Synthesis and Evaluation of Astatinated N-[2-(Maleimido) ethyl]-3

Article pubs.acs.org/bc

Synthesis and Evaluation of Astatinated N‑[2-(Maleimido)ethyl]-3(trimethylstannyl)benzamide Immunoconjugates Emma Aneheim,*,† Anna Gustafsson,† Per Albertsson,‡ Tom Bac̈ k,† Holger Jensen,§ Stig Palm,† Sofia Svedhem,∥ and Sture Lindegren† †

Department of Radiation Physics, Gothenburg University, Gula Stråket 2B, 41345 Gothenburg, Sweden Department of Oncology, Gothenburg University, 41345 Gothenburg, Sweden § PET and Cyclotron Unit, KF3982, Copenhagen University Hospital, DK-2100 Copenhagen, Denmark ∥ Department of Applied Physics, Chalmers University of Technology, 41296, Gothenburg, Sweden ‡

S Supporting Information *

ABSTRACT: Effective treatment of metastasis is a great challenge in the treatment of different types of cancers. Targeted alpha therapy utilizes the short tissue range (50−100 μm) of α particles, making the method suitable for treatment of disseminated occult cancers in the form of microtumors or even single cancer cells. A promising radioactive nuclide for this type of therapy is astatine-211. Astatine-211 attached to tumorspecific antibodies as carrier molecules is a system currently under investigation for use in targeted alpha therapy. In the common radiolabeling procedure, astatine is coupled to the antibody arbitrarily on lysine residues. By instead coupling astatine to disulfide bridges in the antibody structure, the immunoreactivity of the antibody conjugates could possibly be increased. Here, the disulfide-based conjugation was performed using a new coupling reagent, maleimidoethyl 3-(trimethylstannyl)benzamide (MSB), and evaluated for chemical stability in vitro. The immunoconjugates were subsequently astatinated, resulting in both high radiochemical yield and high specific activity. The MSB-conjugate was shown to be stable with a long shelf life prior to the astatination. In a comparison of the in vivo distribution of the new immunoconjugate with other tin-based immunoconjugates in tumor-bearing mice, the MSB conjugation method was found to be a viable option for successful astatine labeling of different monoclonal antibodies.

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character5 prevents it from being attached directly to tyrosine residues, as is iodine. Instead, other types of intermediate linker molecules must be used, such as boron cage structures, nanoparticles, or organic tin derivatives.6−8 One of the more commonly used linker molecules for astatination, N-succinimidyl-3-(trimethylstannyl)benzoate (ATE), employs a reactive trimethyl stannyl group that can be substituted by astatine (Figure 1). In labeling of antibodies and other proteins, the ATE derivatives typically react with lysine residues and hence the modification will become randomly distributed on the surface of the antibody. If the modified lysines are located in or near the antigen binding sites, the immunoreactivity of the antibody may be compromised. Several different methods exist in order to perform labeling distant from the antigen binding site, such as glycosylation and targeting engineered protein scaffolds. One of the most straightforward methods is, however, to target interchain disulfide bridges in the hinge region.9,10 In previous work, we have reported the synthesis and characterization of a new coupling reagent, N-[2-(maleimido)ethyl]-3-

INTRODUCTION Targeted radionuclide therapy with α-emitting nuclides is emerging as a promising option for treatment of microscopic tumors and single cells in disseminated cancer.1 The α emitters have several advantageous physical properties, including a short particle tissue range (50−100 μm), high energy, and consequently high linear energy transfer (LET).2 However, most α-emitting radionuclides are not tumor-specific in vivo and therefore they require a tumor-specific carrier molecule to guide the radiation to the tumor cells. Although the number of available tumor-specific carrier vectors, such as antibodies or peptides, has steadily increased, only a few α-emitting nuclides with suitable physical properties for this type of therapy exist: radium-223, bismuth-213, bismuth-212/lead-212, thorium-227, actinium-225, and astatine-211 (At-211).3−5 Among these, At-211 is promising due to its physical and chemical characteristics, being a halogen with a 7.2 h half-life and 100% α emission in its total decay. In contrast to metallic nuclides such as bismuth, thorium, and actinium, astatine cannot be attached to the biochemical carriers using standard metal chelating agents such as the polydentate oxygen−nitrogen donors DOTA, NOTA, or CHXDTPA. Although astatine is a halogen, its greater metallic © XXXX American Chemical Society

