Biotinylated Polyacrylamide-Based Metal-Chelating Polymers and

Aug 7, 2012 - triazin-2-yl)-4-methylmorpholinium chloride (DMTMM, from Acros. Organic, >99%, Fisher Scientific, Canada). TEA was distilled from...
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Biotinylated Polyacrylamide-Based Metal-Chelating Polymers and Their Influence on Antigen Recognition Following Conjugation to a Trastuzumab Fab Fragment Peng Liu,† Amanda J. Boyle,‡ Yijie Lu,† Raymond M. Reilly,*,‡,§ and Mitchell A. Winnik*,† †

Department of Chemistry, University of Toronto, 80 Saint George Street, Toronto, Ontario, Canada M5S 3H6 Department of Pharmaceutical Sciences, University of Toronto, 144 College Street, Toronto, Ontario, Canada M5S 3M2 § Toronto General Research Institute, University Health Network, Toronto, Ontario, Canada M5G 2M9 ‡

S Supporting Information *

ABSTRACT: We report the synthesis and characterization of metal-chelating polymers (MCPs) with a terminal biotin and a polyacrylamide backbone harboring multiple diethylenetriaminepentaacetic acid (DTPA) chelating sites. These polymers are conjugated to a streptavidin (SAv)-modified Fab fragment of trastuzumab (tmFab) and subsequently complexed with 111In through DTPA. Trastuzumab has specific targeting ability toward human epidermal growth factor receptor-2 (HER2), which is overexpressed on some types of breast cancer cells and ovarian cancer cells. 111In can generate Auger electrons which cause lethal DNA double strand breaks. The radioimmunoconjugates (RICs) were designed to target HER2 overexpressing cancer cells and carry multiple copies of 111In to these cells. The mole maximum specific activities of these polymers were investigated by loading the polymers with 111In at an increasing 111In to polymer ratio. The polymers show 55-fold to 138-fold higher maximum specific activity than DTPA modified tmFab-SAv. Moreover, the HER2 immunoreactivities of these RICs were evaluated by measuring their specific binding ability toward HER2 overexpressing SKOV-3 ovarian cancer cells. The results demonstrate that although in the presence of polymer there is increased nonspecific binding, HER2 targeting ability was retained, ensuring the radionuclide delivery ability of these RICs.

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INTRODUCTION Over the past twenty years there has been growing interest in metal chelating polymers (MCPs). The early work by Torchilin explored applications in biology and medicine, particularly the development MCPs as carriers for Gd3+ as contrast enhancement agents for magnetic resonance imaging.1 Most of these experiments employed polylysine as a framework to which mulitdentate chelators such as diethylenetriaminepentaacetic acid (DTPA) were attached. His review in 1991 provides a useful overview of early contributions to this field.2 In a 1999 paper,3 he showed that these DTPA-containing polylysines could bind a larger number of 111In3+ ions than a biotin DTPA derivative, and he suggested that this kind of polymeric reagent would offer advantages over small molecule chelators in terms of both γ-ray imaging and radiotherapy applications. Over the intervening years, new techniques in synthesis (controlled radical polymerization,4 ring-opening metathesis polymerization,5 and better control over ring-opening polymerization of amino-acid N-carboxyanhydrides6) have led to polymers with a narrow distribution of chain lengths and control over end functionality. In addition, advances in polymer characterization techniques have given new confidence in one’s ability to properly characterize polymers synthesized in this way, and this in turn has led to interest in new applications of © 2012 American Chemical Society

MCPs. For example, a number of authors have introduced pendant groups such as bipyridine7 or terpyridine8,9 as metal binding ligands to promote supramolecular assembly of polymers in solution10 or as coupling agents for noncovalent curing applications.11 These new synthesis techniques allow the preparation of block copolymers with the metal-chelating groups confined to one of the blocks.12 The pendant-group ligands can serve as antennas for light absorption and transfer energy to chelated metal ions, leading to interesting studies of the optical and electrochemical properties of these polymers. For example, the Tew group has developed a series of polymers with terpyridine as ligands to chelate lanthanide ions and investigated their applications as optoelectronic materials and as sensors for nerve gas detection.13,14 We have a twofold interest in developing MCPs for biomedical applications. These polymers are designed to have a narrow distribution of chain lengths, multiple sites for metal chelation, and end-group functionality for attachment to antibodies or other bioaffinity agents. First, we develop polymer reagents for the new technique of mass cytometry. Mass Received: May 30, 2012 Revised: July 18, 2012 Published: August 7, 2012 2831

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Figure 1. Concept of the experiment: (1) A tmFab-SAv conjugate is allowed to interact with and bind a biotin-end-capped metal-chelating polymer. (2) The tmFab-SAv-MCP complex is incubated with 111In. (3) HER2+ cells are treated with the tmFab-SAv-MCP-111In complex in order to test antigen recognition.

transfer due to their nanometer to micrometer range. Release of Auger electrons in close proximity to the cell nucleus causes lethal DNA double-strand breaks. 111In-NLS-trastuzumab is bound, internalized, and transported to the nucleus of HER2+ BC cells, likely due to interaction of the NLS peptides with importins α/β.20 111In-NLS-trastuzumab was 8-fold more potent than trastuzumab at decreasing the clonogenic survival of HER2-amplified SKBR-3 human BC cells,19 and 111In-NLStrastuzumab was able to kill trastuzumab-resistant BC cells, provided that the HER2 extracellular domain (ECD) was preserved.21,6 Nonetheless, the potency of 111In-NLS-trastuzumab was highly dependent on HER2 density, exhibiting its greatest effectiveness on SKBR-3 cells (1.3 × 106 HER2/cell). It was significantly less effective on MDA-MB-361 cells with intermediate HER2 density (5.1 × 105 HER2/cell), and MDAMB-231 cells with low HER2 expression (5.4 × 104 HER2/ cell) were resistant to 111In-NLS-trastuzumab.19 Our interest in developing MCPs for conjugation to trastuzumab is based on the hypothesis that increasing the amount of 111In delivered per HER2 recognition event would enhance the potency of 111In-NLS-trastuzumab for killing HER2-overexpressing BC cells and extend its range to BC cells with lower HER2 density. This idea can be appreciated by the fact that, at the specific activity (SA) achieved for 111In-NLStrastuzumab monosubstituted with DTPA (99%), triethylamine (TEA, >99%), DTPA > 98%, azobis(4-cyanovaleric acid) (ACVA, >98%, from Fluka), pentafluorophenol (pfp-OH, >99%, from Matrix Scientific Inc.), anhydrous pyridine (>99%,), biotin (>99% from Nanjing Tianzun Zezhong Inc., China), 1,2-lutidine (>96%, from Fluka), 2,2′-azobis(2-methylbutyronitrile) (AMBN, from Dupont USA), and 4-(4,6-dimethoxy-1,3,5triazin-2-yl)-4-methylmorpholinium chloride (DMTMM, from Acros Organic, >99%, Fisher Scientific, Canada). TEA was distilled from KOH, dichloromethane (DCM, Aldrich) was distilled from CaH2, and dithiobenzoic acid disulfide was prepared as described previously.30 Other items include silica for chromatography (Merck grade 9385, 230−400 mesh, 60 Å), a Spectra/Por dialysis membrane (MWCO 1 kDA, Spectrum Laboratories, Inc.), and a Millipore Amicon spin filter (3 kDa MWCO, 4 mL, Fisher Science, Canada). The tmFab and tmFab-SAv were prepared and characterized as described previously.29 111 InCl3 was purchased from Nordion, Inc. (Kanata, Canada). Synthesis of the Acrylamide Monomer N-{2-[(BOC)aminoethyl]} Acrylamide (1). tBOC-DE (4.00 g, 25.0 mmol), TEA (3.80 mL, 27.3 mmol), and THF (20 mL) were placed into a 500 mL round-bottom flask (RBF). Acryloyl chloride (2 mL, 25 mmol) was dissolved in THF (180 mL) and added dropwise into the RBF with stirring and cooled with an ice-bath, over 2 h. The solution was allowed to react for another 2 h, during which time some white solids precipitated (salts of TEA), which were removed by filtration. Then the THF was removed with a rotary evaporator. The raw product was dissolved in chloroform (100 mL) and extracted with brine (2 × 100 mL). The organic phase was collected and dried over magnesium sulfate overnight; then after filtration, the solvent was removed with a rotary evaporator. The raw product was purified by chromatography over silica (300 mL) using chloroform/methanol = 20:1 (Rf ≈ 0.3, 1.6 L) as the eluent. The product was obtained as a white solid (3.80 g, 71.8%). 1H NMR (400 MHz, CDCl3, δ (ppm), cf. Figure S1, Supporting Information): 6.25 (t, 1 H, −CH=), 6.09 (t, 1 H, =CHH), 5.51 (t, 1 H, =CHH), 4.91, 6.38 (s, 2 H, −NH), 3.25, 3.41 (t, 4 H, N− CH2), 1.28 (s, 9 H, C(CH3)3). Synthesis of the Biotin Chain Transfer Agent (CTA) 2. Synthesis of Biotinylpentafluorophenyl Ester (Bi-PFP). Bi-PFP was synthesized as described by Korshun et al.31 Biotin (2.00 g, 8.2 mmol) was placed into a 250 mL RBF, and anhydrous pyridine 50 mL was added. The solution was refluxed for 2 h until all the biotin dissolved.

