MONODISPERSED PEG-DOTA CONJUGATED ANTI-TAG-72 DIABODY HAS LOW KIDNEY UPTAKE AND HIGH TUMOR TO BLOOD RATIOS RESULTING IN IMPROVED ⁶⁴Cu PET IMAGING

Abstract

Diabodies are non-covalent dimers of single chain antibody fragments (scFvs) that retain the avidity of intact IgG but have more favorable blood clearance than intact IgGs. Radiometals offer a wide range of half lives and emissions for matching imaging and therapy requirements and provide facile labeling of chelate-antibody conjugates. However, due to their high retention and metabolism in the kidney, use of radiometal labeled diabodies can be problematic for both imaging and therapy.

Methods

Having previously shown that ¹¹¹In-DOTA-PEG3400-anti-CEA-diabody has similarly high tumor uptake and retention and less than 50% as much kidney uptake and retention as non-PEGylated diabody, we synthesized a similar derivative for an anti-TAG-72-diabody. We also reduced the molecular size of the polydispersed PEG3400 to monodispersed PEG27 and PEG12 (nominal masses of 1188 and 528, respectively). We performed biodistributions of their DOTA conjugates radiolabeled with ¹²⁵I, ¹¹¹In, or ⁶⁴Cu in tumor bearing athymic mice.

Results

Addition of PEG3400 to the diabody reduced kidney uptake to a level (≈10 %ID/g) comparable to that obtained with radiometal labeled intact IgG. The PEG27 and PEG12 diabody conjugates also demonstrated low kidney uptake without reduction of tumor uptake or tumor to blood ratios. When radiolabeled with ⁶⁴Cu, the DOTA-PEG12- and PEG27-diabody conjugates gave high contrast PET images of colon cancer xenografts in athymic mice.

Conclusion

PEGylated diabodies may be a valuable platform for delivery of radionuclides and other agents to tumors.

INTRODUCTION

To be effective tumor imaging agents, targeted radiolabeled antibodies must provide both high tumor uptake and high tumor to blood ratios (1, 2). At one extreme, monoclonal antibodies (150 kDa) have a relatively slow blood clearance (t_{1/2 β} = 48-72 h), allowing ample time for accumulation in the tumor, but suffer from low tumor to blood ratios. At the other extreme, single chain (sc) Fv recombinant fragments (25 kDa) are rapidly cleared from the blood (t_{1/2 β} = 0.5-2.0 h) resulting in high tumor to blood ratios but low overall accumulation of radioactivity in tumor (3). After analyzing a series of radioiodinated recombinant antibody fragments (4), we and others concluded that simultaneous achievement of high tumor uptake and high tumor to blood ratio requires a bivalent antibody of size 50-80 kD (1, 3, 4) with diabodies (noncovalent dimers of size 55 kDa) as one platform fulfilling these criteria. For example ¹²⁴I-labeled diabodies have performed well in a pre-clinical model of PET imaging (5). However, a drawback to radioiodinated antibodies is that they may be rapidly metabolized in tissues, including tumor, especially if the antigen undergoes internalization upon antibody binding. Radiometal-labeled recombinant antibody fragments offer a significant improvement in tumor to blood ratio, but this is offset by high kidney retention. Thus, systemic excretion of radiometal metabolites is slow compared to radioiodine metabolic products. We have identified ¹¹¹In-DOTA-ε-lysine as the metabolic endpoint product in the kidney for ¹¹¹In-DOTA anti-CEA antibody fragments (6).