Received: December 11, 2015 Revised: January 19, 2016

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DOI: 10.1021/acs.bioconjchem.5b00664 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry

Figure 1. Top: Reaction between N-[2-(maleimido)ethyl]-3-(trimethylstannyl)benzamide (MSB) and cysteine groups on antibodies (R1) with subsequent astatination. Bottom: Reaction between N-succinimidyl-3-(trimethylstannyl)benzoate (ATE) and lysine groups on antibodies (R2) with subsequent astatination.

immunoconjugates was also compared with traditional ATE immunoconjugates in tumor bearing nude mice.

(trimethylstannyl)benzamide (MSB), for site-selective radiohalogenation of proteins.11 This reagent uses the same leaving group for electrophilic aromatic substitution with astatine as ATE, but reacts with cysteine instead of lysine residues on the biomolecules (Figure 1). The cysteine-cysteine disulfide bridges available for modification are all located in the specific hinge region of the antibody.12 These disulfide bridges in antibodies can be astatinated using MSB, after a mild reduction.12,13 This type of reduction can, however, cause extensive modifications of the antibody if not performed properly. It is therefore important to verify the structural integrity and the retained interaction properties of the formed immunoconjugates after conjugation and labeling. Maleimide-containing molecules similar to MSB have previously been reported for use as iodination reagents of antibodies or fragments thereof with good results.14,15 Also peptides have been successfully iodinated with molecules similar to MSB,16,17 indicating that it could be possible to use MSB for astatination also in such a context. All the iodination studies of maleimide derivatives similar to MSB14−17 have, however, been using an indirect radiolabeling approach, compared to the present study where a direct approach has been used.18 In the indirect approach the linker molecule is radiolabeled before being attached to the protein or peptide, while in the direct approach the bioconjugate is produced before radiolabeling. The direct approach reduces the number of reaction steps and hence also the reaction time including the radioactive nuclide, which minimizes dose to the immunoconjugate solution. In labeling with α particle emitting radionuclides, it is important to keep the absorbed dose in the reaction mixture low, as high-LET radiation can be detrimental for the chemistry, i.e. destroy precursors and products.19 Antibodies have been shown to not tolerate a dose larger than 1000 Gy, while smaller molecules are less sensitive.20 In this work, we present the first investigation of the MSB coupling reagent with respect to the synthesis of immunoconjugates with two different antibodies (trastuzumab and MX35) and their subsequent radiolabeling with astatine. The immunoconjugates were characterized with respect to the number of MSB linkers per antibody, and the radiochemical yield and specific activity for the astatination reaction were evaluated. The in vitro stability of the immunoconjugates before and after astatination and the influence of conjugation on the affinity toward antigen epitopes and intact cells have been assessed. The in vivo distribution of astatinated MSB-

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RESULTS AND DISCUSSION Chemistry. MSB Conjugation and Astatine Labeling. Immunoconjugates with MSB were readily prepared according to the protocol described in Materials and Methods. Using a 10-fold molar excess of MSB to antibody, a yield of 5.8 ± 0.5 MSB linkers per antibody was achieved, determined by analyzing the amount of tin on the conjugate, compared to the added amount. This means that about 6 of the possibly 32 sulfhydryl groups of the IgG1 antibody were modified with the linker. Of the 32 sulfhydryl groups, 8 originate from interchain disulfide bridges, which are exposed to solvent and hence the most easily available for modification. These sulfide groups are thus selectively targeted when performing conjugation with maleimide derivatives after DTT reduction.12,9 The measured number of MSB linkers per antibody was similar to the number of ATE linkers per antibody (6.7 ± 2.4) achieved with the direct ATE conjugation protocol.21 The MSB-immunoconjugates of trastuzumab were labeled with astatine-211 (5−30 MBq), according to Figure 2. An average recovery of 77.2 ± 3.1% (no decay correction) was obtained after size exclusion chromatography purification. The radiochemical purity was generally >98%, resulting in an average radiochemical yield of 76.4 ± 3.1%. It should be noted