fragmentation transfer (RAFT) polymerization that share a common structure and carry a DTPA chelator on each pendant group. The polymers differ in length and were designed to examine how the length of the MCP affects antigen recognition when attached to an antibody. To enable rapid scanning of a broad family of different MCPs, the polymers carry a biotin end group. These are then conjugated to a trastuzumab Fab (tmFab) fragment covalently attached to a streptavidin (SAv) via a polyethylene glycol (PEG) spacer. Recently, a variety of polymers generated by RAFT polymerization have been developed for biomedical applications.22 As in other living/controlled radical polymerization methods, RAFT provides excellent control of polymer molecular weight with narrow molecular weight distribution. Moreover, its versatility for polymer end group functionalization makes RAFT particularly promising in fields such as siRNA delivery,23,24 anticancer drug delivery,25−27 as well as noninvasive biomedical imaging.28 The basic philosophy of our experimental design for rapid screening of different MCPs is presented in Figure 1. We anticipate the synthesis of a broad family of different biotinend-capped MCPs (Bi-MCP). A tmFab-SAv conjugate can be treated with a Bi-MCP, allowing one or more polymer molecules to bind. The tmFab-SAv-Bi-MCP complex can be labeled with 111In to create the radioimmunoconjugate (RIC): tmFab-SAv-Bi-MCP-111In. This RIC would then be tested for HER2 recognition on SKOV-3 (or other HER2 overexpressing) cancer cells. One can construct and examine a large family of Bi-MCPs to assess structure−chain−length−function relationships in terms of ability to recognize HER2 on the surface of BC cells. In this way, one can identify candidate MCPs to develop for covalent conjugation to trastuzumab itself for in vitro and in vivo studies. In this paper our emphasis is on the synthesis and characterization of the biotin-end-labeled MCPs, as well as on the influence of polymer chain length on the antigen recognition efficiency of the tmFab-SAv RIC. We describe RAFT polymerization to generate polymer precursors with biotin end functional groups, followed by postpolymerization to quantitatively introduce DTPA pendant groups as binding sites for 111In. These polymers were then bound to tmFab-SAv through the strong interaction between biotin and SAv (Ka = 1015 M−1). The immunoreactivity of these RICs was then evaluated through cell binding experiments. A detailed study of the biodistribution in mice of tmFab-SAv RICs of a family of biotin-end-labeled MCPs of similar length, including one of the samples described here, is reported elsewhere.29

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EXPERIMENTAL SECTION

Instrumentation. Size exclusion chromatography (SEC) measurements on samples dissolved in N-methylpyrrolidone (NMP) were run on a system consisting of a Viscotek VE1121 solvent pump, an American Standard Corporation gel 10 μm column, and a gel 10 μm guard column and a Viscotek VE 3580 refractive index detector. The analysis was carried out at 80 °C at a flow rate of 0.6 mL/min with NMP containing 0.2 wt % LiCl as the eluent, and the column was calibrated with poly(methyl methacrylate) (PMMA) standards. Watersoluble polymers were analyzed with a Viscotek SEC consisting of a Viscotek VE1122 solvent delivery system and a VE7510 GPC degasser, with Viscotek ViscoGEL G4000PWXL and G2500PWXL columns (kept at 30 °C) connected to a Viscotek VE3210 UV/vis detector and a VE3580 refractive index detector. The eluent was 0.2 M KNO3, 200 ppm NaN3, and 25 mM, pH 8.5 phosphate buffer at a flow rate of 1.0 mL/min. The system was calibrated with poly(methacrylic 2833

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Table 1. Ratio of Reagents Used in the RAFT Polymerization of 1 and Characteristics of the Polymers Obtained Bi-PBocNAAm-20 Bi-PBocNAAm-40f Bi-PBocNAAm-50

[C]/[M]a

[C]/[I]b

reaction time (h)

DPnc

Mn(NMR)d

Mn(SEC)(PDI)e

1/40.0 1/79.6 1/80.0

3.98/1 4.00/1 4.00/1

4 6 8

20.3 ± 1.4 39.7 ± 2.8 50.8 ± 3.6

5.0 kDa 9.1 kDa 11.5 kDa

4.3 kDa (1.16) 11.9 kDa (1.21) 12.8 kDa (1.36)

a

[C]/[M] molar ratios of chain transfer agent to monomer used in the synthesis. b[M]/[I] molar ratios of chain transfer agent to initiator used in the synthesis. cNumber average DPn as determined by 1H NMR. The standard deviation in DPn was estimated assuming a ±5% error in the integration of the aromatic and aliphatic 1H signals being compared. dMn(NMR) values were calculated by multiplying DPn by the molecular weight of the monomers plus the molecular weight of the chain transfer agent end group. eMn(SEC) refers to nominal Mn values determined by SEC in NMP. These values and values of PDI = Mw/Mn were determined by reference to PMMA standards. fUsing the CTA (2) (ε300 = 16.8 M−1 cm−1) as a reference, we determined a value of Mn(UV) = 8.2 kDa.

