Synthesis of Phosphine and Antibody–Azide Probes for in Vivo

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Synthesis of Phosphine and Antibody Azide Probes for in Vivo Staudinger Ligation in a Pretargeted Imaging and Therapy Approach Danielle J. Vugts,*,†,‡ Annelies Vervoort,†,‡ Marijke Stigter-van Walsum,† Gerard W. M. Visser,‡ Marc S. Robillard,§ Ron M. Versteegen,|| Roland C. M. Vulders,§ J. (Koos) D. M. Herscheid,‡ and Guus A. M. S. van Dongen†,‡ Department of Otolaryngology/Head and Neck Surgery, ‡Department of Nuclear Medicine and PET Research, VU University Medical Center, Amsterdam, The Netherlands § Biomolecular Engineering, Philips Research, Eindhoven, The Netherlands SyMO-Chem BV, Eindhoven, The Netherlands

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bS Supporting Information ABSTRACT: The application of intact monoclonal antibodies (mAbs) as targeting agents in nuclear imaging and radioimmunotherapy is hampered by the slow pharmacokinetics of these molecules. Pretargeting with mAbs could be beneficial to reduce the radiation burden to the patient, while using the excellent targeting capacity of the mAbs. In this study, we evaluated the applicability of the Staudinger ligation as pretargeting strategy using an antibody azide conjugate as tumor-targeting molecule in combination with a small phosphine-containing imaging/ therapeutic probe. Up to 8 triazide molecules were attached to the antibody without seriously affecting its immunoreactivity, pharmacokinetics, and tumor uptake in tumor bearing nude mice. In addition, two 89Zr- and 67/68Ga-labeled desferrioxamine (DFO)phosphines, a 177Lu-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-phosphine and a 123I-cubyl phosphine probe were synthesized and characterized for their pharmacokinetic behavior in nude mice. With respect to the phosphine probes, blood levels at 30 min after injection were 80% intact. No in vivo Staudinger ligation was observed in a mouse model after injection of 500 μg antibody azide, followed by 68 μg DFO-phosphine at t = 2 h, and evaluation in blood at t = 7 h. To explain negative results in mice, Staudinger ligation was performed in vitro in mouse serum. Under these conditions, a side product with the phosphine was formed and ligation efficiency was severely reduced. It is concluded that in vivo application of the Staudinger ligation in a pretargeting approach in mice is not feasible, since this ligation reaction is not bioorthogonal and efficient enough. Slow reaction kinetics will also severely restrict the applicability of Staudinger ligation in humans.

’ INTRODUCTION Discovery of molecular targets on, e.g., cancer cells, has boosted the development of targeting agents. mAbs especially are gaining momentum for use in diagnosis and disease-selective therapy. At present, 5 mAbs have been approved by the FDA for diagnosis and 28 mAbs for therapy, either in naked form or conjugated to a toxic drug or radionuclide. Most of the approved therapeutic mAbs are intact immunoglobulins, and the majority are used for systemic treatment of cancer. In addition, hundreds of new mAbs are under development worldwide. Since intact mAbs have slow kinetics, it takes 3 6 days before optimal tumorto-normal tissue ratios are obtained. When intact mAbs are used for nuclear imaging, imaging on the day of injection and at low radiation burden is virtually impossible, because low tumor uptake may be masked by high blood-pool activity. Furthermore, when mAbs are used for delivery of toxic agents like in r 2011 American Chemical Society

radioimmunotherapy, highly radiosensitive bone marrow will be exposed continuously to a large fraction of the injected radioactivity resulting in dose-limiting myelosuppression. Clearly, achieving high tumor-to-blood ratios within a short time period after injection would provide benefit for both imaging and therapeutic applications. Strategies to prevent excessive exposure of normal tissues to toxin mAb or radionuclide mAb conjugates include the use of antibody fragments,1 cleavable linkers,2,3 local delivery,4 antibody-directed enzyme prodrugs (ADEPT),5 and pretargeting.6 11 Several pretargeting approaches are under investigation: (strept)avidin biotin,12 14 hapten/antibody,15 and DNA/DNA interactions.16,17 All these pretargeting strategies Received: June 8, 2011 Revised: August 19, 2011 Published: August 22, 2011 2072

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Scheme 1. Schematic Presentation of a mAb-Based Pretargeting Strategy Using the Staudinger Ligation

