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Improved Biodistribution and Radioimmunoimaging with Poly(ethylene glycol)-DOTA-Conjugated Anti-CEA Diabody Lin Li,† Paul J. Yazaki,‡ Anne-Line Anderson,‡ Desiree Crow,‡ David Colcher,‡ Anna M. Wu,§ Lawrence E. Williams,| Jeffrey Y. C. Wong,⊥ Andrew Raubitschek,‡ and John E. Shively†,* Division of Immunology, Beckman Research Institute of the City of Hope, Duarte, California 91010, Division of Cancer Immunotherapy and Tumor Immunology, City of Hope National Medical Center, Duarte, California 91010, Department of Molecular and Medical Pharmacology, Crump Institute for Molecular Imaging, University of California School of Medicine, Los Angeles, California 90095, Division of Diagnostic Radiology, City of Hope National Medical Center, Duarte, California 91010, and Division of Radiation Oncology, City of Hope National Medical Center, Duarte, California 91010. Received August 25, 2005; Revised Manuscript Received November 20, 2005
Diabodies are single chain antibody fragments (scFvs) that spontaneously form bivalent dimers of molecular size 50-55000. Radiolabeled diabodies are almost ideal tumor targeting agents due to their high avidity (bivalent) binding to tumor antigens and small size (50-55000) that leads to improved tumor-to-blood ratio compared to intact antibodies (150000). However, due to their high retention and metabolism in the kidney, radioiodine is the current radiolabel of choice for diabodies since radioiodine is rapidly excreted from the kidney once metabolized. We have previously shown that 111In-DOTA-diabody gives higher tumor uptake in nude mouse xenografts than 125I-diabody, but has extremely high kidney retention since its 111In-labeled metabolites are retained by and only slowly excreted from the kidney. When a diabody is conjugated to a bifunctional PEG-3400 derivative followed by reaction with cysteinyl-DOTA, the resulting product has an apparent molecular size of 75000 and a Stokes radius of 35 Å on size exclusion chromatography, compared to a Stokes radius of 25 Å for intact diabody. When radiolabeled, the conjugate gives high yields of 111In-labeled product, retains high immunoreactivity, and gives improved biodistributions (30-40%ID/g, 12-48 h) compared to 111In-DOTA-diabody (12-13%ID/g, 6-12 h). We show that the improved biodistribution is due to an increase in Stokes radius caused by the linear PEG-3400 since conjugation of diabody with multiple (PEG)12 linkers followed by reaction with cysteinyl-DOTA does not reduce kidney accumulation. We also show that 111In-cysteinyl-DOTA-PEG3400-diabody gives excellent tumor images in the nude mouse xenograft model and that 125I-PEG3400-diabody gives equivalent images to 125Iminibody (molecular size, 80000), but improved tumor-to-liver ratios, suggesting that this imaging agent can be used to image liver metastases.
INTRODUCTION To be effective tumor imaging agents, tumor-targeted radiolabeled antibodies must give both high tumor uptake and high tumor-to-blood ratios (1). At one extreme, monoclonal antibodies (150 kda) have a relatively slow blood clearance (t1/2 β ) 48-72 h), allowing ample time for high accumulation in the tumor, but suffer from low tumor-to-blood ratios. At the other extreme, single chain (sc) Fv recombinant antibodies (25 kDa) are rapidly cleared from the blood (t1/2 β ) 0.5-2.0 h) resulting in high tumor-to-blood ratios but low overall accumulation of radioactivity in the tumor. To predict optimal tumor imaging, one must account for both parameters using pharmacokinetic analytic indicators such as the Imaging Figure of Merit (IFOM). The IFOM indicator was derived by us by comparing the biodistributions (%ID/g over time) of a series of radioiodinated recombinant anti-CEA antibodies of molecular sizes 25000, * To whom correspondence should be addressed. Tel: 626-359-8111 ext 62601, E-mail:
[email protected]. † Division of Immunology, Beckman Research Institute of the City of Hope. ‡ Division of Cancer Immunotherapy and Tumor Immunology, City of Hope National Medical Center. § University of California School of Medicine. | Division of Diagnostic Radiology, City of Hope National Medical Center. ⊥ Division of Radiation Oncology, City of Hope National Medical Center.
