Metal-Chelating Polymers (MCPs) with Zwitterionic Pendant Groups

Oct 15, 2015 - Terrence Donnelly Center for Cellular and Biomolecular Research, University of Toronto, 160 College Street, Toronto, Ontario Canada M5S...
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Metal-Chelating Polymers (MCPs) with Zwitterionic Pendant Groups Complexed to Trastuzumab Exhibit Decreased Liver Accumulation Compared to Polyanionic MCP Immunoconjugates Peng Liu,†,# Amanda J. Boyle,‡,# Yijie Lu,† Jarrett Adams,§ Yuechuan Chi,† Raymond M. Reilly,*,‡,∥,⊥ and Mitchell A. Winnik*,† †

Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, Ontario Canada, M5S 3H6 Department of Pharmaceutical Sciences, University of Toronto, 144 College Street, Toronto, Ontario Canada M5S 3M2 § Terrence Donnelly Center for Cellular and Biomolecular Research, University of Toronto, 160 College Street, Toronto, Ontario Canada M5S 3E1 ∥ Department of Medical Imaging, University of Toronto, 263 McCaul Street, Toronto, Ontario Canada M5T1W7 ⊥ Toronto General Research Institute and Joint Department of Medical Imaging, University Health Network, Toronto, Ontario Canada, M5G 2M9 ‡

S Supporting Information *

ABSTRACT: Metal-chelating polymers (MCPs) can amplify the radioactivity delivered to cancer cells by monoclonal antibodies or their Fab fragments. We focus on trastuzumab (tmAb), which is used to target cancer cells that overexpress human epidermal growth factor receptor 2 (HER2). We report the synthesis and characterization of a biotin (Bi) end-capped MCP, Bi-PAm(DET-DTPA)36, a polyacrylamide with diethylenetriaminepentaacetic acid (DTPA) groups attached as monoamides to the polymer backbone by diethylenetriamine (DET) pendant groups. We compared its behavior in vivo and in vitro to a similar MCP with ethylenediamine (EDA) pendant groups (Bi-PAm(EDA-DTPA)40). These polymers were complexed to a streptavidin-modified Fab fragment of tmAb, then labeled with 111In to specifically deliver multiple copies of 111In to HER2+ cancer cells. Upon decay, 111In emits γ-rays that can be used in single-photon emission computed tomography radioimaging, as well as Auger electrons that cause lethal double strand breakage of DNA. Our previous studies in Balb/c mice showed that radioimmunoconjugates (RICs) containing the Bi-PAm(EDA-DTPA)40 polymer had extremely short blood circulation time and high liver uptake and were, thus, unsuitable for in vivo studies. The polymer Bi-PAm(EDA-DTPA)40 carries negative charges on each pendant group at neutral pH and a net charge of (−1) on each pendant group when saturated with stable In3+. To test our hypothesis that charge associated with the polymer repeat unit is a key factor affecting its biodistribution profile, we examined the biodistribution of RICs containing Bi-PAm(DET-DTPA)36. While this polymer is also negatively charged at neutral pH, it becomes a zwitterionic MCP upon saturation of the DTPA groups with stable In3+ ions. In both nontumor bearing Balb/c mice and athymic mice implanted with HER2+ SKOV-3 human ovarian cancer tumors, we show that the zwitterionic MCP has improved biodistribution, higher blood levels of radioactivity, lower levels of normal tissue uptake, and higher tumor uptake. Surface plasmon resonance experiments employing the extracellular domain of HER2 show that the MCP immunoconjugates retain high affinity antigen recognition, with dissociation constants in the low nM range. In vitro studies with SKOV-3 cells for both MCP immunoconjugates show a combination of specific binding that can be completed in the presence of excess tmAb IgG and nonspecific binding (NSB) that persists in the presence of tmAb IgG. We conclude that zwitterionic MCPs represent a much better choice than polymers with charges along the backbone for in vivo delivery of RICs to HER2+ cancer cells.