are based on biological interactions and suffer from several disadvantages, like the endogenous nature of the pretargeting ligand, the immunogenicity of the recognizing moiety, the large size of the recognizing moiety preventing fast imaging and effective therapy, and the weak binding of the interacting molecules. Using a covalent, nonbiological recognition system in a pretargeting approach could circumvent most of these drawbacks. In the present study, we evaluated the possibility to use the Staudinger ligation18 23 as the strategy for covalent conjugation in a pretargeting approach. The Staudinger ligation is a bioorthogonal reaction between a phosphine and an azide resulting in a covalent adduct with N2 and methanol as byproducts. Next to the Staudinger ligation, other bioorthogonal reactions,24 26 that might be applied in biological systems, are the ringstrain promoted azide alkyne cycloaddition27 30 and tetrazine ligation,31 34 as published during the course of our studies. To utilize the Staudinger ligation in a pretargeting strategy, a mAb needs to be functionalized with an azide handle. This azide handle should be relatively small and not alter the targeting properties of the mAb. The pretargeted mAb azide will accumulate in target tissue by binding to a disease-specific antigen, while unbound mAb azide will be cleared from the blood via the liver and spleen. Next, a small phosphine-containing diagnostic or therapeutic probe can be injected, which reacts covalently with the azide handle on the mAb (Scheme 1). In this paper, we report on the coupling of triazide 1a (Figure 1) to the anti-CD44v6 chimeric mAb (cmAb) U36 and the determination of the optimal triazide-to-mAb molar ratio for in vivo tumor targeting. To get a variety of imaging and therapeutic possibilities, DOTA and DFO-based phosphine derivatives were synthesized. DOTA can be used for labeling with Gd (MRI), 177Lu (SPECT/therapeutic), 111In (SPECT), 90 Y (therapeutic), and 67/68Ga (SPECT/PET) whereas DFO can be used for labeling with 89Zr (PET), 67/68Ga, and 90Nb (PET). In addition, an iodine-cubyl-phosphine was developed, which can be labeled with different PET, SPECT, or therapeutic iodine radionuclides. 89Zr- and 67/68Ga-DFO-phosphines 2 and 3, 177 Lu-DOTA-phosphine 4, and 123I-cubyl-phosphine 5 were prepared (Figure 1) and evaluated for their in vitro and in vivo ligation characteristics.

’ EXPERIMENTAL PROCEDURES General Methods and Materials. The characteristics of the head and neck squamous cell carcinoma (HNSCC) cell lines HNX-OE and 11B, as well as the selection, production, and characterization of cmAb U36, have been described before.35 cmAb U36 binds to the v6 region of CD44 (CD44v6), and is capable of selective tumor targeting of HNSCC as demonstrated in clinical trials.36 131I (66.4 GBq/mL in 0.1 M NaOH) was obtained from Perkin-Elmer. The syntheses of triazide 1a and phosphines 2 5 are described in the Supporting Information. Dialysis after reaction of triazide 1a with cmAb U36 was performed with a Slide-a-Lyzer cassette from Pierce Biotechnology (cutoff 20 kDa) against PBS. mAb concentrations were measured by using the bicinchoninic acid (BCA) assay (Pierce) (UV-meter: Anthos 2001; data-analysis: Microwin) according to the suppliers instructions using naked cmAb U36 as a standard protein. HPLC analysis of antibody modification, 131I-radiolabeled cmAb U36, antibody-Staudinger product stability, and in vivo Staudinger ligation were performed using a Jasco HPLC system equipped with a Superdex 200 10/30 GL size exclusion column (GE Healthcare Life sciences) using a mixture of 0.05 M sodium phosphate, 0.15 M sodium chloride (pH 6.8), and 0.01 M NaN3 as the eluent at a flow rate of 0.5 mL/min. The radioactivity of the eluate was monitored using an inline NaI(Tl) radiodetector (Raytest Sockett). The ratios of triazide/cmAb U36 and phosphine/cmAb U36-triazide represent an average number of triazides or phosphines per mAb molecule, assuming Poisson distribution as generally observed in these types of mAb modifications.37 Instant thin layer chromatography (iTLC) analysis of radiolabeled antibodies was carried out on silica impregnated glass fiber sheets (Pall Corp., East Hills, NY) using 20 mM citrate buffer (pH 5.0) as the mobile phase. Purification and isolation of 131I-labeled cmAb U36-triazide conjugates and of cmAb U36Staudinger product were performed with PD10 columns (GE Healthcare Life Sciences, NJ, USA). Gel electrophoresis was performed on a Phastgel system (Pharmacia Biotech, Amersham Biosciences) using preformed 7.5% SDS-PAGE gels under nonreducing conditions, followed by phosphor imager analysis (B&L-Isogen Service Laboratory). In vitro binding characteristics of the 131I-cmAb U36-triazide conjugates were determined in an immunoreactivity assay essentially as described by Lindmo et al.,38 using a serial dilution 2073

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Figure 1. Selected pretargeting components for the Staudinger ligation as used in this study: triazide 1 for coupling to a mAb; DFO-phosphines 2 and 3 (for radiolabeling with 89Zr and 67/68Ga), DOTA-phosphine 4 (for radiolabeling with 177Lu), and 123I-cubyl phosphine 5 as phosphine probes.

of 0.2% glutaraldehyde-fixed 11B cells and a fixed amount of 131Ilabeled cmAb U36-triazide conjugate (95 ng). After overnight incubation at 4 °C, the cell suspension was centrifuged and the specific binding calculated as the ratio of cell-bound radioactivity to the total amount of applied radioactivity. This was corrected for nonspecific binding, as determined with a 500-fold excess of nonradioactive U36-triazide. All binding assays were performed in triplicate. Single isotope counting was performed with a γ-well counter (Wallac LKB-CompuGamma 1282; Pharmacia) for 89Zr, 67 Ga, 68Ga, 123I, 131I, and 177Lu. cmAb U36 Modification with Triazide 1a and 131I-Radiolabeling. cmAb U36 (4 mg; 11.53 mg/mL) was mixed with PBS (600 μL) and 1 M carbonate buffer pH 9.6 (30 μL) after which triazide 1a (10, 15, 20, or 40 equiv) in DMF (20 μL) was added and incubated for 30 min at room temperature. The cmAb U36-triazide products were purified by dialysis until all unconjugated triazide was removed (assessed by HPLC-monitoring at 260 nm). After dialysis, the concentration of the cmAb U36-triazide product was determined by BCA assay. The average number of triazide molecules attached per mAb molecule was determined by HPLC using the UV absorption of triazide 1b at 260 nm of the crude conjugation mixture. Next to the conjugation reaction, a mock reaction was performed without cmAb U36 resulting in fully hydrolyzed triazide 1b.