Figure 2. 1H NMR spectrum of biotin-PBocNAA-40 in methanol-d4 with one drop of D2O added. After the solution was cooled to room temperature (RT), PFP-OH (1.54 g, 8.4 mmol) was added into the flask. The temperature was reduced to 0 °C. Then DCC (1.80 g, 8.7 mmol) dissolved in anhydrous pyridine (12 mL) was added dropwise into the flask with stirring. The flask was incubated in an ice-bath for 2 h. Then the icebath was removed and the solution was stirred for another 16 h. The solution was then filtered to remove some of the byproduct, dicyclohexyl urea. Half the solvent was then evaporated on a rotary evaporator, and the solution was allowed to stand for 5 h at RT. The solution was filtered to remove the precipitate, and then most of the solvent was removed by rotary evaporation. The syrup was added dropwise into a mixture of hexane/chloroform (1:1), (200 mL). The precipitate was redissolved in pyridine and washed again with the mixture of hexane and chloroform. The solid was filtered and dried in vacuum. A white solid was obtained (2.60 g, 78%). 1H NMR (400 MHz, d6-DMSO, δ (ppm)): 6.48, 6.41 (2 H, NH); 4.34, 4.16 (m, 2 H, CHNH); 3.16−3.11 (m, 1 H, CHS); 2.85−2.81 (dd, 1 H,CHHexoS); 2.77 (t, 2 H, CH2CO); 2.60 (d, 1 H, CHendoHS); 1.69−1.41 (m, 6 H, CH2-biotin). MS (ESI m/z, [M + H]+ = 411.1, [M + Na] + = 433.1) Synthesis of N-1[-2,2′-(Ethylenedioxy)diethyl-1′-amino]biotinylamide (Bi-EO2NH2). 2,2′-(Ethylenedioxy)diethylamine (6.2 mL, 37.0 mmol) and anhydrous TEA (1.5 mL, 11 mmol) were placed in a 250 mL RBF.32 Bi-PFP (1.50 g, 3.7 mmol) was dissolved in anhydrous DMF (75 mL) and added dropwise into the flask over 1 h. The solution was stirred magnetically for another hour. Most of the solvent was removed with a rotary evaporator, and the raw product

was precipitated into diethyl ether. Then the solid was dissolved in DMF (20 mL) and precipitated again in diethyl ether (150 mL). This product was purified by flash chromatography (400 mL silica) (ethanol/ammonia hydroxide (28%) = 5:1). The solvent was removed in vacuum, and a white solid was obtained (1.14 g, 83%). 1H NMR (400 MHz, deuterium oxide, δ (ppm) cf., Figure S2): 4.48, 4.30 (m, 2 H, CHNH), 3.56 (s, 4 H, OCH2CH2O), 3.48 (m, 4 H, OCH2), 3.26 (m, 2 H, CH2NH), 3.21 (m, 1 H, CHS), 2.87 (dd, 1 H, CHHexoS), 2.72 (t, 2 H, CH2-NH-CO-biotin), 2.65 (d, 1 H, CHHendoS), 2.15 (t, 2 H, biotin-CH2CO), 1.2−1.6 (m, 6 H, biotin-CH2). MS (ESI m/z, [M + H]+ = 375.2). Synthesis of Bis(pentafluorophenyl)azobis(4-cyanovalerate) (Bis-PFP-ACV) and Pentafluorophenyl[4-(phenylthiocarbonylthio)-4-cyanovalerate] (CTA-PFP). These syntheses followed the procedures reported by Roth et al.33 ACVA (5.14 g, 18.3 mmol), pfp-OH (7.90 g, 42.9 mmol), 2,6-lutidine (14.91 g, 139.2 mmol), and dry DCM (105 mL) were placed in a 250 mL RBF. The flask was cooled to 0 °C with an ice bath, and the mixture was bubbled with nitrogen for 10 min. Then TFAA (11.30 g, 53.8 mmol) was injected through a septum. The solution turned yellow. The ice bath was removed. As the reaction was stirred for an additional 24 h, the solution turned brown. Then the solution was extracted with water (2 × 100 mL) to remove the TFAA. The solution was concentrated with a rotary evaporator and precipitated into hexane (70 mL). A white solid was obtained: yield 9.94 g (88%) after drying in vacuum. 1H NMR (400 MHz, CDCl3, δ (ppm)): 3.00−2.48 (m, 8 H, CH2), 1.78, 1.73 (2 s (cis, trans), 6 H, CH3). 2834

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Table 2. Mn Values, Water, and Na+ Ion Content Determined by TGA Analysis, and Effective Molecular Weights of the Three Bi-PAAm(DTPA) Polymers Bi-PAAm(DTPA)-20 Bi-PAAm(DTPA)-40 Bi-PAAm(DTPA)-50

DPnNMR

MnNMR (kDa)a

MnSEC (kDa)b (PDI)

H2O per DTPAc

Na+ ions per DTPAd

effective Mn (kDa)e

20.4 ± 1.4 40 ± 2.0f 51 ± 2.6f

10.0 ± 0.7 19 ± 1.0 23 ± 1.2

14.8 (1.2) 27.0 (1.2) 42.7 (1.4)

3.2 ± 0.16 2.1 ± 0.11 3.6 ± 0.18

2.6 ± 0.13 2.8 ± 0.14 3.1 ± 0.16

12.8 ± 1.0 25.3 ± 2.0 31.7 ± 2.5

a

Calculated from DPn, assuming that the DTPA groups are in the fully protonated form. bNominal Mn values and PDI (Mw/Mn) are from aqueous SEC measurements using poly(methacrylic acid) (PMAA) standards. cDetermined from the mass loss at 100 °C in TGA measurements. d Determined from the ceramic yield (Na2CO3) at 600 °C in TGA measurements. eCalculated from the number average DPn determined by 1H NMR and includes the mean number of Na+ ions and water molecules per repeat unit. fThese values are taken from Table 1 and refer to values determined for the RAFT polymers 3. [monomer 1 (1.50 g, 7.0 mmol), chain transfer agent 2 (112.4 mg, 0.18 mmol), and AMBN (8.45 mg, 0.044 mmol)]. The shortest chain polymer was purified by transferring a solution in methanol into a 1 kDa dialysis membrane and dialyzing against methanol for 5 days. The 1 H NMR spectra for these two polymers are presented in Figures S4A and S4B. Pendant Group Transformations. Removal of the RAFT Agent and Deprotection of the tBoc Groups. Polymer 3 was treated with ethanolamine in THF to remove the RAFT agent, and then the free −SH groups at the chain end were subjected to a Michael reaction with excess 1. These polymers were immediately treated with a mixture of DCM and TFA to remove the tBOC protecting groups. Details of one example are provided below. Bi-PBocNAAm (DPn = 40) (50.10 mg, 0.0057 mmol) was placed in a 20 mL glass vial. The vial was capped with a rubber septum and purged with nitrogen for 5 min. Then anhydrous THF (0.5 mL), which had been purged with nitrogen for 5 min, was injected into the vial to dissolve the polymer. After all the polymer had dissolved, ethanolamine (3.5 μL, ca. 10 equiv to moles of polymer chains) was injected into the vials for aminolysis of the dithiobenzoate groups. After 30 min, the yellow color of the dithiobenzoate ester group disappeared. Monomer 1 (18.01 mg, 0.084 mmol, ca. 15 equiv to moles of polymer chains) was dissolved in anhydrous THF (0.2 mL), which had been degassed with nitrogen, and this solution was injected into the vial to react with −SH groups on the polymer chain ends. The reaction was stirred for 4 h. After the solvent was removed with a rotary evaporator, the solid was dissolved in methanol and dialyzed against methanol in a 1 kDa MWCO dialysis bag for 2 days. Then the solution was concentrated with a rotary evaporator. The solid was dissolved in a mixture of DCM and TFA (7:3) (2 mL) and stirred for 4 h at RT to remove the tBOC groups. After that, the polymer was precipitated into 20 mL of diethyl ether. The supernatant was decanted, and the solid was further dried under vacuum. The amino polymer was obtained as a white solid, as the polytrifluoroacetate salt (38.14 mg, 67%). Synthesis of Poly(acrylamide-diethylene-diamide-DTPA) [BiPAAm(DTPA)]. DTPA (3.76 g, 9.55 mmol, about 80 equiv to the amine groups on the polymer) was dissolved in 5 M aqueous NaOH, adding enough base to adjust the pH to 8.5 (monitored with a pH meter). DMTMM (251 mg, 0.91 mmol, ca. 8 equiv to the amine groups on the polymer) was dissolved in DI water (2 mL) and added into the DTPA solution. The solution was given 5 min to prereact. Then a sample of the amino polymers described above (25.02 mg) was dissolved in DI water (2 mL) and added into the solution containing DTPA and DMTMM. The solution was stirred for 1 h, transferred to a 15 mL, 3 kDA MWCO Millipore Amicon spin filter, and washed with water (9 × 11 mL). Finally, the aqueous solution was freeze-dried to yield the biotin-PAAm (DTPA) polymer (40 mg). Molecular Weight Determination. The number average DPn values were determined by end group analysis using 1H NMR. For the as-prepared RAFT polymer, we compared signals due to the CH2 groups of the biotin as well as the phenyl protons of the RAFT agent to the tert-butyl groups of the repeat units. Polymers (10 mg) were dissolved into CD3OD (1 mL), and 2 drops of D2O were added. 1H NMR spectra were acquired with 10 s delay times and 512 scans. Curiously, spectra of polymers taken in a mixture of methanol-d4 and

