperature, the sample was purified by HPLC on Superdex 75. Peak fractions were selected and diluted in normal saline/1% human serum albumin to prepare doses for injection. The labeling efficiency was 85%. Immunoreactivity and valency were determined by incubation of radiolabeled protein with a 20-fold excess of CEA at 37°C for 15 min, followed by HPLC size-exclusion chromatography on a calibrated Superose 6 column (Amersham Biosciences).
Biodistribution in tumor-bearing mice
Female athymic mice (78 weeks old) were injected subcutaneously in the flank with 106 LS174T human colon carcinoma cells (ATCC CL-188). At 7 days post-inoculation, mice bearing LS174T xenografts were injected with 1 µg of 131I-labeled Cys-diabody (specific activity 1.7 µCi/µg) via the tail vein. Groups of five mice were killed and dissected at 0, 2, 4, 6, 18 and 24 h post-injection. Major organs were weighed and counted in a gamma scintillation counter. Radiouptakes in organs were corrected for decay and expressed as percentage of injected dose per gram of tissue (% ID/g) and as percentage of injected dose per organ (% ID/organ). Tumor masses ranged from an average of 0.580 g (0 h group) to 1.058 g (24 h group). Biodistribution data are summarized as means and corresponding standard errors (SEM). Animal blood curves were calculated using the ADAPT II software (DArgenio and Schumitzky, 1979) to estimate two rate constants (ki) and associated amplitudes (Ai).
Conjugation and radiometal labeling of Cys-diabodies
The VTVS-LGGC Cys-diabody was reduced and conjugated with a bifunctional chelating agent comprised of the macrocyclic chelate DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), a tetrapeptide linker and a hexanevinyl sulfone group for chemical attachment to thiol groups. This compound, DOTA-glycylleucylglycyl(-aminobis-1,6-hexanevinyl sulfone)lysine, abbreviated DOTA-GLGK-HVS, has been described in detail previously (Li et al., 2002
). VTVS-LGGC Cys-diabody (2 mg in 0.5 ml of PBS) was reduced by treatment with 20 µl of 20 mM tris(carboxyethyl)phosphine (TCEP) (Pierce) in PBS for 2 h at 37°C under Ar and centrifuged through a Sephadex G25 spin column. DOTA-GLGK-HVS (58 µl of 20 mM in PBS) was added and the solution rotated at 10 r.p.m. for 4 h at 25°C. The conjugate was dialyzed against 0.25 M NH4OAc, pH 7.0. The extent of modification was evaluated with isoelectric focusing gels (Li et al., 2002
).
Radiolabeling of Cys-diabody conjugates with copper-64
Copper-64 (copper chloride in 0.1 M HCl; radionuclide purity >99%) was produced in a cyclotron from enriched 64Ni targets at the Mallinckrodt Institute of Radiology, Washington University Medical Center (McCarthy et al., 1997). DOTA-GLGK-HVS-conjugated Cys-diabody (200 µg) was incubated with 7.3 mCi of 64Cu in 0.1 M ammonium citrate, pH 5.5, for 1 h at 43°C. The reaction was terminated by addition of EDTA to 1 mM. Labeled protein was purified by size-exclusion HPLC on a TSK2000 column (30 cmx7.5 mm i.d.) (Toso-Haas, Montgomeryville, PA). The radiolabeling efficiency was 56% and the specific activity was 1.7 µCi/µg.
MicroPET imaging
CEA-positive (LS174T) and CEA-negative (C6 rat glioma) xenografts were established in nude mice by subcutaneous injection of (12)x106 cells subcutaneously into the shoulder area 1014 days prior to imaging. Mice were imaged using the dedicated small-animal microPET scanner developed at the Crump Institute for Molecular Imaging (UCLA) (Chatziioannou et al., 1999). Mice were injected in the tail vein with 57 µCi of 64Cu-diabody. After the appropriate time had elapsed, mice were anesthetized with a mixture of ketamine (100 mg/kg) and xylazine (7 mg/kg) injected intraperitoneally, placed in a prone position and imaged using the microPET scanner with the long axis of the mouse parallel to the long axis of the scanner. The acquisition time was 56 min (8 min per bed position; seven bed positions) and images were reconstructed using a MAP reconstruction algorithm (Qi et al., 1998
). Images were displayed and regions of interest (ROIs) were drawn and quantitated using AMIDE (Loening and Gambhir, 2003
).
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Results |
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The Cys-diabodies were expressed in the mouse myeloma cell line NS0. The expression level was low (<1 µg/ml in T-flasks) for the Cys-linker and VTVS-C constructs, whereas the expression level for VTVS-SGGC and VTVS-LGGC was between 5 and 20 µg/ml as determined by ELISA. Cultures were expanded in T-flasks and supernatants collected. The VTVS-SGGC and VTVS-LGGC Cys-diabodies were purified essentially as described (Yazaki et al., 2001a).