The difference in area corresponds to the amount of triazide 1 that was attached to cmAb U36 (see Supporting Information). 131 I-labeling of cmAb U36-triazide conjugates was done according to Tijink et al.39 and is described in the Supporting Information. Radiochemical purity was determined by iTLC analysis, mAb integrity by HPLC and SDS-PAGE analysis followed by phosphor imaging, and immunoreactivity by a cell-binding assay. Radiosynthesis of Phosphine Probes. Radiolabeling yield and percentage of oxidation were determined by HPLC analysis. The radiolabeling of the synthesized phosphine probes required extra precautions, because of the sensitivity of the phosphine for oxidation to phosphine oxide. Therefore, an appropriate antioxidant was used that did not affect the chelation itself. The preparation of 89Zr- and 67/68Ga-DFO-phosphines 2 and 3, 177 Lu-DOTA-phosphine 4, and 123I-cubyl-phosphine 5 are described in the Supporting Information. Biodistribution of 131I-cmAb U36-triazide Conjugates and Radiolabeled Phosphines 2 5. Radiolabeled cmAb U36-triazide conjugates were injected in nude mice bearing subcutaneously implanted xenografts of the tumor line HNX-OE, while radiolabeled phosphines were injected in tumor-free nude mice. Female mice (athymic nu/nu, 21 31 g; Harlan CPB), were 8 10 weeks old at the time of the experiment. All animal 2074

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Bioconjugate Chemistry experiments were performed according to National Institute of Health principles of laboratory animal care and Dutch national law (“Wet op de proefdieren”. Stb 1985, 336). Five groups of four mice were injected via the retroorbital plexus with 185 kBq 131IcmAb U36-triazide conjugate (100 μg in 100 μL), in which the triazide was coupled to the cmAb at different molar ratios (triazide-to-mAb molar ratio 0, 4, 6, 8, and 15). The mean tumor size at start of the experiment was 104 ( 58 mm3. Blood samples were taken at 5, 24, and 48 h post injection (p.i.). At 72 h p.i., the mice were anesthetized, bled, killed, and dissected. Blood, tumor, skin, tongue, sternum, heart, lung, liver, spleen, kidney, bladder, muscle, thighbone, colon (content), ileum (content), stomach (content), and thyroid were weighed and the amount of radioactivity in each tissue was assessed in a γ-well counter. Radioactivity uptake was calculated as the percentage of the injected dose per gram of tissue (%ID/g). Six groups of four mice were injected intravenously (i.v.) via the tail vein with 0.4 MBq 89Zr-DFO-phosphine 2 (1.52 μg, 1.3 nmol), 1.1 MBq 68Ga-DFO-phosphine 2 (1.52 μg, 1.3 nmol), 0.4 MBq 89Zr-DFO-phosphine 3 (1.52 μg, 1.4 nmol), 1.1 MBq 68 Ga-DFO-phosphine 3 (1.52 μg, 1.4 nmol), 0.4 MBq 177LuDOTA phosphine 4 (1.50 μg, 1.7 nmol), or 0.8 MBq 123I-cubylphosphine 5 (1.00 μg, 1.3 nmol). After 5, 10, 15, 30, 45, 60, and 90 min blood samples were taken and after 120 min, the mice were anesthetized, bled, killed, and dissected as described above. In Vitro Staudinger Ligation. Staudinger ligation efficiency was assessed by SDS-PAGE analysis of the reaction mixture followed by phosphor imager analysis. Equivalents of phosphine added for ligation were related to the reacting counterpart, the azide functionality. All Staudinger ligation experiments were performed with a cmAb U36-triazide conjugate containing on average 8 triazide 1 molecules (and thus 24 azide functionalities) and is further designated “cmAb U36-triazide”. Standard reactions were performed with 1.67 nmol (0.25 mg, 40 nmol azide functionalities) cmAb U36-triazide and 1 equiv of phosphine (40 nmol, 44 μg) in 0.5 mL (6 v/v% ethanol) PBS at 37 °C for 2 h. The following characteristics were examined: (a) the in vitro stability of the phosphine probes 2 and 4 expressed as the chemical half-life; (b) the reactivity of phosphines 2 and 4 with cmAb U36-triazide in PBS; (c) the influence of the temperature on Staudinger ligation efficiency; and (d) the influence of the composition of the reaction medium on Staudinger ligation efficiency. The in vitro stability of the phosphine probes was examined by incubating 90 nmol/mL 89 Zr- or 67Ga-DFO-phosphine 2 (10 MBq/mL) or 177LuDOTA-phosphine 4 (7 MBq/mL) in PBS and in 10% or 37% freshly prepared human serum at 37 °C with 6 v/v% ethanol. At different time points (30, 75, and 120 min), aliquots were taken. Serum proteins were precipitated by the addition of a 2-fold excess (v/v) of acetonitrile, and after centrifugation, the supernatant was analyzed by radio-HPLC. The data obtained at 30, 75, and 120 min were plotted on a semi logarithmic scale, whereafter extrapolation to 50% decomposition gave the chemical half-life. In Vitro Stability of cmAb U36-Staudinger Products. For evaluation of the in vitro stability of the cmAb U36-Staudinger ligation products, samples containing 40 μg of the PD10 purified cmAb U36-Staudinger product, resulting from a reaction of cmAb U36-triazide with 89Zr/67Ga-DFO-phosphine 2 or 177 Lu-DOTA-phosphine 4, were added to freshly prepared human serum, mouse serum or 0.9% NaCl (1:4 v/v dilution; sodium azide added to 0.02%) (n = 2). The samples were