Figure 2. Antibody reduction, conjugation with N-[2-(maleimido)ethyl]-3-(trimethylstannyl)benzamide, and astatine labeling of the resulting immunoconjugate. B


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PAGE) was also conducted. The native-PAGE analysis showed a similar migration for the unmodified antibody compared to the MSB conjugated one (Figure S3). As the electrophoretic mobility in native-PAGE depends not only on the charge-tomass ratio but also on the physical shape and size of the protein, this indicates a maintained protein structure upon conjugation. The small difference in retention time could be attributed to the slight difference in charge density between the antibody and the immunoconjugate. In addition, SDS−PAGE analyses (under both reducing and nonreducing conditions) were conducted, but as the denaturation of the protein caused the conjugated antibody to degrade, no useful data were retrieved from these studies. This degradation was most likely triggered by the denaturation process disrupting the chemical interactions that maintain protein conformation after cleavage of the disulfide bridges in the hinge region. For further structural characterization of the immunoconjugate, conformation differential scanning fluorimetry (DSF) measurements were conducted, comparing the nonmodified antibody with the MSB-conjugated antibody (Figure S4). The results showed a similar, single step unfolding behavior without DTT addition, indicating a well-folded protein in both cases. Upon DTT addition a likewise similar double peak unfolding/melting was shown, indicating remaining disulfide bridges being reduced in both the conjugated and unmodified antibody. However, when comparing the final melting point without DTT addition, the MSB-conjugated antibody displayed a higher melting point than the unmodified version (74 and 68 °C respectively), indicating increased protein stability upon MSB modification. This could possibly be attributed to increased stabilization through hydrogen bonding and/or van der Waals forces introduced to the antibody with the MSB molecule. The produced MSB-immunoconjugate in PBS (pH 7.4) was successfully stored for several weeks prior to radiolabeling without any decomposition into fragments, with maintained structural integrity (shown by FPLC and native-PAGE) and without a decrease in labeling performance. The stability of the MSB immunoconjugates was expected according to previous results obtained for ATE-like reagents.21 High radiochemical yields and high specific activities (see Table 1) were obtained using MSB immunoconjugates after refrigerated storage between 12 and 44 days (in air at +4 °C). In addition, the MSB immunoconjugate could be lyophilized and redissolved in buffer prior to successful astatine labeling without any decrease in immunoreactivity (expressed as the immunoreactive fraction, IRF) as compared to a reference immunoconjugate stored in the refrigerator (IRF(Lyophilized) = 0.93; IRF(Reference) = 0.86). The good shelf life of the MSB immunoconjugates and the possibility of lyophilization suggest the possibility of using MSB immunoconjugates prepared in advance of radiolabeling as a kitlike reagent. This approach would be possible with the recently developed direct labeling method for astatine,18 which was employed throughout this work. A kitlike approach is particularly desirable when it comes to the use of astatine, allowing clinical labeling of At-211 even at remote hospitals despite the few cyclotron production facilities that are currently available.27 The astatinated immunoconjugate also displayed chemical stability of the astatine bond in human serum albumin (HSA) at 37 °C, maintaining a radiochemical purity of 99 ± 1% throughout the 48 h duration of the experiment. In Vitro and In Vivo Binding Properties. Immunoreactivity. By reduction of existing disulfide bridges on the

that MSB also allows for the preparation of MSBimmunoconjugates with high specific activity, >0.7 MBq/μg (see Table 1), equaling one or more astatine atoms per 150 Table 1. Results from (1) High Specific Activity Astatinations of Immunoconjugates of MX35 or Trastuzumab with N-[2-(Maleimido)ethyl]-3(trimethylstannyl)benzamide and (2) Astatination of Aged of Immunoconjugates of Trastuzumab and N-[2(Maleimido)ethyl]-3-(trimethylstannyl)benzamide Stored at Different Periods of Time (+4 °C, Air) specific activity (MBq/μg) 0.77 0.70 0.46 0.35