50000, 80000, and 150000 in a nude mouse xenograft model (1). From these studies we concluded that the best combination of high tumor uptake and high tumor-to-blood ratio requires at least a bivalent antibody of size 50-80000. However, a drawback to radioiodinated antibodies is that they may be rapidly metabolized in tissues, including the tumor, especially if the antigen undergoes internalization upon antibody binding. Although most anti-CEA antibodies undergo negligible internalization, we and others have observed higher tumor uptake and persistence at the tumor site for radiometal-labeled anti-CEA antibodies compared to radioioinated anti-CEA antibodies (2). For radiometal-labeled recombinant antibody fragments, this improvement is offset by high kidney retention, indicating that the radiometal-labeled antibody was filtered, reabsorbed, and metabolized by the kidney, but the radiometal metabolic products are retained or only slowly excreted compared to radioiodine metabolic products. Indeed, we have identified 111InDOTA--lysine as the ultimate metabolic product in the kidney when anti-CEA antibody fragments are conjugated to the NHSactive ester of DOTA and radiolabeled with 111InCl3 (3). In light of these findings, we and others have sought to improve the utility of radiometal-labeled recombinant antibody fragments by decreasing kidney retention. In one approach kidney uptake is partially blocked by the administration of intravenous L- or D-lysine (4, 5). In a second approach, we have designed unique metabolizable linkers between the antibody and DOTA that have decreased kidney retention by up to 4-fold
10.1021/bc0502614 CCC: $33.50 © 2006 American Chemical Society Published on Web 12/24/2005
PEG-DOTA Diabody
over NHS-DOTA conjugates (6). A third approach is to increase the blood retention of antibody fragments by increasing their molecular weight through conjugation to poly(ethylene glycol) (PEG) polymers (7). This approach was successfully applied to recombinant cytokines such as IL-2 where the increase in the beta phase of blood clearance was greater than 8-fold (8). In the case of anti-CEA F(ab′) fragment A5B7 conjugated to MPEG6000, tumor retention increased by 5.7-fold and serum half-life by 5-fold (9). In the case of anti-mucin scFv CC49/ 218 conjugated to PEG-5000, a 14-fold increase in serum half was observed (10). In the latter study, the authors conclude that greater improvements were observed with increasing size of PEG polymers rather than total amount of PEG, which in addition, often led to decreased immunoreactivity of the conjugate. In this respect, recombinant antibodies have been designed to allow site-specific attached of PEG to prevent alterations in immunoreactivity (11). As yet, no biodistributions have been published on site-specific PEG- antibody conjugates. In this study we selected a commercially available bifunctional PEG3400 for conjugation to an anti-CEA diabody that we have previously characterized (2). This linear polymer has an NHS-active ester at one end and vinyl sulfone at the other end. Reaction conditions were adjusted to conjugate approximately 1 PEG3400 per diabody molecule (at lysine residues), followed by reaction with cysteinyl-DOTA, a chelate with high metal complex stability that we have previously described (12). The resulting cys-DOTA-PEG3400-diabody conjugate had an apparent molecular size of 75000 and a Stokes radius of 35 Å, compared to an apparent molecular size of 50000 and a Stokes radius of 25 Å for intact diabody, demonstrating a net gain of 50% in its Stokes radius. The conjugate was radiolabeled with high efficiency (>90%) to high specific activity (2-4 µCi/ug) with 111InCl3, retained high immunoreactivity (90%), and showed improved tumor uptake and blood retention compared to 111In-DOTA-diabody. In addition, kidney retention was decreased by over 4-fold compared to 111InDOTA-diabody, and excellent tumor images were obtained. We also conjugated PEG3400 to a site-specific cys-diabody, a diabody with C-terminal cysteines (13) that were reduced and conjugated to DO3A-vinyl sulfone to chemically separate DOTA from PEG3400. This conjugate also gave high efficiency labeling to high specific activity and gave identical biodistributions compared to cys-DOTA-PEG3400-diabody. Since cysDOTA-PEG3400-diabody has an apparent molecular size similar to a “minibody” construct (scFv-hinge-CH3) made from the same parent antibody (14), we decided to compare the two radioiodine-labeled antibodies in the same animals. Within experimental error, the two constructs behaved identically and gave similar images. Since most scFv antibodies are easily expressed as diabodies in bacteria or yeast, while minibodies are best expressed in myeloma cells, we conclude that diabodies conjugated to this bifunctional PEG3400 offer a simple route to the rapid generation of useful imaging agents. A potential drawback is that, due to their small size, conjugation of PEG3400 to a diabody may reduce immunoreactivity.