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incubated at 37 °C in a CO2-enriched atmosphere (5% CO2). After 2, 24, and 48 h incubation, aliquots were taken and analyzed by radio-HPLC. Fractions of 0.5 mL were assessed for radioactivity in a γ-counter and used for determination of the recovery of the HPLC column. In Vivo Staudinger Ligation. Tumor-free nude mice were used for evaluation of in vivo Staudinger ligation in blood. By doing so, quantitative information about the ligation efficiency in vivo can easily be obtained. Three mice were injected with 500 μg cmAb U36-triazide (0.2 mL) via the retroorbital plexus followed by 68 μg 67Ga-DFO-phosphine 2 two hours later via the tail vein. As control, three mice were injected only with 68 μg 67 Ga-DFO-phosphine 2. At 5, 10, 15, 30, 60, and 90 min after injection of the phosphine probe, blood samples were taken; and after 5 h, the mice were anesthetized, bled, killed, and dissected as described above. Blood samples of 5 h p.i. of the phosphine were also analyzed by HPLC. To this end, the serum was separated from the blood by centrifugation and diluted to 25% serum with saline. HPLC-fractions of 0.5 mL were assessed for radioactivity in a γ-counter to determine whether in vivo Staudinger ligation had occurred. Statistical Analysis. All values are given as mean ( SD. Statistical analysis was performed on pharmacokinetics, tissue uptake, and tumor-to-blood ratios between different groups of mice with the Student’s t-test (SPSS) for paired data. Two-sided significance levels were calculated and p < 0.05 was considered statistically significant.

’ RESULTS Synthesis of 131I-cmAb-Triazide Conjugates and Their Biodistribution. cmAb U36 was reacted with 10, 15, 20, or 40

equiv of triazide 1a in order to determine the maximum loading of triazide to cmAb U36 without affecting its integrity, immunoreactivity, and in vivo targeting behavior (Scheme 2). The average number of triazide molecules attached to the antibody was determined by UV-HPLC and appeared to be 4, 6, 8, and 15, respectively, which means that, within the applied stoichiometry, the efficiency of modification was 37 42%. Subsequent radiolabeling of these conjugates with 131I resulted in overall radiolabeling yields of >70% after purification. As a control, cmAb U36 without any triazide groups was also radiolabeled. The radiochemical purity of all 5 products was >99%, and there was a gradual decrease of the immunoreactivity upon increase of the substitution ratio from 91% (131I-cmAb U36 without triazide) to 87%, 85%, 79%, and 46% for cmAb U36 containing, respectively, 4, 6, 8, and 15 triazide molecules. SDS-PAGE analysis (Figure 2) showed an increased percentage of apparent high molecular weight product at higher substitution ratio (20% for cmAb U36 15 triazide), while also a change in the apparent molecular weight of the monomeric cmAb U36 (normally ∼150 kDa) was observed. For assessment of the optimal triazide-to-mAb molar ratio for in vivo tumor targeting, five groups of four mice were injected with 131I-cmAb U36-triazide conjugate with, respectively, 0, 4, 6, 8, and 15 triazide molecules attached. At 5, 24, 48, and 72 h p.i., blood samples were drawn to determine the pharmacokinetics. The blood clearance of the different 131I-labeled cmAb U36triazide conjugates did not show distinctive differences (p > 0.05), albeit the conjugate with the highest triazide-to-cmAb U36 molar ratio (cmAb U36 15 triazide) tended to exhibit a faster blood clearance (Figure 3). At 72 h p.i. the %ID/g in tumor, blood, normal tissue, and gastrointestinal contents was 2075

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Scheme 2. Modification Reaction of cmAb U36 with Triazide 1a Resulting in cmAb U36-Triazide Conjugates with, Respectively, 0, 4, 6, 8, and 15 Triazide 1 Molecules Attached

Figure 2. SDS-PAGE analysis of 131I-cmAb U36-triazide conjugates with, respectively, 0, 4, 6, 8, and 15 triazide 1 molecules attached.