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INTRODUCTION

to tmAb combined with chemotherapy, and even those cancers that do respond, the response diminishes over time, and after about a year, the treatment is no longer effective.2 One

Monoclonal antibodies (mAbs) targeting human epidermal growth factor receptor (HER2), such as trastuzumab (tmAb, Herceptin) and pertuzumab (Perjeta), are used for the treatment of HER2+ breast cancer. Unfortunately, not all cancers respond to treatment with “naked” mAbs.1 This is seen where only 50% of women with HER2+ breast cancers respond © XXXX American Chemical Society

Received: August 6, 2015 Revised: September 24, 2015

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DOI: 10.1021/acs.biomac.5b01066 Biomacromolecules XXXX, XXX, XXX−XXX

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Biomacromolecules

with 111In was used to examine the effects of polymer structure and chain length on normal and tumor distribution in mice, as well as for in vitro studies of immunoreactivity and cellular uptake. These nanoscale structures should be large enough to avoid renal clearance, since the tmAbFab-SAv conjugate itself has an estimated M = 130 kDa, larger than the M < 60 kDa threshold for renal clearance,13 and the polymers add an additional 10 to 25 kDa, not including the chelated metal ions. Because of the immunoreactivity of SAv, these complexes are not suitable to be employed clinically. They are, however, effective preclinically as a rapid method of examining sitedirected MCPs. Ultimately, we intend to link the most promising MCPs to tmAb IgG, where the MCP-tmAb immunoconjugates will be labeled to high specific activity for enhanced Auger electron radioimmunotherapy of HER2+ breast cancer.14 In one example, we described a site-specific conjugation of a polyglutamide MCP to oxidized glycans on the Fc domain of tmAb. This study found that the specific activity of 111In-labeled tmAb could be increased 90-fold through this covalent attachment of the MCP to the antibody, which greatly amplified the cytotoxic effect against HER2+ breast cancer cells.10 Thus far, in examining the traits of MCPs, we have explored three different Bi end-capped MCPs of similar length, one with a polyaspartamide backbone and DPn = 33 [Bi-PAsp(DETDTPA)33], one with a polyglutamide backbone and DPn = 28 [Bi-Glu(DET-DTPA)28], and one with a polyacrylamide backbone and DPn = 40 [Bi-PAm(EDA-DTPA)40], where DET (diethylenetriamine diamide) and EDA (ethylenediamine diamide) refer to the linkers used to connect the DTPA chelators to the polymer backbone.7 In comparing the total percent radioactivity recovery (percentage of radioactivity remaining in the mice at 24 h compared to the injected radioactivity) from sampled tissues, the polyglutamide conjugate had the lowest total recovered radioactivity (22.5 ± 1.5%) at 24 h post-injection (p.i.), approximately 2-fold lower than that for the polyaspartamide conjugate (40.6 ± 3.1%). A low value of the total recovered radioactivity implies that radioactive species were excreted by the animal. These values were not significantly affected by saturation of the uncomplexed DTPA with stable 115InCl3. However, both polypeptide conjugates not saturated with indium showed a strong tendency to accumulate in the kidney. Kidney uptake was reduced 3-fold (p < 0.001) for tmAbFab-SAv-Bi-PGlu(DET-DTPA)28 with indium saturation, but there was no corresponding significant effect with tmAbFab-SAv-Bi-PAsp(DET-DTPA)33. The most striking effect was seen with the polyacrylamide tmAbFab-SAvBi-PAm(EDA-DTPA)40. This RIC had the highest total recovered radioactivity, 91 ± 4% with the polymer complexed with a low level of 111In, and this retention of radioactivity was associated with a high level of uptake in the liver, suggesting that liver uptake decreased the elimination of radioactivity from the body. Saturation of the DTPA groups in the polymer with indium lowered the total recovered radioactivity by 1.4-fold (p = 0.016) and the liver uptake by 1.5-fold (p < 0.05). This tendency for accumulation of the RIC in the liver was the reason that we did not use this polymer in the tumor uptake experiments also reported in ref 7. An important feature of the system to keep in mind when considering the experiments described below is that the number of negative charges along the polymer backbone is affected by the chelation state of the DTPA groups. From the pKa values of DTPA monoamide, one can estimate that at