Analysis of the purified proteins by SDSPAGE demonstrated that the two-step purification scheme yielded VTVS-SGGC and VTVS-LGGC diabodies that were >95% pure (Figure 2A), whereas the other two constructs could not be readily separated from other proteins present in the growth medium (not shown). Under non-reducing conditions both proteins migrated as single species in the range 5560 kDa with the VTVS-LGGC version exhibiting slightly lower mobility (Figure 2A, lanes 1 and 2). Under reducing conditions both proteins demonstrated the presence of the expected 25 kDa monomer (Figure 2A, lanes 4 and 5). Thus, when purified under native conditions, both of these Cys-diabodies existed as essentially pure disulfide-bonded homodimers. Small amounts of the Cys-linker diabody were purified on protein L. It migrated as higher molecular weight proteins (aggregates) as well as monomers under non-reducing conditions. The aggregates were only partially reduced under reducing conditions. The VTVS-C version of Cys-diabody was never expressed in sufficient quantities to permit purification and biochemical characterization. The remainder of the work was focused on the VTVS-LGGC Cys-diabody as it expressed very well and existed as a homogenous covalent homodimer.
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The binding activity of the Cys-diabody to CEA was initially demonstrated by ELISA. Affinity was measured by competition ELISA in the presence of competitors at different concentrations. As shown in Figure 2B, by competition assay the affinities of the Cys-diabody and parental diabody were essentially the same as that of the intact chimeric T84.66 antibody.
The immunoreactivity and valency of the Cys-diabody were analyzed following radioiodination by solution-phase incubation in the presence of excess CEA. Size-exclusion HPLC analysis demonstrated that 90% of the Cys-diabody shifted to high molecular weight complexes indicated by two peaks, suggesting that the Cys-diabody was bound to one and two CEA molecules (not shown).
In vivo biodistribution and targeting
The 131I-labeled Cys-diabody was assessed for its ability to target tumor in athymic mice bearing xenografts of LS174T human colon carcinoma cells. As can be seen in Table I, the accumulation of the 131I-labeled Cys-diabody reached 9.32% ID/g at 2 h and this level of localization was maintained at 4 and 6 h post-injection. Blood clearance was rapid and nearly complete by 18 h (0.55% ID/g), with the half-life in the beta-phase being 2.68 h, essentially the same as that observed with the non-covalently bound iodinated diabody (2.89 h) (Yazaki et al., 2001b). Activities in other normal organs (liver, spleen, lung, kidneys) also fell rapidly and were below 1% ID/g by 18 h. These biodistribution results were essentially identical with those observed for the iodinated parental anti-CEA diabody (Wu et al., 1999
).
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Cys-diabody was conjugated with the macrocyclic chelate DOTA using a novel peptidehexanevinyl sulfone derivative described in detail elsewhere (Li et al., 2002). This allowed efficient radiolabeling with 64Cu, a positron-emitting radionuclide with a 12.7 h half-life, well matched to the targeting and clearance kinetics observed for diabodies in murine systems in vivo. MicroPET imaging studies were conducted on athymic mice bearing LS174T (CEA-positive human colorectal carcinoma) and C6 (CEA-negative rat glioma) xenografts. Specific targeting to the CEA-positive xenograft was observed at 4 and 18 h post-injection, with little evidence of activity in the CEA-negative tumor at 18 h (Figure 3). The positive tumor/control tumor uptake ratio deducted from ROI analysis was 4.6:1 at 18 h, demonstrating specificity. This ratio is comparable to that observed with the 64Cu anti-CEA mininbody (3.4:1) (Wu et al., 2000
). However, this particular proteinchelateradionuclide combination (64Cu-DOTA-GLGK-HVS Cys-diabody) resulted in elevated liver activity (19.4% ID/g at 4 h) in addition to kidney activity (55.1% ID/g at 4 h) (Li et al., 2002
).
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Discussion |
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The crystal structure of the parental T84.66/GS8 diabody has recently been solved (Carmichael et al., 2003). In the crystal the Fv units of the diabody assumed a very compact, twisted structure, with the binding sites oriented in a skewed orientation at a tight 70° angle. The C-termini of the heavy chain variable regions (where we have appended the Cys residues) are
60 Å apart. However, the structure that was solved is likely to represent one of many conformations that the parental diabody can adopt. The fact that the Cys-diabody forms with such high efficiency implies that the parental diabody is flexible and that the Fv domains can swivel such that the C-termini are juxtaposed. Figure 4 shows the crystal structure of the parental diabody (A) and a model where the Fvs have been rotated to bring the C-termini close enough for disulfide bridge formation [represented by the blue (Gly) and yellow (Cys) residues in the model] (B). Size-exclusion HPLC analysis confirms the notion that the native, non-covalent diabody is fairly flexible; it elutes at an earlier retention time, suggesting a larger Stokes radius. By contrast, the covalently linked Cys-diabody elutes at a later retention time, implying a more compact, more highly constrained molecule. Taken together, these results suggest that the native diabody has an open and flexible structure.
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The novel covalently-linked Cys-diabody described here provides a platform for ready conjugation of a wide variety of effector moieties to this rapid targeting anti-tumor molecule. These would include alternative radiolabeling approaches; for example, we are developing an F-18 tag with thiol-specific chemistry for generating additional PET tracers. Other tags such as optical or fluorescent probes can be site-specifically attached. In addition, the C-terminal cysteine residues can be used for conjugation of other small molecules such as chemotherapeutic drugs. This engineered antibody fragment has a unique advantage: the presence of the cysteine thiol groups in an internal, protected format, which can be released when required for chemical modification.
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Acknowledgements |
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Received October 15, 2003; accepted October 15, 2003 Edited by Greg Winter