Figure 3. Pharmacokinetics of 131I-cmAb U36-triazide conjugates containing, respectively, 0, 4, 6, 8, or 15 triazide 1 molecules in nude mice bearing HNX-OE tumors.

determined and depicted in Figure 4 for a selected panel of tissues. The tumor uptake of unconjugated 131I-labeled cmAb U36 was 23.0 ( 3.5%ID/g, and was at the same level when 4 triazide groups were attached (23.1 ( 3.4%ID/g). However, tumor uptake tended to decrease at higher triazide-to-cmAb U36 molar ratio. A tumor uptake of 16.4 ( 3.7 and 18.7 ( 6.3%ID/g, respectively, was observed when 6 or 8 triazide groups were attached to cmAb U36. Tumor uptake of conjugates containing

15 triazide groups was 10.5 ( 1.9%ID/g, which is significantly lower than for all the other four conjugates (p < 0.05). No distinctive increased uptake in any of the normal organs was observed at higher triazide-to-cmAb U36 molar ratios. The 131IcmAb U36 conjugate with 15 triazide groups showed a significantly lower (1.05 ( 0.20) tumor-to-blood ratio than the other 131 I-cmAb U36-triazide conjugates (1.49 ( 0.24 to 1.81 ( 0.19) (p < 0.05). This parameter also indicates worse tumor targeting when too many triazide groups are attached to the mAb. The cmAb U36-triazide conjugate with 8 triazide groups was used in subsequent in vitro Staudinger ligation experiments and will be designated “cmAb U36-triazide” hereafter. Biodistribution of Phosphine Probes. The pharmacokinetics and biodistribution of 89Zr- and 68Ga-DFO-phosphines 2 and 3, 177Lu-DOTA phosphine 4, and 123I-cubyl-phosphine 5 were examined in tumor-free nude mice. All six probes showed fast blood clearance, and after 30 min, less than 5%ID/g was still present in the blood (Figure 5). The phosphines are mainly excreted via the gastrointestinal and urinary tract with elevated uptake in liver (8.1 ( 1.8%ID/g) and spleen (3.2 ( 1.3%ID/g) for 177Lu-DOTA phosphine 4, and in kidney for 89ZrDFO-phosphine 2 (4.1 ( 0.6%ID/g), 89Zr-DFO-phosphine 3 (2.0 ( 0.2%ID/g), and 177Lu-DOTA-phosphine 4 (1.4 ( 0.2% ID/g) at 2 h p.i (Figure 6). The blood kinetics of 68Ga-DFOphosphine 2 and 68Ga-DFO-phosphine 3 were comparable, while there was a difference between 89Zr-DFO-phosphine 2 and 89Zr-DFO-phosphine 3, the latter clearing faster. 177LuDOTA-phosphine 4 showed comparable blood kinetics to 89ZrDFO-phosphine 3. 123I-cubyl-phosphine 5 cleared significantly faster than the other probes and therefore this probe was not further evaluated in Staudinger ligation experiments. Because the labeling of DFO-phosphine 3 resulted in a higher percentage of oxidation of the phosphine, it was decided to select DFO-phosphine 2 for Staudinger ligation experiments. For evaluation of the effect of the chelate on ligation efficiency, DOTA-phosphine 4 was also included. In Vitro Staudinger Ligation in PBS. The chemical half-life of 89 Zr- and 67Ga-DFO-phosphines 2 and 177Lu-DOTA-phosphine 4 in PBS in the absence of cmAb U36-triazide was examined at 37 °C. All three phosphines were only slowly oxidized in PBS with a chemical half-life of about 10 h. Staudinger ligation efficiency was investigated using cmAb U36-triazide in combination with each of the phosphine probes in aqueous solution. No significant difference in reactivity was observed for the three phosphines; Staudinger ligation efficiency was always between 20% and 25% after 2 h at 37 °C. The kinetics of 2076

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Figure 4. Biodistribution of 131I-cmAb U36-triazide conjugates with, respectively, 0, 4, 6, 8, or 15 equiv of triazide 1 attached in nude mice bearing HNX-OE tumors at 72 h after injection.

Figure 5. Pharmacokinetics of 89Zr- and 68Ga-DFO-phosphines 2, 89 Zr- and 68Ga-DFO-phosphine 3, 177Lu-DOTA phosphine 4, and 123 I-cubylphosphine 5 in tumor-free nude mice.

the Staudinger ligation were determined with 67Ga-DFOphosphine 2 under standard conditions for shorter and longer periods. After 10 min, about 5% Staudinger ligation efficiency was observed, which increased to 32% after 5 h. Temperature dependency of the Staudinger ligation was examined with 177 Lu-DOTA phosphine 4 after 2 h incubation at 4 °C, room temperature, and 37 °C (standard temperature). The Staudinger ligation efficiency was found to be strongly temperature dependent, being 5% at 4 °C, 8% at room temperature, and 20% at 37 °C. Interestingly, when the reaction mixture contained 0.04% sodium dodecyl sulfate (SDS), the ligation efficiency was nearly twice as high (40 45% after 2 h at 37 °C). 24 Once formed, the Staudinger products (89Zr- and 67Ga-DFOStaudinger-cmAb U36 and 177Lu-DOTA-Staudinger-cmAb U36) remained more than 93% intact upon 48 h incubation at 37 °C