We and others have sought to improve the utility of radiometal labeled recombinant antibody fragments by decreasing kidney uptake/retention. In one approach kidney uptake is partially blocked by the administration of intravenous L- or D-Lysine (7, 8). Alternatively, we have designed unique metabolizable linkers between the antibody and DOTA that have decreased kidney retention up to four-fold compared with NHS-DOTA conjugates (9). A third approach is to decrease kidney retention of antibody fragments by increasing their apparent molecular weight by PEGylation (10). This approach was successfully applied to anti-CEA F(ab’) fragment A5B7 by conjugating it to MPEG6000, which increased tumor retention and serum half life by 6- and 5-fold, respectively (11). In the case of anti-mucin scFv CC49/218 conjugated to PEG5000, a 14-fold increase in serum half life was observed (12). In the latter study, the authors concluded that greater improvements were observed with increasing size of PEG polymers rather than total amount of PEG, which often led to decreased immunoreactivity of the conjugate. In another study, investigators engineered an anti-MUC-1 dimeric scFv-SH (di-scFv, 55 kDa) with C-terminal cysteine residues for site specific conjugation to maleimide-PEGs of different sizes (13). They concluded that conjugation yields needed improvement prior to clinical application. Interestingly, a recent report (14) evaluated a 40 kDa branched chain PEG conjugated to a bispecific diabody that bound covalently to CEA and CD3 and showed that PEGylation doubled tumor uptake and increased circulation time (t_1/2β of 50 h). Along the same line of investigation, Leong et al. (15) utilized the natural thiol group of an engineered Fab’ fragment to IL-8 for conjugation to maleimide-PEGs of different sizes. They found that conjugation to maleimide PEG 10 kDa resulted in an apparent molecular size of 180 kDa, comparable to the size of an intact antibody. Importantly, blood clearance studies revealed a t_1/2β of 44 h compared to 3 h for the unmodified Fab’. Also, Yang et al. (10) conjugated maleimide PEGs of different sizes to an anti-TNF-α scFv (half the size of a diabody) with a cysteine engineered into the linker region or C-terminus. They found that conjugation to maleimide PEG (20 kDa) gave a blood clearance half life similar to intact IgG but offered no conclusion regarding the effect of the PEG modification on kidney retention.

Recently, we have shown that adding PEG3400 to ¹¹¹In-DOTA-anti-CEA diabody reduced kidney uptake at 24-48 h from over 200 %ID/g for the non-PEGylated diabody to 50 %ID/g, while preserving high tumor uptake (16). However, further reduction in kidney uptake is required. To study diabody dependence of DOTA-PEG3400 conjugation, we generated and PEGylated an anti-TAG-72-diabody for which kidney retention was reduced to levels (10 %ID/g at 1-24 h) usually seen with intact IgG. This suggested that kidney uptake is a function of both apparent molecular size (as modified by PEG) and other less-defined factors intrinsic to the diabody (e.g., modification of pI has been shown to influence kidney uptake (17, 18)).

Having reduced the kidney uptake for radiometal labeled diabody by conjugation to PEG3400, we sought to further investigate the approach by using monodispersed DOTA-PEG conjugates. We synthesized DOTA-PEG-27-Cys-VS and DOTA-PEG-12-Cys-VS, where Cys-VS = vinyl sulfone conjugated to the thiol of cysteine, and conjugated these monodispersed PEGs to amino groups on the diabody at pH 9.0, a reaction we have previous described (19). The conjugates were characterized by mass spectrometry, IEF, and size exclusion chromatography. Biodistributions of ¹¹¹In-labeled conjugates in athymic mice bearing LS-174T colon tumor xenografts gave tumor uptakes of 40-45 %ID/g, tumor/blood ratios of 8-10 and a kidney uptake of 10 %ID/g at 24 h. Furthermore, PET imaging with ⁶⁴Cu-labeled conjugates provided high-contrast tumor images within 24 h after injection (tumor to blood = 8:1).

EXPERIMENTAL PROCEDURES

Materials, radiolabels, mass spectrometry

LS-174T cells were obtained from ATCC and maintained as previously described (16). 1,4,7,10-Tetraazacyclododecane-1,4,7-tris(t-butyl acetate)-10-acetic acid was obtained from Macrocyclics, Inc., Dallas, TX. NHS-PEG3400-VS (Cat No 4M5B0F02) was purchased from Nektar (San Carlos, CA). N-FMOC-amido-dPEG-27 acid was obtained from Novabiochem (EMD Biosciences, San Diego, CA) and N-FMOC-amido-dPEG-12 acid was purchased from Quanta Biodesign Ltd (Powell, OH). ¹¹¹In chloride was from Amersham, ⁶⁴Cu from Washington University School of Medicine, St. Louis, MO., and ¹²⁵I from Perkin Elmer. Mass spectra were recorded on an Agilent 6520 Q-TOF LC/MS.