Bis-PFP-ACV (3.10 g, 5.1 mmol), dithiobenzoic acid disulfide (1.06 g, 3.4 mmol), and ethyl acetate (50 mL) were placed into a 250 mL RBF, which was then capped with a septum and bubbled with nitrogen for 20 min. The reaction mixture was refluxed for 16 h. Then the solvent was removed on a rotary evaporator, and the remaining solid was purified by flash chromatography (hexane/chloroform = 4:1, silica 400 mL). Using 3 L eluent, we obtained 2.83 g of CTA-PFP (63%). 1 H NMR (400 MHz, CDCl3, δ (ppm), cf., Figure S3A): 7.91 (d, 2 H, o-Ar), 7.57 (t, 1 H, p-Ar), 7.39 (t, 2 H, m-Ar), 3.08−3.01 (m, 2 H, O C−CH2), 2.80−2.70 (m, 1 H, OCH2−CHH), 2.59−2.49 (m, 1 H, OCH2−CHH), 1.97 (s, 3 H, CH3). MS (ESI m/z, [M + H]+ = 446.0). Synthesis of N-Biotinyl-N′-(3-(4-phenylthiocarbonylthio-4cyanovaleryl)-3,6-dioxaoctane-1,8-diamine CTA (2). This synthesis followed a procedure similar to that reported by Bathfield et al.34 CTA-PFP (0.38 g, 0.85 mmol) was placed in a RBF and dissolved with dry DCM (40 mL). Bi-PFP (0.30 g, 0.8 mmol) was suspended in dry DCM (27 mL) and added to the flask in three portions at 20 min intervals. After 4 h, the solution was extracted with water (3 × 70 mL). All solvent was removed on a rotary evaporator. Then the raw compound was purified by two consecutive column chromatography steps (initially (silica 200 mL, DCM eluent 1 L), then DCM/methanol = 20:1 (silica 200 mL, eluent 3 L)). The product (0.25 g, 50%) was obtained as a purple solid. 1H NMR (400 MHz, CDCl3, δ (ppm) cf., Figure S3B): 7.91 (d, 2 H, o-Ar), 7.57 (t, 1 H, p-Ar), 7.39 (t, 2 H, mAr), 6.99 (t, 1 H, NHC(O)C2H4C(CN)(CH3)S), 6.20, 5.70 (s, 2 H, NH), 4.82 (t, 1 H, NHC(O)(C4H8)), 4.48, 4.30 (m, 2 H, CHNH), 3.63−3.39 (m, 12 H, CH2CH2OCH2CH2OCH2CH2), 3.14 (m, 1 H, CHS), 2.90 (dd, 1 H, CHHexoS), 2.76−2.54 (m, 4 H, OCH2− CH2C(CN)(CH3)S), 2.43 (d, 1 H, CHHendoS), 2.22 (t, 2 H, biotinCH2CO), 1.95 (s, 3 H, CH3), 1.68−1.46 (m, 6 H, biotin-CH2). Elem. Anal.: theor C 54.78, H 6.50, N 11.01; found C 53.63, H 6.58, N 10.60. MS (ESI m/z, [M + H]+) Cal: 636.2342. Exp: 636.2346. Synthesis of Polymers through RAFT Polymerization. RAFT Polymerization To Form Biotin-poly-(N-{2-[(BOC)aminoethyl]} Acrylamide) (Bi-PBocNAAm). In these polymerizations, 1 was used as the monomer and 2 was used as the chain transfer agent, with AMBN as the initiator. Polymers with different chain lengths were synthesized by varying the ratio of monomer to chain transfer agent and by varying the reaction time (Table 1). A representative example is described below. Bi-PBocNAAm with DPn = 40. Monomer 1 (1.50 g, 7.0 mmol), chain transfer agent 2 (55.62 mg, 0.088 mmol), and AMBN (4.19 mg, 0.022 mmol) were dissolved in dioxane (10 mL) and transferred to a 50 mL Schlenk flask. The solution was degassed by five freeze− pump−thaw cycles and then sealed. The solution was stirred at 70 °C for 4 h and then quenched by cooling the reaction flask with liquid nitrogen. The polymer was purified by precipitating into a mixture of hexane/diethyl ether (1:2, 40 mL). The solid obtained was redissolved in THF and precipitated into 40 mL of a mixture of hexane and diethyl ether. This process was repeated five times. A 1H NMR spectrum of this polymer is presented in Figure 2 of the Results and Discussion section. In a similar manner, polymers were synthesized with DPn = 50 [monomer 1 (2.00 g, 9.35 mmol), chain transfer agent 2 (74.28 mg, 0.11 mmol), and AMBN (5.58 mg, 0.029 mmol)] and with DPn = 20 2835

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Scheme 1. Synthesis of the Biotin Chain Transfer Agent 2

D2O show what appear to be two discrete -OH peaks at 4.6 and 4.8 ppm (Results and Discussion; Figure 2). Examination of the solvent mixture itself also showed these two peaks in addition to the CHD2Osignal at 3.3 ppm. Following subsequent transformations of the polymer, values of DPn were estimated by comparing integrations of the CH group of the biotin at the peak at 4.3 ppm to that of signals characteristic of the pendant groups. Polymers were isolated by freeze-drying from solution at pH 8.5, containing sodium ions as well as small traces of water. To calculate micromoles of polymer for weighed samples, we needed to determine an “effective molecular weight” for each sample. This was accomplished by subjecting the polymers to thermal gravimetric analysis (TGA) as described by Majonis et al.35 Carefully weighed samples (ca. 5 mg) were heated at 100 °C to remove moisture and then at 600 °C to convert the remaining polymer to Na2CO3. From the ceramic yield, we determined that each polymer contained between 2 and 3 Na+ ions per DTPA group. These values and other characteristics of the polymers are collected in Table 2 below. As in ref 17, standard error propagation expressions were used to calculate the standard errors. We assumed an uncertainty of ±5% in NMR integrations and an inherent ±5% error for each water/DTPA value and sodium/DTPA value from the TGA measurements. A value of Mn (UV) for Bi-PBocNAAm-40 was estimated as follows: A sample of Bi-PBocNAAm-40 (2.14 mg) was dissolved in CHCl3 and diluted to 25.0 mL. Two dilutions were also prepared; then UV spectra were obtained using 1.00 cm cells. From the plot (not shown) of absorbance at λmax = 300 nm vs polymer concentration, using ε300 = 1.68 × 104 M−1 cm−1 determined from CTA 2 as the model compound, we calculated Mn (UV) = 8.2 kDa. Preparation of Immunoconjugates. Conjugation of Trastuzumab Fab Fragments to SAv. The details of covalent attachment of SAv to trastuzumab Fab fragments (tmFab-SAv) and trastuzumab Fab fragments with DTPA chelators (tmFab(DTPA)-SAv) are described elsewhere.29 Briefly, SAv was pretreated with Traut's reagent to convert a lysine amino group to a thiol for reaction with the maleimide of a heterotelechelic oligoethylene glycol with a maleimide at one end and an N-hydroxysuccinimide ester (NHS) at the other.29 This, in turn, was reacted with a lysine amine on the Fab fragments. For tmFab(DTPA)-SAv, the Fab fragment was pretreated with DTPA dianhydride to introduce the chelator; then the streptavidin was attached via an oligoethylene glycol spacer as described above. Complexation of Bi-MCPs to tmFab-SAv. Experiments were designed to obtain a molar ratio of tmFab-SAv/Bi-MCP of 1:1