under the applied conditions (0.9% NaCl or 80% mouse or human serum). The 67Ga-DFO-Staudinger cmAb-U36 product was the least stable in mouse serum (93% intact after 48 h incubation), while in human serum and 0.9% NaCl, the product was over 96% intact after 48 h incubation. The other two Staudinger ligation products were very stable in all three media for 48 h (>98% intact). In Vitro Staudinger Ligation in Human Serum. In the absence of cmAb U36-triazide, all three phosphine probes remained >90% intact in 10% human serum and >80% intact in 37% human serum after 2 h at 37 °C. Staudinger ligation took place in the presence of 10% and 37% human serum; however, the kinetics of all probes were slowed down with 30% compared with the reaction in PBS and with 50% for 89Zr-DFO phosphine 2 in 37% human serum. In Vivo Staudinger Ligation. In vivo Staudinger ligation in blood of tumor-free mice was studied via the sequential administration of cmAb U36-triazide and 67Ga-DFO-phosphine 2. Blood kinetics and biodistribution of 67Ga were not significantly different for mice which received cmAb U36-triazide plus phosphine in comparison with mice which did not receive cmAb U36-triazide, e.g., the blood levels at 5 h p.i. of the phosphine were 0.20 ( 0.04 vs 0.14 ( 0.05%ID/g. HPLC analysis of these blood samples did not reveal evidence for the presence of the cmAb U36-Staudinger product (Figure 7). In Vitro Staudinger Ligation in Serum of Different Animal Species. To explain the absence of Staudinger product formation in mice, in vitro Staudinger ligations experiments were performed in mouse serum and in serum of a panel of other animal species. 67 Ga-DFO-phosphine 2 was reacted with cmAb U36-triazide in 50% pig, goat, rabbit, human, and mouse serum for 2 h at 37 °C. In 50% mouse serum, Staudinger ligation was virtually absent (∼95% reduction), and a side product was observed (Figure 8). The other sera did allow Staudinger ligation, although not as efficient as in PBS. Staudinger ligation in 50% human and goat 2077

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Figure 6. Biodistribution of 89Zr-DFO-phosphine 2 (red bars), 68Ga-DFO-phosphine 2 (green bars), 89Zr-DFO-phosphine 3 (light blue bars), 68GaDFO-phosphine 3 (purple bars), 177Lu-DOTA-phosphine 4 (blue bars), and 123I-cubyl-phosphine 5 (light salmon bars) in nude mice at 2 h after injection for a larger selection of tissues and a smaller selection of tissues (inset).

Figure 7. Representative HPLC diagram of mouse blood taken 5 h p.i. of the phosphine of a mouse that received cmAb U36-triazide and 67GaDFO-phosphine 2 (black line). As a reference, the HPLC diagram of the in vitro prepared Staudinger product of this reaction is depicted (red line).

Figure 8. SDS-PAGE analysis of a Staudinger ligation reaction of cmAb U36-triazide with 1 equivalent of 67Ga-DFO-phosphine 2 in the presence of 50% serum of different sources after 2 h at 37 °C: (A) PBS, no serum, (B) pig serum, (C) goat serum, (D) rabbit serum, (E) human serum, (F) mouse serum.

serum gave 30% reduction of ligation efficiency compared to PBS. Ligation efficiency in 50% pig and rabbit serum was, respectively, 50% and 75% reduced compared to PBS (Figure 8),

the latter also showing the extra band on SDS-PAGE. Reduced ligation efficiency and the extra band were also observed when Staudinger ligation was performed in rat serum. Decreasing the percentage of mouse serum resulted in an increase in Staudinger ligation up to 4% efficiency in the presence of 37% mouse serum and 8% efficiency in 10% mouse serum. Reduced Staudinger ligation efficiency in mice was also observed when 177Lu-DOTAphosphine 4 instead of 67Ga-DFO-phoshine 2 was used. In search for an explanation for the extra band on SDS-PAGE and the reduced Staudinger ligation efficiency in mouse serum, Staudinger ligation was also performed in the presence of mouse serum albumin. The ligation, however, was as efficient as in PBS. Dialysis of mouse serum before use (4.5 and 20 kDa cutoff) did not improve the low ligation efficiency and still gave the prominent extra band on SDS-PAGE. Finally, upon oxidation of the phosphine in advance, followed by incubation in mouse serum, no side product was observed by SDS-PAGE analysis.

’ DISCUSSION The Staudinger ligation has until now been applied in the field of chemical biology and organic chemistry, but not in the field of radiochemistry nor in a pretargeting approach with mAbs. The Staudinger ligation is a bioorthogonal reaction, which implies that phosphine and azide react with each other and not with other bioavailable molecules in the body. Therefore, we investigated the applicability of this bioorthogonal reaction in a pretargeting concept with antibodies for tumor diagnosis and therapy. In the present paper, we developed the synthesis of mAb azide conjugates and radiolabeled phosphines and evaluated their in vivo pharmacokinetic behavior as well as their in vitro and in vivo reactivity in Staudinger ligation. cmAb U36 was chosen as the model mAb, because this mAb has shown high and selective tumor uptake in cancer patients. Moreover, cmAb U36 internalizes just to a limited extent, and this is expected to be a favorable characteristic for pretargeting. On the basis of the work of Bertozzi,23 the amide containing azide 1a was designed having a trivalent azide branch to increase the number of azides that are attached per lysine.19,24 cmAb U36 was reacted with different amounts of triazide 1a and subsequently radiolabeled with 131I to evaluate the in vivo behavior. Since the 2078