antibody

radiochemical purity (%)

labeling yield (%)

immunoconjugate age (days)

trastuzumab MX35 trastuzumab trastuzumab

98 96 99 99

69 72 79 76

44 1 16 12

antibody molecules. High specific activity labeling is often a prerequisite for efficient therapy of microtumors with αemitters, e.g., in an intraperitoneal setting.22 Other radioimmunotherapies with β-emitters exist where predosing with unlabeled antibody or the use of higher quantities of antibody results in better tissue distribution and tumor uptake.23−25 Whether such therapies can be improved by using α-emitters remains to be seen. Even if α-emitters may not display maximum potential in a low specific activity setting, it is, however, always possible to decrease an already high specific activity, while the opposite is impossible if the labeling method is not good enough. The results obtained for the MSBimmunoconjugates were similar to those for astatination of ATE-immunoconjugates using the same labeling procedure.18 As the trimethyl tin groups on MSB can be toxic, the extent of tin group removal by N-iodosuccinimide (NIS) was analyzed with ICP-MS. It was found that only 16.8 ± 1.5% of the tin groups remained after NIS treatment, corresponding to ∼1 per antibody. In a future patient treated with 1 GBq of At-211 with a specific activity of 0.5 GBq/mg of antibody, this corresponds to ∼13 nmol of trimethyl tin, which is >25 000 times lower than the toxic level of triethyl tin in humans, which in turn is more toxic than trimethyl tin.26 Characterization and Stability. The exposure of antibodies to reducing agents carries the risk of both decomposition of the antibody and the formation of oligomeric species through recombination of reduced disulfide bridges from different antibodies. No decomposition of the antibody (MX35 or trastuzumab) into larger protein fragments or oligomer formation could be found after conjugation with MSB and the subsequent astatination. This was determined by FPLC measurements using a size exclusion column with sample collection for activity measurements. The single peak chromatograms displayed nearly identical retention times for both unmodified antibodies and antibody conjugates (25.7 ± 0.9 min for trastuzumab and MX35 conjugates, Figure S1 in Supporting Information). To validate the FPLC results, an antibody sample (150 kDa) spiked with a F(ab)2 fragment (100 kDa) of the same antibody was analyzed. The chromatogram in this case clearly showed two partly separated peaks (Figure S2). No tendency of such a double peak could be seen for the MSB immunoconjugate. In order to further investigate the structural integrity of the MSB-immunoconjugate, nonreducing nondenaturing polyacrylamide gradient gel electrophoresis (nativeC


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Table 2. Fitted Dissociation Rate Constants (kd) and Equilibrium Constants (KD) with Uncertainties (χ2) for the Interactions between the Immobilized Peptide Epitope and Unmodified MX35, as Well as the MX35 Immunoconjugates with ATE and MSB Using Two Different Interaction Models Langmuir (1:1) −1

kd (s ) MX35 unmodified MX35-ATE MX35-MSB

bivalent analyte (1:2) χ

2

KD (M) −4

3.39 × 10 3.04 × 10−4 1.14 × 10−4

−9

4.06 × 10 4.04 × 10−9 1.99 × 10−9

12.8 11.6 9.57

−1

kd1 (s ) −4

8.56 × 10 7.7 × 10−4 2.13 × 10−4

kd2 (s−1)

χ2

7.9 4.41 0.34

1.59 1.4 1.47

Figure 3. Interaction analysis using SPR-based sensing of MX35 and MX35 conjugated with both ATE and MSB (5, 10, 3 × 20, 40, and 80 nM), using 100 RU of immobilized peptide (the chip was regenerated with 10 mM glycine (pH 2.3) and 10 mM NaOH between runs). Fitted curves resulting from Langmuir and bivalent fit interaction models are displayed for the highest concentration (80 nM) of ATE- and MSB-conjugated MX35.