based on the effective molecular weight (Mn) of the MCPs. For HER2 binding experiments, 0.04 nmol of tmFab-SAv (4.4 μg) was incubated with 0.04 nmol of Bi-PAAm(DTPA)-20, Bi-PAAm(DTPA)-40, or BiPAAm(DTPA)-50 (0.5 μg, 1.0 μg, and 1.3 μg, respectively) in a final volume of 15 μL of PBS for 30 min at RT.29 The resulting complexes were buffer-exchanged into 100 mM sodium acetate buffer, pH 6.0, by ultrafiltration on an Amicon Ultra device (MWCO = 50 kDa). Radiolabeling with 111In and Determination of Maximum SA Achievable. The maximum SA that could be achieved for labeling with 111In was identified by incubating decreasing amounts of BiMCPs (2, 0.2, 0.02, or 0.002 μg) or tmFab(DTPA)-SAv (10, 1.0, 0.1, and 0.01 μg) incorporating 2.1 ± 0.1 DTPA groups/molecule, with a constant amount of 111InCl3 (3.7 MBq) in 100 mM sodium acetate, pH 6.0, for 1 h at RT. The final volume was 15 μL. The percent labeling efficiency (LE) was measured by instant thin layer silica gel chromatography (ITLC-SG) in 100 mM sodium citrate, pH 5.0, as previously reported.29 The Rf values of 111In-labeled Bi-MCPs or 111IntmFab(DTPA)-SAv and free 111In in this system are 0.0 and 1.0, respectively. The SA obtained (MBq/μg and MBq/μmol) with the minimum mass of Bi-MCP or tmFab(DTPA)-SAv that provided a LE >80% was taken as the maximum SA practically achievable. Determination of the maximum SA achievable for Bi-MCPs complexed to tmFab-SAv was not performed, since this varies with the number of Bi-MCPs complexed to the immunoconjugates due to the tetravalency of SAv, and this was not known. For evaluation of HER2 binding, tmFab-SAv-Bi-MCP complexes and tmFab-SAv derivatized with DTPA were radiolabeled by incubation of 4 nmol of each in 30 μL of 100 mM sodium acetate buffer, pH 6.0, with 2 MBq 111InCl3 for 1 h at RT, as previously reported.29 The samples were then buffer-exchanged into PBS, pH 7.4, by ultrafiltration on an Amicon Ultra device (MWCO = 30 kDa). The final radiochemical purity was >95%, measured by ITLC-SG in 100 mM sodium citrate, pH 5.0. The Rf values of the 111In-complexes and free 111In were 0.0 and 1.0, respectively. HER2 Binding. HER2 binding of 111In-labeled tmFab(DTPA)-SAv or tmFab-SAv complexed to Bi-MCPs was measured using SKOV-3 human ovarian cancer cells (1 × 106 HER2/cell). Briefly, 1.5 × 105 cells were cultured overnight in wells in a 24-well plate (Sarstedt, QC) in RPMI-1640 growth media (Sigma-Aldrich, USA) containing 10% fetal bovine serum. Following removal of the media, the cells in each well were incubated with 0.5 mL of 10 nM of RICs (0.25 MBq/μg) in serum-free medium for 3 h at 4 °C. Nonspecific binding was evaluated by repeating the assay in the presence of a 100-fold excess of unlabeled 2836

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Scheme 2. RAFT Polymerization of Monomer 1 and Subsequent Transformation of the Polymer To Remove the Dithiobenzoate End Group and To Introduce DTPA Chelating Groups on Each Repeat Unit

trastuzumab IgG. The media was removed, and the adherent cells were rinsed with PBS, pH 7.4, and then solubilized in 100 mM NaOH at 37 °C for 30 min. The dissolved cells were transferred to γ-counting tubes, and the total cell-bound radioactivity was measured in a γcounter. Specific binding was calculated by subtracting nonspecific binding from total binding. HER2 binding was expressed as percentage of RICs bound per 1.5 × 105 SKOV-3 cells.

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necessary to assume that the modified polymers have DPn values very similar to those of their precursors. Synthesis of the Biotin Chain Transfer Agent. Our synthesis of the biotin chain transfer agent is outlined in Scheme 1. It is based on the idea of Bathfield et al.34 for preparing a dithiobenzoate derivative that also contains an active ester and reacting it with a biotin-amine reagent. We employed CTA-PFP, first reported by Theato and coworkers,33 and reacted it with Bi-EO2NH2. In this reaction sequence, both the carboxylic groups of the biotin and ACVA were activated by the formation of PFP activated ester groups. While the Bi-PFP activated ester was synthesized effectively through traditional DCC coupling chemistry (yield 78%), we employed TFAA as a coupling agent, as performed previously by Green et al.,36 to synthesize the PFP-activated ACVA (BisPFP-ACV). Here the yield was somewhat higher (88%), and we avoided the cumbersome removal of dicyclohexylurea as a byproduct in the DCC coupling reaction. CTA-PFP itself was synthesized by refluxing Bis-PFP-ACV with dithiobenzoate disulfide in ethyl acetate. In the reaction of CTA-PFP with BiEO2NH2, we chose to employ a small excess of CTA-PFP. While the PFP ester is more reactive to amines than the dithiobenzoate, we were concerned that an excess of amine might lead to some aminolysis of the CTA. The biotin functionalized CTA was then purified by column chromatography. 1H NMR spectra of the biotin-CTA 2 and several of its precursors are presented in Figures S2−S4. RAFT Polymerization. The synthesis of the Bi-MCPs described here began with RAFT polymerization of the monomer N-{2-[(BOC)aminoethyl]} acrylamide (1) in dioxane in the presence of the biotin-RAFT agent N-biotinylN′-(3-(4-phenylthiocarbonylthio-4-cyanovaleryl)-3,6-dioxaoctane)-1,8-diamine (2), initiated by AMBN. Our target was the

RESULTS AND DISCUSSION

Our philosophy for the synthesis of end-functional MCPs is based on the idea that one should begin with the polymerization of a readily available monomer to obtain a polymer for which the number average of DPn can be determined with confidence by 1H NMR. Once the base polymer is synthesized and characterized, we carry out a series of pendant group transformations, using reactions that occur in close to quantitative yield, to introduce metal chelating groups such as DTPA on each of the repeat units. Polydispersity can be checked by SEC, and we monitor DPn by end group analysis after each step. In our experience, however, pendant group transformations introduce new signals to the 1H NMR. The NMR spectra become more complex, and some of the new signals may overlap signals associated with the end group. Thus, it becomes more difficult to quantitatively compare the integrals of peaks associated with the end group to those associated with backbone or pendant group protons. This problem is particularly acute here, where, in the initial polymer synthesis, the dithiobenzoate end group allows values of DPn to be determined with confidence by 1H NMR. But in later steps of the synthesis, after removal of the dithiobenzoate, we have only individual protons of a biotin end group as a reference peak for estimating DPn by 1H NMR. Then it becomes 2837