Construction, cloning, expression and purification of the anti-TAG-72 diabody

The anti-TAG72 diabody (AVP04-07) was based on V-domains from the parent CC49 antibody and derivative scFv fragments. The gene had codons optimized for E. coli expression with the orientation V_H-V_L joined by a G₄S linker and included a C-terminal His₆ tail. AVP04-07 was subcloned into E. coli BL21 (DE3)(F^– ompT hsdS_B(r_B^- m_B^-) gal dcm (DE3)) (Novagen) and produced in an 18L fermentor. Bacterial lysate cleared by centrifugation (16,000 × g, 30 min) and filtration at 0.45 μm was purified using a three-step procedure (HisTrap, HiTrap SP HP, and Superdex 75 prep chromatography) on an AKTA Purifier 10 (GE Healthcare, Uppsala, Sweden).

Analytical ultracentrifugation

Sedimentation velocity experiments were conducted using a Beckman model XL-I analytical ultracentrifuge (Fullerton, CA, USA) at 20°C. Data were collected at A_290nm in continuous mode, using a time interval of 300 sec and a step-size of 0.003cm, without averaging. Solvent density (1.00499 g/mL at 20°C), solvent viscosity (1.0214 cp), and partial specific volume for AVP04-07 were computed using the program SEDNTERP. Sedimentation velocity data were fitted to a continuous size-distribution model using the program SEDFIT.

Immunoreactivity and isoelectric focusing

Binding of AVP04-07 to the TAG-72 antigen was determined using bovine submaxillary mucin (BSM, Aldrich-Sigma (St. Louis, MO) in a column shift assay (Superdex 200). Isoelectric focusing gel electrophoresis was run either on an IEF Precast gel (pH 3 – 10) (Novex) or on a Pharmacia PhastGel as per the manufacturer’s instructions.

Conjugation of AVP04-07

NHS-DOTA was conjugated to AVP04-07 diabody as previously described (16). NHS-PEG3400-VS was conjugated to AVP04-07 diabody at a molar ratio of 30:1 and pH 6.0 as previously described (19). The conjugate was reacted with Cysteineamido-DOTA (20) at a molar ratio of 50. N-FMOC-amido-PEG-27-acid was coupled to Cys-polystyrene Wang resin, cleaved and purified by RP-HPLC, reacted with a 10 molar excess of vinyl sulfone and repurified by RP-HPLC. DOTA-PEG-27-Cys-VS was reacted with AVP04-07 diabody at a molar ratio of 50. Synthesis and conjugation of DOTA-PEG12-Cys-VS to diabody at molar ratios of 20:1 and 50:1 were performed as described above.

Radiolabeling of AVP04-07 and its conjugates

Radioiodination of AVP04-07 and its conjugates with ¹²⁵I (Perkin Elmer) was performed using the Iodogen method (21). Na ¹²⁵I (5-10 μL, 26 mBq) was added to 200 μg AVP04-07 in a tube pre-coated with 20 μg Iodogen (Pierce), and incubated at RT for 3 min. Radiolabeling yields were 80-100%. Radiometal labeling of DOTA-AVP04-07 was performed using ¹¹¹InCl₃ (0.15 mBq/μg) or ⁶⁴CuCl₂ (0.37 mBq/μg) as previously described (19). Radiolabeling yields were typically 70-90%. The radiolabeled material was purified on Superdex-75 or 200 columns.

LS-174T Xenograft Model

Female, athymic nu/nu mice (Charles River Laboratories), 6 – 8 weeks old, were injected with LS-174T cells (10⁶) s.c. in the flank. After 10 days mice were injected i.v. with a mixture (200 μl) of 370 kBq of ¹²⁵I- and 150 kBq of ¹¹¹In-labelled AVP04-07 (2-6 μg of total protein) for biodistribution studies. Mice were euthanized at various time points: tumor, blood and major organs were collected, weighed and counted with correction of for crossover of ¹¹¹In counts into the ¹²⁵I channel. Time-activity curves were corrected for radioactive decay and presented as percentage of injected dose/g tissue.

PET imaging

Tumor-bearing mice were injected i.v. with ⁶⁴Cu-labeled diabody or diabody-PEG conjugates and imaged at 1, 4 , 21-22 and 45-46 h with a small-animal PET scanner (microPET Model R4; Siemens/CTIMI, Knoxville, TN). Mice anesthetized with isoflurane, were scanned for 20 min for the 1 and 4 h time points, 45 min at 21-22 h and 60 min at 45-46 h. Data were sorted into two-dimensional sinograms using the Fourier rebinning method and corrected for intrascan radiodecay, detector non-uniformity and random coincidence noise. Images were reconstructed by the iterative ordered subsets expectation maximization (OSEM) method (4 iterations, 16 subsets).