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Bioconjugate Chemistry applied radiolabeling method does not impair the immunoreactivity and in vivo pharmacokinetic behavior of cmAb U36,40 any change is caused by the number of triazide 1 groups that are attached to cmAb U36. A series of conjugates with, on average, 4, 6, 8, and 15 equiv of triazide 1 attached to cmAb U36 was produced (Scheme 2). The in vitro and in vivo evaluation revealed that up to 8 triazide 1 groups (24 azide functionalities) could be attached to cmAb U36 without substantially affecting the immunoreactivity, pharmacokinetics, tumor uptake, and tumor-toblood ratio. These characteristics became clearly impaired, however, when 15 triazide 1 groups were coupled per mAb molecule, a phenomenon also observed earlier in the development of 186 Re-MAG3-labeled conjugates.37 Several radiolabeled phosphine probes were synthesized, differing in radioisotope, chelate, and linker. The major radiochemical challenge was the protection of the phosphine moiety against oxidation to phosphine oxide (see Supporting Information for experimental details). In the radiolabeling of DFOphosphine 2 and 3 with 89Zr, radiolytically formed oxidative species in the 89Zr stock solution and in the reaction mixture needed to be scavenged. Sodium sulfite proved to be an excellent antioxidant. In this way, 45 μg/12 MBq/mL 89Zr-DFO-phosphine 2 and 3 could be prepared with less than 10% oxidation caused by radiolabeling. Because 67Ga was purified over a chromafix column just prior to use, the addition of an antioxidant was found to be unnecessary during the preparation of 67GaDFO-phosphine 2. However, for the in vivo experiment with a high dose 68Ga-DFO-phosphine 2 and 3, addition of an antioxidant to the reaction mixture appeared necessary. In this case, not sodium sulfite but a stannous sulfate containing gentisic acid solution proved adequate. A concentration of 270 μg/314 MBq/mL 68Ga-DFOphosphine 2 with 8% oxidation or 68Ga-DFO-phosphine 3 with 13% oxidation could be prepared. Since addition of antioxidants like gentisic acid or ascorbic acid did not inhibit oxidation, the preparation of 177Lu-DOTA phosphine 4 was restricted to the use of fresh 177 Lu and execution of the labeling at pH 8. In this way, oxidation caused by radiolabeling was limited to 15% and 91 μg/25 MBq/mL 177 Lu-DOTA-phosphine 4 could be prepared. 123I-cubyl-phosphine 5 had to be synthesized from 123I-cubyl carboxylic acid and linker phosphine because direct labeling of a Br-analogue of 5 resulted in completely oxidized phosphine. Following this procedure, 95 μg/72 MBq/mL 123I-cubyl-phosphine 5 could be prepared with less than 10% oxidation. In vivo evaluation of the six radiolabeled phosphine probes revealed that blood clearance was very fast with a half-life time of less than 15 min. This means that the reaction of the phosphine probes with pretargeted mAb triazide conjugate should be very fast for making Staudinger ligation an efficient option for in vivo pretargeting strategies. The stability of 89Zr- and 67Ga-DFO-phosphine 2 and 177LuDOTA-phosphine 4 in PBS was satisfactory, since the chemical halflife was about 10 h at 37 °C. However, their reactivity with cmAb U36-triazide was rather slow, taking the observed fast pharmacokinetics into account: after 2 h, Staudinger ligation efficiency was 20 25% at 37 °C, which means that per cmAb U36 molecule on average 5 to 6 out of 24 azide 1 groups have reacted. The presence of human serum did not affect the stability of the three probes, but their reactivity was reduced compared to PBS. Although the circulation time of the probes was less than the reaction time in vitro, we speculated that it may still be enough to achieve conjugation in vivo. Therefore, we attempted to demonstrate in vivo Staudinger ligation in a pretargeting approach in