EXPERIMENTAL PROCEDURES Materials, Radiolabeling, Immunoreactivity, and Mass Spectrometry. NHS-PEG3400-VS (Cat No 4M5B0F02) was obtained from Nektar (San Carlos, CA). MAL-dPEG12-NHS ester (Cat No 10284) was obtained from Quanta Biodesign Ltd. (Powell, OH). The synthesis of cysteinyl-DOTA (cys-DOTA) and DO3A-VS were previously described (12, 15). All other reagents were obtained from Aldrich-Sigma (St. Louis, MO). LS-174T cells were obtained from ATCC and maintained in maintained in sterile growth media consisted of Eagle’s Minimal Essential Media 1X (EMEM) (Cellgro, Herndon, VA) supple-
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mented with 10% heat inactivated fetal bovine serum (FBS) (Omega Scientific, Tarzana, CA), and 1% L-glutamine, 10 mM sodium pyruvate, 0.1 mM nonessential AA. The production and purification of anti-CEA T84.66 diabody, Cys-Diabody, and Minibody have been previously described (13, 16). Chelateconjugated antibodies were radiolabeled either with 111In (Amersham, 2-3 mCi/mg of protein) as previously described (17) or with 125I or 131I by the iodogen method (2). Percent labeling was determined by ITLC or by size exclusion chromatography (SEC) on a Superose 6 column (1 × 30 cm, Amersham). Radiolabeled antibody was purified by SEC on the same column (in saline, flow rate 0.5 mL/min, fraction size was 0.5 mL). Immunoreactivity was determined by mixing the radiolabeled sample with a 20-fold excess by mass of CEA and running the sample over a Superose 6 column. Percent immunoreactivity was calculated as counts in CEA conjugate complex/ total counts × 100. MALDI-MS was performed on Perkin-Elmer-Sciex prOTOF 2000 using sinapinic acid (10 mg/ mL in TFA/water/acetonitrile (0.1/99/100 v/v/v) as a matrix. Prior to mixing with matrix the sample was desalted on a Sephadex G25 spin column (100-200 mg in a 250 µL pipet tip), equilibrated in water. Cys-DOTA-PEG3400-Diabody Conjugate. The reaction conditions for a molar ratio of 20:1 NHS-PEG3400-VS to diabody at pH 6.0 are given here. NHS-PEG3400-VS (3.0 mg, 0.8 µmol) was mixed with 2 mg (0.04 µmol) of diabody in 0.62 mL of pH 7.0 PBS, the pH was adjusted to 6.0 with 0.1 M NaOH, and allowed to react for 2 h at RT. In other reactions, the molar ratios and reaction pH were varied (see Results). When the reaction was monitored by SDS gels, it appeared to be >70% complete at the end of 2 h. At the end of 2 h the entire reaction mixture was applied to a Superdex 75 column (1 × 30 cm, Pharmacia), monitored at 214 and 280 nm, and eluted at a flow rate of 0.5 mL/min with PBS. The first peak that eluted at 18 min was the conjugate followed by unconjugated diabody at 22 min and free NHS-PEG3400-VS at 35-40 min (see Results). The conjugate (4 mL) was concentrated to 0.3 mL in a Amicon Ultra-4 (Millipore, Bedford, MA) and mixed with 1.0 mg (2 µmol) of cys-DOTA, the pH adjusted to 8.5, and reaction continued on a sample rotator for 17 h at RT. The product was dialyzed vs 3 × 1.5 L of PBS containing 1.5 g Chelex 100, followed by 2 × 1.5 L of 0.9% NaCl or 0.25 M ammonium acetate, pH 7.0. The dialysate was concentrated to 2-4 mg/L on a Centricon 10 and sterile filtered. The conjugate was characterized by both SDS and IEF gel electrophoresis (see Results). Cys-DOTA-Mal-PEG12-Diabody