direction for overcoming these limitations is the construction of antibody−drug conjugates (ADCs), in which a drug too toxic to apply on its own is covalently attached to the mAb for release once the ADC is internalized into the target cancer cell. For example, ado-tmAb emtansine or T-DM1 was approved in 2013 following the completion of clinical trials for the treatment of tmAb-resistant metastatic HER2+ breast cancer.3 Another direction involves conjugating metal-chelators to mAbs for binding radioisotopes for a targeted cytotoxic effect.4 We have been interested in the use of Auger electronemitting radionuclides to increase the cytotoxicity of mAbs used in the treatment of HER2+ breast cancers. Auger electrons are low energy (99%, Fisher Scientific, Ottawa, Canada) and 111InCl3 (Nordion, Inc., Kanata, ON, Canada). The tmAbFab(DTPA)-SAv conjugate, covalently coupled with a 24-mer PEG spacer, and the tmAbFab-DTPA conjugate were prepared as previously described.7 Details are provided in Supporting Information (SI). The Bi end-capped MCP Bi-poly(3,6,9-triscarboxymethyl-3,6,9triazaundecandioicacid-amido-N-ethylamidoacrylamide) [Bi-PAm(EDA-DTPA)40], with a mean degree of polymerization of DPn = 40, is the same sample as that reported in ref 7. The synthesis of Bi end-capped P(PFPA) (1) by reversible addition−fragmentation transfer (RAFT) polymerization and the subsequent removal of the dithiobenzoate end group to obtain the corresponding polymer (2) are described in the SI. As shown in the SI, the DPn of this polymer was determined by 1H NMR to be 36. This polymer is denoted BiP(PFPA)36, and we assume that the value of DPn did not change during the subsequent pendant group modifications of the polymer. Synthesis of Biotin-Poly(3,6,9-triscarboxymethyl-3,6,9-triazaundecandioicacid-amido-N-2′ethyl(2-ethylamido)acrylamide) (Bi-PAm(DET-DTPA)36) (4). Formation of the DET Polymer Biotin-Poly(2′-aminoethyl-(2-aminoethyl)acrylamide) (BiPAm(DET)36) (3). Bi-P(PFPA)36 (polymer 2, 28.8 mg) was dissolved in a dimethylformamide (DMF)/dioxane solvent mixture (1/1 v/v, 0.8 mL) and subsequently transferred to a glass vial containing DET (0.9 mL). This solution was then stirred overnight at room temperature (RT). The DET polymer was precipitated by adding the reaction solution to diethyl ether (40 mL). The precipitate was dissolved in methanol and reprecipitated into diethyl ether, and this process was repeated 3 times. The Bi-PAm(DET)36 polymer (3) was obtained as a white solid (15.2 mg, yield: 80.0%). 1H NMR (D2O, δ, ppm): 3.45 (polymer pendant group, NH2CH2CH2NHCH2CH2NH−), 3.30 (polymer pendant group, NH2CH2CH2NHCH2CH2NH−), 3.10