Comparison of Imaging Characteristics Among Diabody and Derivatives

An Imaging Figure of Merit (IFOM) was applied to both to planar and PET images (1, 2, 16). The IFOM at time t after injection is defined as:

$$\begin{array}{cc}\hfill & \mathit{IFOM}\left(t\right)={(-1)}^a{\mathit{CV}}_0{\left(z\right)}^2\epsilon u_T\mathit{exp}(-\lambda t)A_0\mathit{Vol}{[1-1∕r]}^2∕[1+1∕r];\hfill \\ \hfill & r=u_T∕u_B;a=0\text{when}r\ge 1,a=1\text{when}r<1,\hfill \end{array}$$

with CV₀ being the coefficient of variation of the difference in tumor and background counts (z), ε the detector efficiency factor, λ the decay constant, A₀, the total injected activity and Vol the tumor volume. Uptake (%ID/g) was denoted as u_T (tumor) and u_B (background), respectively. In plotting this function, we set the product of CV₀, ε, A₀ and Vol equal to unity. The only sources of variation are tumor uptake, background tissue uptake and physical decay. Note that IFOM(t) < 0 implies that background tissue (blood or kidney) has greater uptake than tumor at time t.

RESULTS

Characterization of AVP04-07 Diabody

AVP04-07 diabody expressed in E. coli and purified by a three-step method eluted as a single species on gel filtration (Suppl Fig 1A) as a monodispersed dimer with an apparent molecular mass of 52.5 kDa by analytical untracentrifugation (Suppl Fig 1B). It had >99% immunoreactivity as judged by a column shift assay (Suppl Fig 1C).

Biodistribution of ¹¹¹In-DOTA- and ¹²⁵I- AVP04-07 in an Athymic Mouse LS-174T Xenograft Model

¹²⁵I-DOTA-AVP04-07 and ¹¹¹In-DOTA-AVP04-07 had similar blood clearances, with about 50% of radiolabel removed by 1h post-injection and 10% in circulation at 4 h (Fig. 1A, B). Kidney uptake was high (100 %ID/g at 24 h) for ¹¹¹In-labeled, but not for ¹²⁵I-labeled diabody, demonstrating that kidney was the major route of clearance. For the ¹¹¹In-labeled diabody there was significant tumor uptake, with over 25 %ID/g observed as early as 4 h post injection and more than 20 %ID/g retained at 48 h. Tumor to blood ratio for ¹¹¹In-AVP04-07 was >50:1 at 24 h. ¹²⁵I-labeled AVP04-07 gave tumor uptakes of 17 and 10 %ID/g at 4 and 48 h respectively.

Figure 1. Biodistributions of unconjugated and DOTA-Cys-VS-PEG3400 conjugated AVP04-07.

Biodistributions were measured in athymic mice bearing LS-174T xenografts as described in Methods. A. ¹¹¹In-labeled non-PEGylated diabody. B. ¹²⁵I-labeled intact diabody. C. ¹¹¹In-labeled PEG3400 conjugated diabody.

Synthesis and biodistribution of DOTA-Cys-VS-PEG3400-AVP04-07

Since PEGylation reduces kidney uptake of radiometal labeled diabodies, we first conjugated AVP04 -07 to DOTA-Cys-VS-PEG3400-NHS as previously described for a anti-CEA diabody (16). VS-PEG3400-NHS was conjugated to surface lysines of the diabody (via active ester), followed by reaction with DOTA-Cys (20). Analysis by SDS and IEF gel electrophoresis indicate an expected shift to lower pI (IEF gel, Suppl Fig 2A, lane 5) as well as a shift to higher apparent molecular size due to the addition of PEG3400 (SDS gel, Suppl Fig 2B, lane 5). The conjugate was radiolabeled with ¹¹¹InCl₃, chromatographed on Superdex 75 and compared to unconjugated AVP04-07 (Suppl Fig. 2A-B). The apparent molecular weight of the PEG3400 derivative was 105 kDa, versus 52.5 kDa. The increase can be attributed to the effect of PEGylation on Stokes radius and is similar to the shift in size we observed for PEG3400 derivatized anti-CEA diabody (16).