Conjugate. Diabody (0.5 mL of 4 mg/mL; 0.04 µmol)) in PBS was mixed with 0.93 mg (0.8 µmol) of NHS-PEG12-maleimide, the pH was adjusted to 6.0 with 0.1 M NaOH, reacted for 2.5 h at RT, and the conjugate purified by SEC on a Superdex 75 column as above. The major peak (see Results) was collected and concentrated to 0.7 mL by centrifugation on an Amicon Ultra-4. The conjugate was mixed with 1 mg (1.0 µmol) of cys-DOTA, the pH adjusted to 8.3, and reaction continued overnight at RT. The product was characterized by SDS and IEF gel electrophoresis (see Results) and dialyzed vs PBS (1.5 L), saline (1.5 L) and then concentrated to 0.3 mL (3.6 mg/mL) on an Amicon Ultra-4. DO3A-VS-cys-Diabody-PEG3400 Conjugate. Cys-diabody (2 mg, 0.04 µmol) in 0.38 mL of pH 7.5 PBS was reduced with 6 µL of freshly made 0.2 M TCEP (tris-carboxyethylphosphine, 0.12 µmol) for 2 h at 37 °C. The sample was run through a 1 mL spin column (Sephadex G25 in PBS) to remove excess TCEP and run on a nonreducing SDS gel to verify complete reduction from 50 kDa to 25 kDa. The reduced sample was mixed with 0.9 mg (2.0 µmol) of DO3A-VS and reacted at 25 °C for 2 h. Reaction with DO3A-VS was verified by running
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an IEF gel where the conjugate was shifted to a lower pI (see Results). The conjugate was reacted with 3.0 mg (0.8 µmol) of NHS-PEG3400-VS at pH 6.0 for 2 h at RT and purified on a Superdex 75 column as described above. The final product (0.28 mL) was dialyzed into 0.25 M, pH 7.0 ammonium acetate and concentrated to 2.0 mg/mL. Determination of Stokes Radius of Cys-DOTA-PEG3400Diabody Conjugate. The elution volumes (Ve) of proteins of known Stokes radii were measured by SEC on a Superdex 75 column (1 × 30 cm, Pharmacia) in PBS with a flow rate of 0.5 mL/min. The standards were catalase (250000, 52 Å), alcohol dehydrogenase (150000, 46 Å), bovine serum albumin (65000, 35 Å), carbonic anhydrase (29000, 24 Å), myogobin (19000, 19 Å), and cytochrome c (17000, 12.4 Å). Stokes radii were plotted vs Kd1/3 where Kd) (Ve - Vo)/(Vt - Vg - Vo) (18). The void volume (Vo) was determined as 7.71 mL using Dextran Blue. The values for Vt (24 mL) and Vg (0.84 mL) were obtained from Pharmacia. The Ve for diabody was 11.0 mL, corresponding to a Stokes radius of 25 Å, and 9.8 mL for Cys-DOTAPEG3400-diabody conjugate, corresponding to a Stokes radius of 35 Å. LS-174T Xenograft Model. Female, athymic nu/nu mice (Charles River), 10-12 weeks old, were injected with LS-174T cells (5 × 106) sc in the flank. Tumors were established within 7-10 days postinjection. Athymic mice bearing LS-174T xenografts were tail vein injected with 200 µL of 4 µCi/2-3 µg 111In-labeled DOTA-conjugates, 4 µCi/2 µg I-labeled minibody, or 4 µCi/1 µg of 125I-labeled PEG3400 diabody. Five mice per time point were sacrificed at 0, 2, 4, 6, and 18 h postinjection and biodistributions performed (blood, liver, spleen, kidneys, lungs, tumor, and carcass). Results were calculated as percent injected dose per gram (%ID/g) vs time. For radioimaging, mice were injected with 40 µCi/20 µg 111In-labeled DOTA conjugates or 20 µCi/5µg of 125I PEG3400 diabody and imaged using a BIOSPACE γ Imager instrument with settings of 154-188 keV and 220-270 keV, respectively.