as low as possible. The difference in KD was caused by the slower kd for the MSB-immunoconjugate (Figure 3). The lower value of the equilibrium constant, which indicates a stronger binding to the peptide epitope for the MSB-immunoconjugate compared to the ATE counterpart, does not, as such, mean an improved in vivo behavior. In fact, the unmodified antibody and the ATE-immunoconjugate interact similarly with the immobilized peptide, indicating a better preserved immunoreactivity. The higher affinity for the interaction between the MSBimmunoconjugate and the peptide hence signals a difference in binding rather than an improvement, likely due to slight structural changes induced by modification of the disulfide bridges. In vitro cell assays with astatinated immunoconjugates using both trastuzumab and SKOV-3 cells and MX35 and OVCAR-3 cells (Figure 4) were performed to investigate whether the new conjugation method provided a better antigen interaction compared to the regular method where lysine is targeted. There was no marked difference between the two methods, since the behaviors of the two different astatinated immunoconjugates were very similar, as shown by the immunoreactive fraction values obtained through nonlinear regression analyses: IRFATE‑Trastuzumab = 1.05 ± 0.08, IRFMSB‑Trastuzumab = 1.02 ± 0.07, IRFATE‑MX35 = 0.79 ± 0.01 IRFMSB‑MX35 = 0.83 ± 0.01 (uncertainties derived from the data fitting). Biodistribution. Direct comparisons of the organ biodistribution between ATE- and MSB-immunoconjugates (MX35) labeled with astatine were performed on tumor-bearing nude

antibody, thiol groups become accessible for reagent (MSB) modification. Disulfide bond reduction allows modification that is distant from the antibody binding regions. Thus, MSB modification could potentially better preserve the immunoreactivity as compared to the use of reagents that result in more randomly distributed modifications, e.g., succinimide derivatives, which could lead to modification of residues in the antigen-binding region. To evaluate the interaction properties of the MSB-immunoconjugate, biosensing methodology based on surface plasmon resonance (SPR) was employed to compare the MSB- and ATE-immunoconjugates. SPR-based interaction analysis provides label-free, real-time monitoring of biomolecular interactions, thereby permitting the study of interaction kinetics and equilibria.28,29 The interaction analysis of the OVCAR-3-specific murine antibody MX35 and an antigen-specific peptide epitope was used to calculate affinity constants and to compare the two different immunoconjugates to the unmodified antibody. The obtained KD values were in the same nanomolar range as previous results using saturation curve cell-assays for ATEmodified MX35.18 However, a difference in KD between the two different immunoconjugates of a factor 2 was observed when using immobilized peptide epitope and a 1:1 Langmuir binding model for evaluation (Table 2). The same behavior and a better curve fit were obtained when using a bivalent binding model (Table 2, Figure 3). This could be a result of too close immobilization of the small peptide epitopes on the chip surface, despite the efforts to keep the level of immobilization D