The biodistribution of ¹¹¹In-AVP04-07-PEG3400 is shown in Fig 1C. Kidney uptake was 98 %ID/g at 24 h for non-PEGylated diabody (Fig 1A) and 8.4 %ID/g at 24 h for PEGylated diabody (Fig 1C). The large reduction of kidney uptake with PEGylation was accompanied by an increase in tumor uptake from 22 to 46 %ID/g at 24 h and a reduction in tumor/blood ratio (> 46:1 to 2:1 at 24 h). The increase in tumor retention is evidently due to the prolonged blood clearance of PEGylated diabody (t_1/2β = 36 h) versus non-PEGylated diabody (t_1/2β = 18 h). The reduction in kidney uptake was much greater than previously observed with ¹¹¹In-DOTA-Cys-VS-PEG3400 anti-CEA-diabody (16). Possible explanations for the observed differences will be discussed later.

Synthesis and biodistribution of a monodispersed PEG27 AVP04-07conjugate

Although conjugation of AVP04-07 to a PEG3400 moiety achieved reduced kidney uptake, PEG3400 would have major drawbacks as a clinical product, due to its inherent polydispersity and the consequent inability to manufacture products that are easy to characterize chemically. Several monodispersed PEG building blocks are commercially available that can be converted into heterobifunctional reagents using peptide synthesis methodology. Thus, we first synthesized a DOTA-PEG27-Cys-VS derivative as summarized in Suppl Scheme 1. Second, we conjugated DOTA-PEG27-Cys-VS to surface lysines of the diabody at pH 9.0 at a molar ratio of 50:1 (22). When the conjugate was characterized by IEF and SDS gel electrophoresis (Suppl Fig 2), the expected shifts in pI and molecular size were observed. The ¹¹¹In (Fig 2, green trace) and ¹²⁵I radiolabeled products were purified on Superdex 75, the major peak collected and biodistribution studies performed. Inspection of the chromatogram and comparison to non-PEGylated diabody reveal a shift in apparent molecular size.

Figure 2. Size exclusion chromatography of radiolabeled non-PEGylated AVP04-07 diabody and its PEG conjugates.

All samples were radiolabeled with ¹¹¹In and run on a Superdex 75 size exclusion column as described in Methods (radioactivity in arbitrary units). Black: DOTA- non-PEGylated diabody conjugate. Red: DOTA-Cys-VS-PEG3400-diabody conjugate. Green: DOTA-PEG27-Cys-VS-diabody conjugate. Blue: DOTA-PEG12-Cys-VS-diabody conjugate.

Biodistribution results are shown in Fig 3A-B. Despite the lower molecular weight of the PEG27 derivative compared to the PEG3400 derivative, nearly equivalent results were obtained (compare with Fig 1C). At 24 h, kidney uptake of ¹¹¹In-labeled conjugate was 8.3 %ID/g, tumor uptake 49 %ID/g, and the tumor to blood ratio 4.2:1. Given the encouraging reduction in kidney uptake for the PEG27 derivative, we decided to synthesize and test the analogous PEG12 derivative.

Figure 3. Biodistributions of DOTA-PEG27-Cys-VS-AVP04-07 and DOTA-PEG12-Cys-VS-AVP04-07.

Biodistributions were measured in athymic mice bearing LS-174T xenografts as described in Methods. A. ¹¹¹In-labeled PEG27 conjugate. B. ¹²⁵I-labeled PEG27 conjugate. C. ¹¹¹In-labeled PEG12 conjugate. D. ¹²⁵I-labeled PEG12 conjugate.

Synthesis and biodistribution of monodispersed PEG12 AVP04-07

We synthesized DOTA-PEG12-Cys-VS (Suppl Scheme 1) and conjugated it to AVP04-07 at pH 9.0 using molar ratios of 20:1 and 50:1. Unmodified AVP04-07 and the two conjugates were analyzed by high-resolution nanospray mass spectrometry to determine their degrees of substitution (Fig 4A-C). The unmodified diabody gave a series of m/z species that, when deconvoluted, had a calculated mass of 26,869 (Fig 4A), in good agreement with that predicted by the amino acid sequence. For conjugation to DOTA-PEG12-Cys-VS at a molar ratio of 20:1, the deconvoluted peaks all differ from each other by multiples of 1225 mass units (Fig 4B), as expected for conjugation with DOTA-PEG12-Cys-VS. The mass differences are the same for the 50:1 molar ratio conjugation, but are shifted to a higher degree of substitution (Fig 4C). Using peak heights as an estimate, we calculate an average of 1.7 PEGs per monomer for the 20:1 conjugate, and an average of 3.0 PEGs for the 50:1 conjugate. When each conjugate was test labeled with ¹¹¹In (178 mBq/mg), the 20:1 conjugate gave 8.3% incorporation and the 50:1 92% incorporation of radiolabel.