RESULTS Optimization of Cys-DOTA-PEG3400-Diabody Conjugate. PEG-antibody conjugates have demonstrated the ability for improved blood retention over their unconjugated cognates but have the drawback of reducing immunoreactivity if overconjugated (10). We searched for a commercially available PEG reagent of moderate size that could be both conjugated to diabody and to a chelate without over-derivatizing the diabody. We chose the bifunctional reagent NHS-PEG3400-VS because, in theory, it could first be conjugated to the -amino group of lysines in the diabody, followed by reaction with a chelate possessing a free thiol group (Scheme 1). Indeed, we had such a chelate (cysteinyl DOTA) in hand that previously had been shown to have high metal binding stability when conjugated via linkers to antibody (12). We were further interested in the use of vinyl sulfone as a functional group because its reaction toward thiols is rather pH specific (15) and the products are more stable than maleimide derivatives (12) which are also commercially available. The bifunctional reagent NHS-PEG3400VS was first reacted with diabody over a range of conditions to determine the optimal reaction conditions to retain maximal immunoreactivity (Table 1). From these studies we determined that a NHS-PEG3400-VS to diabody ratio of 20:1 at pH 6.0 was a good compromise since >80% of the diabody reacted under these conditions (Figure 1) retaining 90% of its immunoreactivity (Table 1). At higher ratios (e.g., 50:1), more PEG3400 reacted with diabody resulting in lowered immunoreactivity (e.g., 40-54%, Table 1). In addition, we found that the reaction products of the first step must be purified by SEC
Li et al. Table 1. Optimization of Reaction Conditions on the Formation of Diabody-PEG Conjugatesa conjugate Cys-DOTA-VS-PEG3400-DB Cys-DOTA-VS-PEG3400-DB Cys-DOTA-VS-PEG3400-DB Cys-DOTA-VS-PEG3400-DB Cys-DOTA-Mal-PEG12-DB DO3A-VS-cys-DB-PEG3400
conj conj labeling pH ratio ratio mCi/mg 7 6 6 6 6 6
50:1 50:1 20:1 10:1 20:1 20:1
97 96 60-70 70-94 95 95-98
3 3 2-3 3 2-3 2-8
% IR 40 54 83-95 88 85 90-97
a Reaction conditions include pH and molar ratio of PEG bifunctional reagent to diabody. The products were compared after further conjugation to cys-DOTA by labeling efficiency, labeling ratio (mCi/mg), and % immunoreactivity (IR).
and not by dialysis because NHS-PEG3400-VS remained in the dialysate (10 kDa cutoff) and interfered with the subsequent reaction with cys-DOTA. Although the diabody-PEG3400-VS conjugate could not be completely separated from unreacted diabody by SEC at this ratio (Figure 1A vs 1C), we were able to estimate the apparent molecular size of the conjugate as 75000 with a Stokes radius of 35 Å by SEC. In contrast, intact diabody has an apparent molecular weight of 50000 with a Stokes radius of 25 Å. Since SDS gel electrophoresis revealed that the PEG3400 conjugated diabody was >90% monosubstituted (Figure 1B and 1D), and the predicted molecular size of the monosubstituted product was only 28400 (diabody plus PEG3400), we conclude that the addition of the linear PEG polymer to the diabody led to a dramatic change in its Stokes radius as analyzed by SEC. The final product was also characterized by MALDI-MS. In this analysis intact diabody gave three peaks corresponding to monomer, dimer, and trimer species in the gas phase (Figure 1E). The PEG3400-modified monomer peak increased in mass from 25771 Da to 29944 Da, an increase in agreement with the addition of PEG3400 and Cys-DOTA (507 Da). Cys-DOTA-PEG3400-diabody (pH 6.0, 20:1 ratio) was radiolabeled with 111InCl3 in good yield (60-90% efficiency, 2-3 mCi/mg labeling ratio, and immunoreactivity 83-95%, Table 1), suitable for biodistribution studies. Stability studies performed by radiolabeling the sample (stored at 4 °C) and remeasuring immunoreactivity and molecular size over a 3 month interval demonstrated no loss in activity or aggregation over this period (data not shown). Biodistribution and Imaging of 111In-Cys-DOTA-PEG3400Diabody Conjugate in a Nude Mouse LS174T Xenograft Model. The cys-DOTA-PEG3400-diabody conjugate obtained from the pH 6.0, 20:1 ratio reaction was radiolabeled with 111InCl3, purified by SEC, and injected into nude mice bearing LS174T (CEA positive) xenografts. The results of the biodistributions shown in