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The polymers were then treated with TFA in DCM to remove the tBOC groups and to yield the deprotected polymer 5 as its polytrifluoroacetate salt. In the 1H NMR spectra, the peaks for the tert-butyl groups at ca. 1.4 ppm disappeared, and two sharp peaks appeared at 2.9 and 3.3 ppm, representing the CH2 groups on the pendant protonated aminoethyl amide substituents. The 1H NMR spectrum of 5 (DPn = 40) is presented in Figure S8. The pendant amino groups on these polymers were converted to DTPA monoamide derivatives following the procedure developed by Majonis et al.17 In this approach, a large excess of DTPA is prereacted with a limiting amount of the coupling agent DMTMM in aqueous solution at pH 8.5. The polymer in water is then added, and the reaction is complete in about an hour. These conditions are effective at quantitative conversion of polymer primary amines to DTPA monoamides without any detectable cross-linking that would appear as peak broadening in SEC measurements. The polymers were subsequently purified from water-soluble low molecular weight side products and excess reactants by dialysis or spin filters.39 The polymers were then isolated as powders by lyophilization. SEC traces (in water) of the three polymers 6 obtained in this way are presented in Figure 3, and values of Mn and PDI

synthesis of three samples of Bi-PBocNAAm with DPn of approximately 20, 40, and 50. The reaction was stopped by cooling the reaction vessel and then pouring the contents into a mixture of diethyl ether and hexane to precipitate the polymer. Ether itself was ineffective because the polymers show some solubility in this solvent. In Table 1 we present the ratios of reactants used in these syntheses and the values of the number average of DPn determined by 1H NMR. By varying the ratio of reactants and reaction time, polymers with different chain lengths were achieved, and the number average DPn values determined by 1 H NMR corresponded closely with the target values. The polymer with the DPn of 20 had to be purified by dialysis in methanol because we were unable to find a ratio of hexane to diethyl ether suitable to separate this short chain polymer from unreacted monomer. The 1H NMR spectrum of Bi-PBocNAAm-40 with DPn = 40 is presented in Figure 2. The signals from the protons on the dithiobenzoate end groups appear at 7.91, 7.57, and 7.39 ppm. To determine DPn, the integral of these signals was compared with the integral of peaks appearing in the range 2.59−0.38 ppm. These peaks contain the signals of protons of the polymer backbone (d and e in Figure 2) and protons from the tert-butyl (Boc) groups (h). The signals in the region of ca. 3−4 ppm represent protons on the ethylene diamine linkers overlapped with the peaks of methanol protons. Peaks m, n, and i−l from the biotin-EO2NH-end group are resolved in the spectrum, and they confirm the presence of biotin groups on the polymers. Analysis of all three RAFT polymers in this way gave the values of DPn collected in Table 1. SEC traces for all three polymers are presented in Figure S5 (Supporting Information). Calculated values of Mn and the polydispersity index PDI based on PMMA standards are also presented in Table 1. Postpolymerization Modification. In Scheme 2, we present the chemical reactions for the RAFT polymerization and for all the postpolymerization modifications of the polymers we prepared. For the applications described below, we wanted to avoid any complications that might be caused either by the chain transfer group or by free thiol groups that might be liberated in situ due to reactions of this group. Following the lead of others,37 we removed the dithiobenzoate group of the RAFT polymer 3 by aminolysis with ethanolamine and then trapped the free thiols by Michael addition with the acrylamide monomer 1. In the 1H NMR spectra of 4, we note the disappearance of the aromatic protons of the chain transfer agent (Figure S6). We also observed a new peak at δ = 2.9 ppm due to protons on the carbon adjacent to the thiol ether end group.38 An example of the polymer with DPn = 20 is shown in Figure S6. With the removal of the dithiobenzoate group, it becomes significantly more difficult to use 1H NMR to obtain reliable values of DPn. Signals associated with the biotin end group can be resolved, but it is difficult to compare the peak integrations of the pendant group signals with those of the end group. At this stage of the synthesis, we prefer to rely on SEC chromatograms to monitor chain length and polydispersity. In SEC chromatograms (Figure S7), the transformation of 3 to 4 largely preserved the shape of the peak for each polymer, with a small peak shift for the sample with DPn = 20. These SEC traces provide evidence that end group transformation did not significantly change the chain length or polydispersity of the polymers at this stage.

Figure 3. SEC chromatograms of Bi-PAAm(DTPA)-50, Bi-PAAm(DTPA)-40, and Bi-PAAm(DTPA)-20 in water at 30 °C containing 0.2 M KNO3, 25 mM phosphate buffer (pH 8.5), and 200 ppm NaN3. The traces were analyzed by comparison to PMAA standards.

calculated by reference to poly(methacrylic acid) standards are collected in Table 2. For the shortest chain polymer (DPn = 20), it is possible to compare 1H NMR pendant group signals to those of peaks from the biotin. In this analysis, the integral of peak a (1 H) from the biotin (Figure 4) at 4.3 ppm was compared to the integral of the signal at 1.0−2.25 ppm associated with the backbone protons d and e. In this way we determined DPn(NMR) = 20.4 ± 1.4 (Table 2). By comparing the integrals of peaks at 2.8−3.4 ppm associated with the protons of the pendant groups and DTPAs with the integrals of the protons on the backbone, we determined that there are 0.99 DTPA per repeat unit, i.e., quantitative attachment of DTPA units to each pendant group. The corresponding spectra of BiPAAm(DTPA)-40 and Bi-PAAm(DTPA)-50 are presented in Figure S9. While the DPn can be measured by 1H NMR for BiPAAm(DTPA)-20 and estimated for Bi-PAAm(DTPA)-40 and 2838

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Figure 4. 1H NMR spectrum of Bi-PAAm(DTPA)-20 in D2O. DPn = 20.4 ± 1.4 was calculated by comparing the integral for peaks d + e to that of a (1 H) from the biotin at 4.3 ppm.

Bi-PAAm(DTPA)-50, converting weighed amounts of these samples to moles of polymers requires knowledge of the effective molecular weight of the samples. These samples, obtained by DTPA attachment at pH 8.5, followed by washing with DI water and lyophilization, contain traces of residual moisture and sodium ions associated with partial neutralization of the −COOH groups of the DTPA units. In order to determine the moisture and Na+ ion content of each sample, aliquots of each powder were subjected to two-step TGA. As shown by Majonis et al.,35 heating at 100 °C leads to loss of moisture without subsequent decomposition of the polymer. Subsequent heating in air at 600 °C converts all nonvolatile material to Na2CO3. The TGA traces are presented in Figure S10 of the Supporting Information. The results of this analysis and calculated effective Mn values are shown in Table 2. Complexation of Biotin-MCPs with tmFab-SAv and Radiolabeling with 111In. While SAv can in principle bind up to four MCPs, we did not attempt to saturate the binding of polymer to tmFab-SAv, due to possible interference with the tertiary structure of the binding epitope of the trastuzumab Fab fragment for HER2. 111In labeling was conducted under slightly acidic conditions (100 mM acetate buffer, pH 6.0), as is routinely performed,40 to inhibit possible hydrolysis of the In3+ ions. The binding of the Bi-MCP to tmFab-SAv after labeling with 111 In was examined by HPLC-SEC using UV/vis (280 nm) and radioactivity detectors. The results for Bi-PAAm(DTPA)-40 are presented in Figure 5, with the corresponding chromatograms for Bi-PAAm(DTPA)-20 and Bi-PAAm(DTPA)-50 provided in Figure S11. The superposition of the UV signal, which detects the tmFab-SAv, and the radioactivity signal, which detects the 111 In bound to the Bi-MCP, confirms that the polymer was complexed to tmFab-SAv. In order to verify complexation of Bi-MCPs to tmFab-SAv, we compared the HPLC-SEC chromatograms using UV detection only at 280 nm of tmFab-SAv-BiPAAm(DTPA)-20