physiological pH (7.4) each DTPA not bound to a metal ion carries a charge of −2 or −3,15 whereas the monoamide DTPA4−−In3+ complex has a charge of −1. Thus, saturation of the uncomplexed DTPA with InCl3, following labeling with trace quantities of 111In, reduces the net charge of the polymer component of the conjugate. For the tmAbFab−SAv conjugates, each polymer had its own unique behavior. In this paper, we test the hypothesis that the most important factor that contributed to the rapid elimination from the blood and high liver uptake of tmAbFab-SAv-Bi-PAm(EDA-DTPA)40 in Balb/c mice was the polyanionic character of the MCP. In the absence of indium-saturation, there were multiple negative charges on each pendant group. When the polymer was saturated with In3+, the DTPA4−−In3+ complex had a charge of −1, as indicated in Figure 1A. Thus, the indium-saturated tmAbFab-SAv-Bi-PAm(EDA-DTPA)40 was also an anionic polyelectrolyte. In contrast, the DET spacer in the pendant group contains a secondary amine that is protonated at neutral pH. Thus, polymers with DET-DTPA pendant groups are zwitterionic when saturated with trivalent metal ions (c.f., Figure 1B). It is known that zwitterionic polymers can reduce protein adsorption to surfaces, for example, as a promising alternative to PEGylation. Incorporation of zwitterionic polymers into micelles and other colloidal particles enhances the blood circulation time of these synthetic materials.16−19 Here we describe the synthesis of a Bi end-capped polyacrylamide [Bi-PAm(DET-DTPA)36] with a DET spacer between the polymer backbone and the DTPA pendant groups. This polymer is zwitterionic when saturated with In3+ and allows a direct comparison with the Bi-PAm(EDA-DTPA)40 synthesized previously. We prepared complexes of both polymers with tmAbFab-SAv, labeled them with 111In and examined their chelator-saturated and unsaturated states in in vivo and in vitro studies.

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EXPERIMENTAL SECTION

Instrumentation. Gel permeation chromatography (GPC) analysis of poly(pentafluorophenyl acrylate) [P(PFPA)] samples, with tetrahydrofuran (THF) at 23 °C as the eluent, employed a system equipped with a Waters (Milford, USA) 515 HPLC Pump, a Polymer Laboratories gel 5 μm Mixed-D (300*7.5 mm) column protected by a gel 5 μm guard column, and a Viscotek VE 3580 RI detector. The flow rate was 0.6 mL/min, and the column was calibrated with polystyrene standards. Aqueous size exclusion chromatography (SEC) measurements employed a Viscotek (Malvern Instrument, Worcestershire, UK) VE1122 solvent delivery system equipped with a VE7510 GPC degasser, Viscotek ViscoGEL G4000PWXL and G2500 PWXL columns, which were connected to a Viscotek VE3210 UV/vis detector and a VE3580 refractive index C

DOI: 10.1021/acs.biomac.5b01066 Biomacromolecules XXXX, XXX, XXX−XXX

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Biomacromolecules

absolute binding (not corrected for SB) of RICs and unconjugated polymers was expressed as pmol bound. Tissue Biodistribution and Imaging Studies. The biodistribution of RICs consisting of 111In-labeled tmAbFab-SAv-Bi-PAm(EDADTPA)40-111In/115In and tmAbFab-SAv-Bi-PAm(DETDTPA)36111In/115In was determined in nontumor-bearing female Balb/c mice following procedures previously described.7 Groups of 3 Balb/c mice were injected intravenously (tail vein) with 10 μg 111Inlabeled polymer RICs (0.2−0.4 MBq), with the DTPA groups of the polymer backbone saturated with stable indium. Mice were sacrificed at 24 h p.i. at which time the organs were harvested. Samples of blood and selected normal tissues were collected, rinsed briefly in DI water, then air-dried, weighed, and transferred to γ-counting tubes. The tissue concentration of radioactivity was measured in a γ-counter and expressed as percent injected dose/g (% ID/g). MicroSPECT/CT imaging was performed up to 72 h p.i. in Balb/c mice injected i.v. with 2 0− 3 7 M B q ( 1 0 μg ) o f t m A b F a b - S A v - B i - P A m ( E D A DTPA) 40 - 111 In/ 115 In and tmAbFab-SAv-Bi-PAm(DET-DTPA) 36 -111In/115In. Mice were anesthetized by inhalation of 2% isoflurane in O2. Imaging was performed on a NanoSPECT/CT tomograph (Bioscan) equipped with four NaI detectors and fitted with 1.4 mm multipinhole collimators (full width at half-maximum ≤1.2 mm). A total of 24 projections were acquired in a 256 × 256 acquisition matrix with a minimum of 80000 counts per projection. Image reconstruction was performed using InvivoScope software (version 1.34β6; Bioscan) with an ordered-subset expectation maximization algorithm (9 iterations). Cone-beam CT images were acquired (180 projections, 1 s/projection, 45 kVp) before microSPECT images. Co-registration of microSPECT and CT images was performed using InvivoScope software. Tumor and Normal Tissue Localization. The tumor and normal tissue localization of tmAbFab-SAv-Bi-PAm(DET-DTPA)36-111In and tmAbFab-SAv-Bi-PAm(DET-DTPA)36-111In/115In were determined in female CD1 athymic mice (Charles River, Boston, MA) with subcutaneous (s.c.), HER2+ SKOV-3 tumors (8 mm in diameter). Tumor-xenograft mouse models were established by inoculating 1 × 107 HER2+ SKOV-3 cells with 100 μL of serum-free media s.c. on the right flank. Groups of four tumor-bearing CD1 mice were injected i.v. with 0.2−0.4 MBq (0.5 mg/kg) of one of the RICs and then sacrificed at 48 h p.i. Tumor and normal tissue uptake were measured by γcounting and expressed as % ID/g. All animal studies were carried out by following the “Principles of Laboratory Animal Care” (NIH publication #85−23, revised in 1985) and under a protocol (No. 282.9) approved by the Animal Care Committee at the University Health Network (Toronto, ON, Canada) following Canadian Council on Animal Care (CCAC) guidelines. Statistical Analysis. Data are presented as mean ± SEM. Statistical analyses were performed with an unpaired t-test Prism Ver 4.0 software (GraphPad Software Inc., San Diego, CA). Values of p < 0.05 were considered significant.