Figure 4. High resolution nanospray mass spectrometry analysis of PEG12 AVP04-07 conjugates.

A. Raw mass spectrum for non-PEGylated diabody (inset: deconvoluted spectrum). B. Deconvolved spectrum for PEG12 conjugate 20:1. C. Deconvolved spectrum for PEG12 conjugate 50:1.

Biodistribution data for ¹¹¹In- and ¹²⁵I-labeled DOTA-PEG12-Cys-VS-AVP04-07 were similar to those for the PEG3400 and PEG27 derivatives (Fig 3C-D). Notably, kidney uptake was initially higher (13.4 %ID/g) for the PEG12 than the PEG27 conjugate, but fell to lower levels (6.1 %ID/g) by 96 h, while tumor uptake and blood clearance are nearly identical for PEG12 and PEG27 conjugates.

⁶⁴Cu PET imaging of DOTA-PEG conjugates of AVP04-07

PET imaging of ⁶⁴Cu-labeled AVP04-07 (Fig 5A) demonstrated relatively modest tumor uptake and very high kidney uptake over 2 days for the non-PEGylated diabody. In contrast, the PEG12 and 27 conjugates showed relatively little kidney uptake throughout and high tumor uptake as early as 21-22h (Fig 5B-C). The measured tumor uptake for PEG12 was 49 ± 3 %ID/g at 46 h with a tumor to blood ratio of (9 ± 4):1. These results are similar to those for the ¹¹¹In-labeled conjugate and suggest that choice of radiometal has little effect on the biodistribution, an observation we made previously for an anti-CEA diabody (16).

Figure 5. Serial PET imaging of ⁶⁴Cu AVP04-07 and PEGylated AVP04-07 in athymic mice peripherally xenografted with TAG-72 expressing LS-174T colon tumors.

Images are PET anterior-view maximum intensity projections (MIPs) normalized to reflect radiodecay-corrected relative image intensity per unit injected activity. Labels in red, green, yellow and turquoise respectively show %ID/g in blood (heart), liver, kidney and tumor as measured by direct assay following the final scan. A: ⁶⁴Cu-AVP04-07. B: ⁶⁴Cu-PEG12 AVP04-07 C: ⁶⁴Cu PEG27 AVP04-07.

Comparative Imaging Characteristics of ⁶⁴Cu- and ¹¹¹In-Labeled AVP04-07and Its DOTA-PEG conjugates

In order to compare different PEGylated versions of AVP04-07 radiolabeled with either ¹¹¹In or ⁶⁴Cu, we examined two parameters- (a) absolute tumor uptake and (b) tumor to non-tumor ratios using an Imaging Figure of Merit (23). The biodistribution data were also replotted on the same graph for comparisons (Suppl Fig 3). The PEGylated versions outperform the non-PEGylated diabody in terms of both absolute tumor uptake and tumor to blood ratios for both ¹¹¹In and ⁶⁴Cu. The IFOM plots presented in Suppl Fig 4 predict best imaging with either the ⁶⁴Cu-labeled non-PEGylated diabody at 10 h or the ⁶⁴Cu-labeled PEG12 version at 20 h. With ¹¹¹In, imaging is optimal with the PEG12 conjugate at 30-40 h. However, when the background tissue is kidney, non-PEGylated diabody shows negative contrast out to 50 h due to high renal uptake and retention of radiolabel. All PEGylated diabodies had similar image quality through 50 h with a slight edge to the PEG27 conjugate when imaged at 10-15 h for ⁶⁴Cu and 20-30 h for ¹¹¹In.