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bond within the maleimide linker molecule and that this destabilization of the conjugate was increased in blood due to the lack of antibody−antigen binding. The MSB molecules, however, do not contain any such ester bond, and the stability test in HSA with methanol precipitation confirms that there was no increased presence of free activity or activity connected to small molecules or protein fragments of 70 cycles (standard deviation at 3 × 20 nM < 1%). Double referencing was used to correct for experimental artifacts during sample injection. Cell Assays To Determine Immunoreactivity. Trastuzumab (IgG1) is ErbB2 (Her2) specific, and hence a Her2 expressing human tumor cell line, SKOV-3, was used for the trastuzumab experiments. MX35 (IgG1) targets the membrane transporter NaPi2b (SLC34A2) in human carcinomas, and hence the human tumor cell line OVCAR-3 was used for MX35 experiments. Both SKOV-3 and OVCAR-3 were obtained from ATTC (Rockville, MD, USA). The cell culture conditions were in both cases 10% FBS (fetal bovine serum), 1% L-glutamine, and 1% PEST (penicillin and streptomycin) in RPMI media for OVCAR-3 and McCoy’s media for SKOV-3 (Invitrogen, Life Technologies). Both cultures were adherent, fed twice a week, and split once a week. The immunoreactive fractions of astatinated ATE- and MSBimmunoconjugates were determined using a serially diluted (1:2, six times) single-cell suspension of SKOV-3 or OVCAR-3 cells (5 × 106 cells/mL). The cell samples were prepared in duplicate for each immunoconjugate, and a constant amount (5 ng) of 211At-labeled immunoconjugate was added to every sample. The reactions were allowed to proceed for 180 min under gentle agitation. The cells were then pelleted via centrifugation (3500 rpm, 5 min) and subsequently washed with PBS. The bound immunoconjugate fraction of each sample was determined by comparing the total radioactivity (T) added to each sample (determined through reference samples) with the activity associated with the cells (B). The immunoreactive fraction (Bmax) was then determined by nonlinear regression analysis of the average data points from the duplicate series. Immunoconjugate Biodistribution. Following 1 week of acclimatization, nude mice (BALB/c, 4 weeks old, n = 26) were inoculated with 1 × 107 OVCAR-3 cells via subcutaneous double sided injection in the scapular region. After 4 weeks, when tumors had established, 24 of the animals were divided in two groups and given intravenous treatment with two different 211 At-labeled immunoconjugates. Group A was treated with astatinated ATE-immunoconjugates of MX35 and group B was treated with astatinated MSB-immunoconjugates of the same antibody. Each mouse was given 700 kBq of astatine in 150 μL of solution with a specific activity of 0.70 GBq/mg. The mice were then sacrificed (by cervical dislocation) in groups of four animals and at three time-points (1, 5, and 25 h). No thyroid blocking was conducted in order to use thyroid uptake as an indicator of the presence of free astatine. The mice were dissected and tumors and normal tissues (blood, salivary glands, neck (thyroid), lungs, stomach, (small intestine), spleen, liver, kidneys, heart, muscle, tumor, and bone marrow (first two time points only)) were removed, weighed, and measured for activity content. The organ samples were measured using a NaI(Tl) γ-counter (Wizard 1480, Wallac, Finland). One kidney per animal for the first two time points was also removed specifically for α camera analysis. Analysis of Immunoconjugate Stability in Serum. Astatinated MSB-immunoconjugates of MX35 (produced as described above) in PBS (100 μL) were added to human serum H


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Bioconjugate Chemistry derived from blood (1 mL) and stored in 37 °C. After 20.75, 21.22, and 21.93 h, the samples were removed from the heat, diluted 1:4 with binding buffer (Thermo Scientific Protein A IgG binding buffer), and added to a 1 mL Protein A column pre-equilibrated with binding buffer. The activity of the samples was measured before addition to the column. The column was washed with 7−10 column volumes of binding buffer, and the washing solution was collected. Subsequently, 5−6 column volumes of elution buffer (Thermo Scientific Protein A IgG elution buffer) were allowed to pass the column and the eluate was collected. The empty sample vial, the washing solution, and the eluate were measured for activity content with a NaI(Tl) detector. The radiochemical purity of the serum samples was also measured using methanol precipitation as described before. As a reference, fresh astatinated MSB-immunoconjugates of MX35 and astatinated ATE-Fab2 fragments of MX35, both added to HSA, were allowed to pass the column in the same way to observe the activity retentions.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.5b00664. Additional characterization data; FPLC chromatograms, native PAGE gel photograph and DSF measurements. Tables of complete biodistribution data (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +46313429736. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by grants from the Swedish Research Council (Grant K 2015-99X-22704-01-5), the King Gustaf V Jubilee Clinic Cancer Research Foundation in Göteborg, Sweden (Grant 234/14), the Västra Götalands Regional Agreement on Medical Training and Clinical Research (ALF) (Grant 165261), and the Swedish Cancer Society (Grants 2015/450, 2013/642, and 2013/501).

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REFERENCES

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