Figure 5. HPLC-SEC chromatogram of tmFab-SAv after treatment with 1 equiv of Bi-PAAm(DTPA)-40-111In. The narrower peak (green) is the response of the UV detector at 280 nm. The larger diameter of the flow cell in the radioactivity detector leads to peak broadening. [Flow rate, 0.35 mL/min; BioSep-SEC-4000 column (300 mm × 4.6 mm). Eluent 0.1 M NaH2PO4, pH 7.0; injection volume 20 μL; UV detector at 280 nm.]

with tmFab-SAv (Figure 6). The Bi-MCP-tmFab-SAv complex eluted about 2 min earlier than tmFab-SAv not complexed to Bi-MCP. The shift in elution to shorter retention times for a polymer conjugate is similar to results reported by Zheng et al.41 This shift is too large to be caused only by the increase of molecular weight of the Bi-MCP-tmFab-SAv complex compared to the case of tmFab-SAv. Bi-PAAm(DTPA)-20, BiPAAm(DTPA)-40, and Bi-PAAm(DTPA)-50 are negatively charged polyelectrolytes. In their random conformation state in 100 mM phosphate buffer, these Bi-MCPs may make a disproportionately large contribution to the hydrodynamic volume of the Bi-MCP-tmFab-SAv complex, resulting in a shorter retention time. In Figure 7, HPLC-SEC chromatograms of RICs formed from the three Bi-MCPs with different DPn values are shown. Although Bi-MCP-tmFab-SAv complexes with longer polymer 2839

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Bi-PAAm(DTPA)-50 has the largest polydispersity, and tmFabSAv-Bi-PAAm(DTPA)-50 shows the broadest peak in the HPLC-SEC analysis. Evaluation of Maximum Achieved Specific Activity of Bi-MCPs. To examine the maximum SA of the three Bi-MCPs for complexing 111In, a comparison of the SA achievable was made with that for tmFab(DTPA)-SAv (Table 3). The maximum SA achieved was calculated based on the minimum mass which provided a LE > 80%. The SA values achieved for all three polymers were very similar (175 ± 2 MBq/μg) with a LE ≥ 92%. The maximum SA achieved on a per mole basis was 223 ± 20, 429 ± 50, and 560 ± 60 MBq/μmol, respectively, for Bi-MCPs harboring 20, 40, or 51 pendant DTPA chelators. These results indicate that the 111In3+ binding capacity increases linearly with the number of DTPA groups on the polymer. They further suggest that there are no negative cooperativity effects that prevent very small amounts of these polymers from complexing 111In. The maximum SA achieved on a per mole basis for the different polymers also indicates that SA is dependent on chain length. Particularly notable is that the maximum SA achieved with tmFab-SAv modified with two DTPA chelators per molecule was only 0.3 MBq/μg (3.4 MBq/ μmol), which is 55-fold to 138-fold lower than that for BiMCPs. These results not only confirm Torchilin’s suggestion that polymers with multiple chelators conjugated to a bioaffinity agent can achieve a higher per mole SA than one bearing a small number of these groups,3 but they show for these three polymers that the SA increases linearly with the number of DTPA groups, which corresponds to the chain length. HER2 Binding. To assess the HER2 binding of 111In-labeled tmFab-SAv-Bi-MCP complexes, their specific binding to SKOV-3 human ovarian cancer cells (1 × 106 HER2/cell) was determined and compared to that of 111In-labeled tmFab(DTPA)-SAv. The results of these assays are shown in Figure 8, presented as percentage of RICs bound per 1.5 × 105 cells. For 111Inlabeled tmFab(DTPA)-SAv, the proportion of specific binding was (88.8 ± 2.1)%. In comparison, the proportion of specific binding for 111 In-labeled tmFab-SAv-PAAm(DTPA)-20, tmFab-SAv-PAAm(DTPA)-40, and tmFab-SAv-PAAm(DTPA)-50 was (40.93 ± 1.6)%, (26.5 ± 5.4)%, and (26.3 ± 1.4)%, respectively.

Figure 6. HPLC-SEC chromatogram of tmFab-SAv-Bi-PAAm (DTPA)-20-111In and tmFab-SAv. Flow rate, 0.35 mL/min; BioSepSEC-4000 column (300 mm × 4.6 mm). Eluent 0.1 M NaH2PO4, pH 7.0; injection volume 20 μL; UV detector at 280 nm. The large shift between the tmFab-SAv and tmFab-SAv-Bi-PAAm(DTPA)-20 confirms the complexation of polymer to tmFab-SAv.

Figure 7. Comparison of HPLC-SEC chromatograms (UV detection) for tmFab-SAv conjugated to the three different Bi-MCPs of different chain lengths: tmFab-SAv-Bi-PAAm(DTPA)-20-111In, tmFab-SAv-BiPAAm(DTPA)-40-111In, tmFab-SAv-Bi-PAAm(DTPA)-50-111In. Flow rate, 0.35 mL/min; BioSep-SEC-4000 column (300 mm × 4.6 mm). Eluent: 0.1 M NaH2PO4, pH 7.0; injection volume 20 μL; UV detector at 280 nm.

chains show slightly earlier retention times, the differences among these three complexes are not as great as the differences in retention times between the Bi-MCP-tmFab-SAv complexes and tmFab-SAv. The differences in peak broadening of the three Bi-MCP-tmFab-SAv complexes (Figure 7) compared to tmFab-SAv (Figure 6) are likely due to the differences in polydispersity of the MCPs (Table 2). Of the three polymers,

Table 3. Maximum Achieved Specific Activity (SA) for 111In Labeling of Metal-Chelating Polymers (MCPs) of Varying Chain Lengths and tmFab(DTPA)-SAv Which Harbors 2 DTPA Chelators complex

massa (μg)

amount of DTPA (nmol)

Bi-PAAm(DTPA)20

2 0.2 0.02 2 0.2 0.02 2 0.2 0.02 10

3.28 0.328 0.0328 3.28 0.328 0.0328 3.22 0.322 0.0322 0.16

Bi-PAAm(DTPA)40

Bi-PAAm(DTPA)50

tmFab(DTPA)-SAv

labelingb efficiency (%)

specific activity (MBq/μg)

specific activity (MBq/μmol × 104)

± ± ± ± ± ± ± ± ± ±

1.80 ± 0.001 18.0 ± 0.02 174 ± 1.5 1.80 ± 0.01 18.0 ± 0.01 172 ± 2.0 1.80 ± 0.03 18.1 ± 0.03 177 ± 1.8 0.31 ± 0.003

2.33 ± 0.01 23.1 ± 0.3 223 ± 20 4.53 ± 0.1 45.0 ± 0.2 429 ± 50 5.65 ± 0.8 57.4 ± 0.9 560 ± 60 4.05 ± 0.04

98.6 97.5 94.1 97.9 97.2 92.8 96.3 98.0 95.5 84.4

0.1 0.1 1.0 0.3 0.1 1.0 1.4 0.2 1.0 0.9

a

. Decreasing amounts of Bi-MCP or tmFab(DTPA)-SAv were labeled with a constant amount of 111In (3.7 MBq). Each experiment was done in triplicate; the standard error of the mean is reported. b. The labeling efficiency was determined by ITLC and used to calculate the achievable activity for the polymers. 2840

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binding to HER2+ SKOV-3 cells was decreased approximately 2-fold. The shortest chain polymer (DPn = 20) yielded modestly higher specific HER2 binding complexed to tmFabSAv than the two longer polymers (DPn = 40, 50), but the differences were not significant. The maximum achievable SA of these polymers for complexing 111In was 55- to 138-fold higher than that for DTPA-tmFab-SAv, and it was linearly correlated with chain length and the number of pendant DTPA groups. These polymers provide an attractive route to increasing the amount of 111In delivered to HER2+ tumor cells per receptor recognition event, but the effects of polymer composition on nonspecific binding to cells should be further investigated.