Figure 2 demonstrate high tumor uptake, reaching a maximum of about 40% ID/g at 12 h that is sustained through 48 h. Over the same period blood levels drop to about 10% ID/g at 12 h, and to about 2% ID/g by 48 h. Thus, the tumor-to-blood ratios increase from 5:1 at 12 h to 25:1 at 48 h. The combination of high tumor uptake and high tumor-to-blood ratios gives exceptionally high IFOM values (see below). As expected, the uptake in kidney is high but, unlike unmodified 111In-DOTA-diabody (2), is similar to tumor levels at all time points tested. Kidney reaches a maximum of about 50% ID/g at 24 h and remains high at 48 h. Thus, the tumor-to-kidney ratio at 24 h is about 0.8, a significant improvement over that seen for 111In-labeled DOTA-diabody at 12 h (0.08, see ref 2). Importantly, low liver levels (90% labeling) to suitably high specific activities (2-3 mCi/mg) for both biodistribution and radioimmunoimaging studies in nude mice bearing CEA-positive xenografts. Although the radiolabeled conjugate contains up to 20% unconjugated diabody, the presence of unconjugated diabody was not expected to adversely
PEG-DOTA Diabody
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to tumor uptake, the problem is somewhat lessened compared to the use of 111In-DOTA diabody where kidney uptake is roughly 10-fold higher than tumor uptake at 12 h (2). Nonetheless, we are committed to further reducing the kidney uptake by combining other approaches such as the use of metabolizable linkers (6) and adminstration of intravenous lysine (20). If successful, we may have a fully optimized approach to radioimmunoimaging with radiometal-labeled antibody fragments.
ACKNOWLEDGMENT This research was supported by NCI grant CA43904.
LITERATURE CITED
Figure 11. Biodistribution of 111In-DO3A-VS-cys-diabody-PEG3400 in nude mice bearing LS174T xenografts. The average %ID/g (( std dev) of five animals per tissue analyzed is shown.
affect tumor targeting because nonsaturating amounts of total diabody were injected into animals and intact diabody was expected to clear more rapidly from the blood than conjugated diabody. The results were very encouraging in that tumor accumulation was higher than that seen for 111In-DOTA-diabody and essentially equivalent to that seen for 111In-DOTA minibody which has a molecular mass of 80 kDa. Since the molecular masses of the minibody and PEG3400-diabody are almost equivalent, we decided to compare their biodistributions side by side in the same animals. In this comparison, one was radiolabeled with 125I and the other with 131I to take advantage of discriminatory counting and the wealth of data (including IFOM analysis) we have on our radioiodine-labeled recombinant antibody fragments. As expected, the biodistributions were essentially equivalent. However, the question remained as to whether the radiometal-labeled conjugates were equivalent. This was an important question since 111In-DOTA minibody has a problem with high liver uptake and 111In-cys-DOTA-PEG3400diabody has a problem with high kidney uptake. When the IFOM analysis was performed on the two, the 111In-cys-DOTAPEG3400-diabody was clearly superior to 111In-DOTA minibody, in fact, the tumor to liver IFOMs of 111In-cys-DOTAPEG3400-diabody at 24 h were equivalent to the tumor to blood IFOMs of 111In-DOTA minibody at 24 h. Since our experience with imaging tumors in patients with 111In-DOTA minibody have been excellent (unpublished data), based on these IFOM calculations, we expect that 111In-cys-DOTA-PEG3400-diabody will be superior to 111In-DOTA minibody in areas both outside and inside the liver. Since a nude mouse, liver metastasis model is available for CEA positive LS174T cells (19), we are currently testing this hypothesis. If these predictions are verified in the clinic, then 111In-cys-DOTA-PEG3400-diabody will be a decided improvement as a radioimmunoimaging agent over all the agents we have tested so far and may permit us to positively image liver metastases with a radiometal-labeled imaging agent for the first time. The remaining problem with the high kidney uptake should not be an issue with imaging CEA positive colorectal or breast cancer patients where kidney metastases are rarely observed. Since the kidney uptake is roughly equivalent
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