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tumor-free mice. HPLC analysis of the mouse blood revealed that the activity still present in the blood was not from cmAb U36-Staudinger product (Figure 7). To explain the complete absence of in vivo Staudinger ligation, we performed in vitro Staudinger ligation in mouse serum as well as in serum of other animal species. In the presence of mouse serum, the Staudinger ligation efficiency was severely reduced. SDS-PAGE analysis revealed that Staudinger ligation efficiency was primarily hampered by the formation of a side product. This side product was only formed when the phosphine was intact and not oxidized in advance. Identification of this side product was without success, and the molecular basis for the formation of this product is still not known. Apparently, larger molecules in mouse serum are involved, since in dialyzed mouse serum, the extra band on SDS-PAGE was still formed with concomitant reduction of Staudinger ligation efficiency. Tests in serum of other animals revealed that reduced Staudinger ligation efficiency and the formation of the extra band was not limited to mouse serum. These results seem to be in contrast with those of the group of Bertozzi who reported on successful in vivo Staudinger ligation in mice.20 In this study, azide groups were introduced on target cells by daily injection of the unnatural sugar peracetylated N-αazidoacetylmannosamine (Ac4ManNAz, 300 mg/kg) to mice for 7 days resulting in a total dose of about 52 mg azide per mouse (122 μmol), which is ∼3000 times more than we used in our research. Staudinger ligation in the splenocytes was confirmed by administration of 16 μmol phosphine-FLAG (∼300 times more than used here) 24 h after the last injection of Ac4ManNAz. In this in vivo model system, in which pretargeting is not disease selective, the side reaction of the phosphine as we observed in mouse serum might be less dominant due to the very large amounts of azide and phosphine-FLAG used and the fact that intraperitoneal injections were applied. Therefore, in our opinion the results obtained in this way can not be directly translated to pretargeting approaches with mAbs. Apart from not being bioorthogonal in rodents, chemical possibilities to increase the Staudinger ligation efficiency seem to be rather limited, and we have strong scientific doubts whether the Staudinger ligation can be made more efficient. Its kinetics are a function of subtle folding/defolding of the azide functionality at the mAb molecule and the accessibility and reactivity of the phosphorus atom within the triphenylphosphine methyl ester moiety. The mAb triazide conjugate used was the best in the series of mono- to nonavalent azide, whereas removal of the amide next to the azide resulted in a 2-fold decrease of the efficiency (unpublished results). Variation around the phosphorus atom is very limited, since the triphenyl moiety is necessary for Staudinger ligation and to protect against oxidation of the phosphorus atom. And finally, introduction of an SDS moiety either in the triazide handle or in the phosphine probe is not expected to lead to a major breakthrough, because the observed increase in the presence of SDS in the reaction medium was only 2-fold. Therefore, we conclude that the Staudinger ligation is not the method of choice for pretargeting approaches in humans. We foresee that this also holds for the very recently reported so-called traceless Staudinger ligation.41 This approach makes use of diphenylphosphines which are even more prone to oxidation than the triphenylphosphines used here for in vivo pretargeting. Possibly, the tetrazine ligation34 is, because the reaction kinetics of this bioorthogonal reaction are reported to be at least 1000 faster than the Staudinger ligation. 2079

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’ ASSOCIATED CONTENT

bS

Supporting Information. Synthesis and characterization of triazide 1a and phosphine 2 5, conjugation of triazide to cmAb U36 and determination of the average triazide to cmAb U36 ratio, and radiolabeling of cmAb U36 conjugates and phosphines. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Danielle J. Vugts, Ph.D., Dept. of Otolaryngology/Head and Neck Surgery and Nuclear Medicine and PET research, VU University Medical Center, De Boelelaan 1085c, 1081 HV Amsterdam, The Netherlands, Tel +31-20-4445699, Fax +3120-4449121, E-mail: [email protected].

’ ACKNOWLEDGMENT We thank Marek Smoluch for measuring HRMS, Elwin Jansen for HPLC measurements of the modification of cmAb U36 with triazide 1a, and Joost Verbeek for his help with the preparation of 123 I-cubyl-phosphine 5. ’ ABBREVIATIONS DFO, desferrioxamine; DOTA, 1,4,7,10-tetraazacyclododecane1,4,7,10-tetraacetic acid; mAb, monoclonal antibody; PBS, phosphate buffered saline; PET, positron-emission tomography; SPECT, single-photon emission computed tomography; % ID/g, injected dose per gram tissue ’ REFERENCES (1) Dancey, G., Begent, R. H., and Meyer, T. (2009) Imaging in targeted delivery of therapy to cancer. Targ. Oncol. 4, 201–217. (2) Arano, Y., Wakisaka, K., Mukai, T., Uezono, T., Motonari, H., Akizawa, H., Kairiyama, C., Ohmomo, Y., Tanaka, C., Ishiyama, M., Sakahara, H., Konishi, J., and Yokoyama, A. (1996) Stability of a metabolizable ester bond in radioimmunoconjugates. Nucl. Med. Biol. 23, 129–136. (3) Akizawa, H., and Arano, Y. (2002) Altering pharmacokinetics of radiolabeled antibodies by the interposition of metabolizable linkages Metabolizable linkers and pharmacokinetics of monoclonal antibodies. Q. J. Nucl. Med. 46, 206–223. (4) Grainger, D. W. (2004) Controlled-release and local delivery of therapeutic antibodies. Expert Opin. Biol. Ther. 4, 1029–1044. (5) Tietze, L. F., and Krewer, B. (2009) Antibody-directed enzyme prodrug therapy: a promising approach for a selective treatment of cancer based on prodrugs and monoclonal antibodies. Chem. Biol. Drug Des. 74, 205–211. (6) Goldenberg, D. M., Sharkey, R. M., Paganelli, G., Barbet, J., and Chatal, J. F. (2006) Antibody pretargeting advances cancer radioimmunodetection and radioimmunotherapy. J. Clin. Oncol. 24, 823–834. (7) Boerman, O. C., van Schaijk, F. G., Oyen, W. J. G., and Corstens, F. H. M. (2003) Pretargeted radioimmunotherapy of cancer: Progress step by step. J. Nucl. Med. 44, 400–411. (8) Liu, G., and Hnatowich, D. J. (2008) A semiempirical model of tumor pretargeting. Bioconjugate Chem. 19, 2095–2104. (9) Goodwin, D. A., and Meares, C. F. (2001) Advances in pretargeting biotechnology. Biotechnol. Adv. 19, 435–450. (10) Goldenberg, D. M., and Sharkey, R. M. (2007) Novel radiolabeled antibody conjugates. Oncogene 26, 3734–3744. (11) Wu, A. M., and Senter, P. D. (2005) Arming antibodies: prospects and challenges for immunoconjugates. Nat. Biotechnol. 23, 1137–1146.

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