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ASSOCIATED CONTENT

* Supporting Information S

1

Figure 8. Percent binding of RICs bound to HER2+ SKOV-3 human ovarian cancer cells for 111In-labeled tmFab(DTPA)-SAv and for 111Inlabeled tmFab-SAv-Bi-PAAm(DTPA)-20, tmFab-SAv-Bi-PAAm(DTPA)-40, and tmFab-SAv-Bi-PAAm(DTPA)-50. Error bars represent the mean ± SEM, n = 3. The asterisk represents p < 0.0001 for comparison with polymer RICs.

H NMR spectra of monomer 1, of the precursors to the CTA 2, of Bi-PBocNAAm-20 and Bi-PBocNAAm-50, of BiPBocNAAm-20 after end group modification, of the (deprotected) polyamino-polymer 5 (DPn = 40) after removal of the tBOC groups, and of Bi-PAAm(DTPA)-40 and Bi-PAAm(DTPA)-50; size exclusion chromatograms of Bi-PBocNAAm20, Bi-PBocNAAm-40, and Bi-PBocNAAm-50 at 80 °C in NMP; TGA traces of Bi-PAAm(DTPA)-n with n = 20, 40, 50; and HPLC-SEC chromatograms of tmFab-SAv-Bi-PAAm(DTPA)-20-111In and tmFab-SAv-Bi-PAAm(DTPA)-50-111In with UV and radioactivity detection. This material is available free of charge via the Internet at http://pubs.acs.org.

These results revealed that complexation of tmFab-SAv with Bi-MCPs increased nonspecific binding to SKOV-3 cells (i.e., binding that could not be displaced by an excess of unlabeled trastuzumab IgG) such that the proportion of specific binding was lower than that found with tmFab(DTPA)-SAv. There was a modest chain length effect observed for the Bi-MCPs, in that the polymers with DPn = 40 and 50 exhibited a slightly lower proportion of HER2 specific binding. Although there was an increase in the nonspecific binding of tmFab-SAv complexed to Bi-MCPs, these complexes retained specific binding to HER2. The reasons for the increase in nonspecific binding are not currently known and are an area for further investigation. Possible explanations include steric effects of the hydrodynamic volume of the polymer and the electrostatic effects associated with the negative charges along the polymer backbone. We anticipate that further studies with polymers of different backbone structure, polymers with different pendant groups (for example, zwitterionic pendant groups with no net charge), polymers with a broader range of chain lengths, and variation of the site of attachment on Fab or an intact antibody will provide a richer appreciation of these effects from which we expect to develop mechanistic explanations of how the immunoreactivity of antibodies is related to polymer conjugation.

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AUTHOR INFORMATION

Corresponding Authors

*[email protected] (R.M.R.). *[email protected] (M.A.W.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors gratefully acknowledge financial support from the Canadian Institutes of Health Research/Natural Sciences and the Engineering Research Council Collaborative Health Research Program (Grant No. CHRPJ 365423-09; CPG95268), as well as from DVS Sciences. Y.L. thanks the Chinese Scholarship Council for a scholarship to come to the University of Toronto. We also thank Dr. M. Soleimani for assistance with the TGA measurements, as well as D. Majonis for helpful discussions.

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ABBREVIATIONS ACVA, azobis(4-cyanovaleric acid); AMBN, 2,2′-azobis(2methylbutyronitrile); BC, breast cancer; Bi-EO2NH2, N-1[2,2′-(ethylenedioxy)diethyl-1′-amino]biotinylamide; Bi-MCP, biotin-end-capped metal-chelating polymer; Bi-PAAm(DTPA), poly(acrylamide-diethylene-diamide-DTPA; Bi-PFP, biotinylpentafluorophenyl ester; Bis-PFP-ACV, bis(pentafluorophenyl)azobis(4-cyanovalerate); Bi-PBocNAAm, biotin-poly-(N-{2-[(BOC) aminoethyl]} acrylamide); CTA, chain transfer agent; CTA-PFP, pentafluorophenyl-[4-(phenylthiocarbonylthio)-4-cyanovalerate]; DCC, N,N′-dicyclohexylcarbodiimide; DCM, dichloromethane; DMF, dimethylformamide; DMTMM, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride; DPn, degree of polymerization; DI, deionized; DTPA, diethylenetriaminepentaacetic acid; ECD, extracellular domain; HER2, human epidermal growth factor

CONCLUSIONS Using RAFT polymerization, we synthesized three well-defined polyacrylamides of different chain length, all with a biotin end group. Through postpolymerization modification, metal-chelating (DTPA) pendant groups were attached to each repeat unit. The polymers were characterized by a combination of 1H NMR and SEC to determine DPn and polydispersity, and then by TGA to determine the moisture and Na+ ion content of the polymers obtained by lyophilization at pH 8.5. These MCPs were conjugated to tmFab-SAv through the strong affinity (Ka = 1015 M−1) between biotin and SAv. After labeling with 111In, these RICs were evaluated by HPLC-SEC to confirm the conjugation of MCPs. Cell binding assays revealed that the tmFab-SAv-MCP complexes have a higher proportion of nonspecific binding compared to tmFab-SAv modified with DTPA and labeled with 111In. Thus, the proportion of specific 2841

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Biomacromolecules

Article

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receptor-2; HPLC, high pressure liquid chromatography; ICPMS, inductively coupled plasma mass spectrometry; ITLC-SG, instant thin layer silica gel chromatography; LE, labeling efficiency; mAb, monoclonal antibody; MCP, metal-chelating polymer; MWCO, molecular weight cut-off; NHS, Nhydroxysuccinimide ester; NLS, nuclear localization sequence; NMP, N-methylpyrrolidone; PBS, phosphate buffered saline; PEG, polyethylene glycol; PFP, pentafluorophenyl; pfp-OH, pentafluorophenol; PMMA, poly(methyl methacrylate); RAFT, reversible addition−fragmentation transfer; RIC, radioimmunoconjugate; RBF, round-bottom flask; RT, room temperature; SA, specific activity; SAv, streptavidin; SEC, size exclusion chromatography; tBoc-DE, N-(tert-butoxycarbonyl)-1,2-diaminoethane; TE, triethylamine; TFA, trifluoroacetic acid; TFAA, trifluoroacetic anhydride; TGA, thermal gravimetric analysis; THF, tetrahydrofuran; tmFab, trastuzumab Fab fragment; TOF, time-of-flight

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dx.doi.org/10.1021/bm300843u | Biomacromolecules 2012, 13, 2831−2842