DISCUSSION

The search for an effective radiolabeled antibody based imaging agent has focused on antibody fragments for their high tumor to blood ratios and on PET imaging for its high sensitivity and quantitative results. We chose the diabody construct because it is the smallest bivalent recombinant antibody that can be produced directly in large quantities (24). However, if renal clearance of diabody is too rapid, the absolute tumor uptake will be low. In the case of radiometals such as ⁶⁴Cu where tumor retention is greater than for radioiodine labels, its advantage is offset by prolonged kidney retention. Use of ¹²⁴I (5) avoids the kidney uptake issue due to metabolism and redistribution, but at the expense of lower tumor uptake.

In this study our goal was to optimize the clearance parameters for ⁶⁴Cu which has an appropriate half-life (12 h), comparable to that of the diabody itself. PEGylation of diabody was selected because it has biodistributions similar to whole IgG or F(ab’)2 fragments but has the advantages that it is engineered from a single gene rather than two and does not need to be treated with a protease to reduce its molecular size. We previously approached this problem by conjugating a DOTA-PEG3400 derivative to an anti-CEA diabody, and although kidney uptake was reduced from 200 to 50 %ID/g at 24 h, we were unable to lower it to the levels (10-12 %ID/g at 24-48 h) seen with intact anti-CEA IgG (21, 25). Factors that could explain the remaining high kidney uptake included the pI of the diabody (17, 18), the effect of binding to circulating antigen, or some unforeseen result of the conjugation chemistry. To gain insight into the problem, we selected a different diabody that, like anti-CEA diabody, shows strong tumor targeting in athymic mice bearing LS-174T xenografts. The anti-TAG-72 antibody CC49 and a diabody derivative of CC49 have been previously described (26-29) and CC49 intact IgG has been used in many clinical studies (25, 30, 31), making CC49 a good choice for a comparative study. Using an hexahistidine-tagged, E. coli produced version of the CC49 diabody, namely AVP04-07, we found that an absolute tumor uptake in the LS-174T mouse model at 24 h of 25 %ID/g (Fig 1A), superior to that previously reported for a (scFv)₂ version of CC49 (4 %ID/g) (32).

For purposes of comparison, our first PEG construct (DOTA-Cys-VS-PEG3400) was identical to the one used with anti-CEA diabody (16). DOTA-Cys-VS-PEG3400 is a linear, bifunctional, polydispersed PEG that attaches to amino groups of diabodies via an active ester at one end and to DOTA-Cys via vinyl sulfone at the other end. In contrast to anti-CEA diabody, kidney uptake was significantly lower (10-12 %ID/g) than normally seen for intact IgGs. It is unlikely that the reduction in kidney uptake was due either to the conjugation chemistry or the nature of the PEG agent (identical in both the anti-CEA and anti-CC49 cases); however, the reduction in kidney uptake could be due to either the final pI of the DOTA-PEG-diabody conjugate or binding to circulating antigen. Regarding the pI, both antibodies have a net negative charge after conjugation. Although negatively charged proteins are cleared less rapidly by the kidney compared to positively charged proteins (18), the difference in pIs between the anti-CEA and anti-CC49 diabodies seems insufficient to account for their rather large differences in kidney uptake. Likewise, the role of circulating antigen in the kidney seems an unlikely explanation. While it is well known that CEA is cleared rapidly by Kupffer cells in the liver (33), thus explaining the accumulation of ¹¹¹In-DOTA-anti-CEA IgG in liver (34, 35), there is no obvious connection to the kidney. While less is known about clearance of TAG-72, this high molecular weight acidic mucin is, like CEA, released into circulation and its binding to diabody may be expected to protect the diabody from kidney filtration. The main argument against this idea is that only the PEGylated diabody has reduced kidney accumulation. Thus, further studies will have to be performed to determine the mechanism(s) causing the observed differences between the PEGylated anti-CEA and PEGylated anti-TAG-72 diabodies.

Although PEGylation is an attractive method for reducing kidney accumulation, over-substitution may inhibit immunoreactivity (10-12, 36). In the case of anti-CEA diabody, it was necessary to limit the number of PEG3400s to about one per monomer (16). This fact, plus the polydispersed nature of PEG3400, prompted us to evaluate the effect of smaller DOTA-PEGs on kidney uptake. In the case of DOTA-PEG12-Cys-VS-diabody, the degree of substitution was 3 PEG moieties per monomer as determined by high-resolution nanospray mass spectrometry. Notably, the biodistribution results for ¹¹¹In-labeled PEG12 conjugate were similar to both the PEG3400 and PEG27 conjugates, indicating that even PEGs of modest molecular size are efficient in reducing kidney uptake.

Based on the satisfactory biodistribution results with ¹¹¹In, we went on to demonstrate high quality PET imaging of tumors both with ⁶⁴Cu-labeled PEG12 and PEG27 conjugates by 24 h, a time point that is well matched with the half-lives of both blood clearance and radionuclide.

All of the DOTA-PEG-diabody conjugates had dramatically reduced kidney uptake compared to non-PEGylated diabody and were similar among themselves with regard to biodistribution (Fig 1 and Suppl Fig 3). In order to further refine the comparison among the derivatives, we employed a previously developed Imaging Figure of Merit (4, 16, 23, 37). The analysis indicates modest advantages for the PEG12 conjugate when tumor-to-blood ratio is of primary concern, and for the PEG27 conjugate when tumor-to-kidney contrast is the chief criterion. Based on their excellent tumor and minimal kidney uptakes, we predict that these monodispersed DOTA-PEGylated diabody conjugates will be suitable for human imaging of TAG-72-positive tumors. Further, we suggest that PEGylated diabodies may be an ideal platform for delivering other conjugated payloads to tumor.

Supplementary Material

Fig 1

Supplemental Figure 1. Characterization of AVP04-07 Diabody. AVP04-07 expressed in E. coli was purified to homogeneity as indicated by a single elution peak on Superdex 200 gel filtration (A) and was further shown to be both dimeric and monodispersed by analytical ultracentrifugation (B), with an apparent molecular weight of 52 kDa. AVP04-07 diabody was able to bind antigen in vitro as shown by reduced elution time (i.e.; increases in apparent molecular weight) of diabody-antigen complexes (C, black solid line) when compared to AVP04-07 alone (C, grey dotted line) and antigen alone (C, gray solid line).

Fig 2

Supplemental Figure 2. Isoelectric focusing and SDS gel electrophoresis of AVP04-07 and its PEG conjugates. A. Isoelectric focusing: 1, non-PEGylated diabody; 2, PEG27 conjugate, 50:1 (ratio of PEGylating agent to diabody); 3, PEG12 conjugate, 20:1 (ratio of PEGylating agent to diabody); 4, PEG12 conjugate, 50:1 (ratio of PEGylating agent to diabody); 5, PEG3400 conjugate, 30:1 (ratio of PEGylating agent to diabody). B. SDS gel electrophoresis: 1, intact diabody; 2, PEG27 conjugate; 3, PEG12 conjugate; 4, PEG12 conjugate; 5, PEG3400 conjugate.

Fig 3

Supplemental Figure 3. Comparative blood, tumor, kidney and liver curves for ¹¹¹In-DOTA labeled PEGylated diabody derivatives and intact diabody. A. Blood clearance curves. B. Tumor uptake curves. C. Kidney uptake curves. D. Liver uptake curves. Red: intact diabody. Magenta: PEG3400 diabody conjugate, 15:1 molar ratio. Black: PEG3400 diabody conjugate, 60:1 molar ratio. Blue: PEG27 diabody conjugate. Green: PEG12 diabody conjugate.

Fig 4

Supplemental Fig 4. IFOM plots for tumor vs. blood and tumor vs. kidney for non-PEGylated diabody and its PEGylated conjugates. A. Tumor vs. Blood, ⁶⁴Cu-labeled AVP04-07 and its derivatives. B. Tumor vs. kidney, ⁶⁴Cu-labeled AVP04-07 and its derivatives. C. Tumor vs. blood, ¹¹¹In-labeled AVP04-07 and its derivatives. D. Tumor vs. kidney, ¹¹¹In-labeled AVP04-07 and its derivatives. Dark Blue: unmodified diabody. Light Blue: PEG3400 diabody conjugate. Yellow: PEG27 diabody conjugate. Magenta: PEG12 diabody conjugate.

Scheme 1

Supplemental Scheme 1. Synthesis of DOTA-PEG-Cys-VS. FMOC-amido-PEG-acid was conjugated to S-t-butyl cysteine on Wang resin using standard activation chemistry (DCC/HOBt). The FMOC was removed with piperazine and conjugated to DO3AtBuAc using standard activation chemistry (DCC/HOBt). The product was removed from the resin with TFA, purified by reverse phase HPLC, reacted with excess vinyl sulfone in DMF, and repurified by reverse phase HPLC.