(polymer pendant group, NH2CH2CH2NHCH2CH2NH−), 2.22− 1.22 (backbone protons). Introduction of Pendant DTPA Groups to Form Bi-PAm(DETDTPA)36 (4). An aqueous solution of Bi-PAm(DET)36 polymer (3) (7 mg) was prepared by dissolving the polymer in deionized (DI) water (1 mL). DTPA (1.40 g, 80 equiv to the primary amino groups of the polymer) was solubilized by the continuous addition of 5 M aqueous NaOH (about 4.5 mL). The pH of final DTPA solution was controlled to be about 8.5 (monitored with a pH meter). DMTMM (98 mg, 8 equiv to the primary amino groups of the polymer) was dissolved in DI water (2 mL) and immediately transferred to the DTPA solution to preactivate the DTPA. After 5 min, the aqueous solution of BiPAm(DET)36 was added into the solution of DMTMM-activated DTPA, and the mixture was stirred for 1 h at RT. Then the solution was transferred to a 25 mL, 3 kDa MWCO Millipore Amicon spin filter and washed with DI water (9 washes of 11 mL each). The residual aqueous solution containing Bi-PAm(DETDTPA)36 (4) was freeze-dried to yield a white solid (20.2 mg, yield: 85.2%). 1H NMR (D2O, δ, ppm): 4.02−2.82 (protons from DET and DTPA pendant groups), 2.22−1.20 (protons on the polymer backbone). Complexation of MCPs with tmAbFab-SAv. Trastuzumab Fab fragments (tmAbFab) were covalently linked to SAv with a PEG24 spacer (tmAbFab-SAv) as described in ref 7. To complex MCPs to tmAbFab-SAv, the polymer and protein complex in a 1:1 molar ratio were incubated in PBS at pH 7.4 at RT for 1 h. In the SPR experiments described below, as well as in the biodistribution studies, the tmAbFab-SAv-Bi-MCP complexes were all prepared in the same manner. SPR Measurements for the tmAbFab-Containing Solution. The metal-free complex of tmAbFab-SAv-Bi-PAm(DET-DTPA)36 was prepared with a concentration of 200 nM in PBS buffer (pH = 7.4) containing 0.05% Tween 20 (PBST). Solutions of tmAbFab and tmAbFab-SAv were also prepared with concentrations of 200 nM in PBST. In the binding assays, analyte solutions were applied to the chips at a flow rate of 100 μL/min for 1 min at 25 °C. The dissociation step was monitored for 10 min. ProteOn manager software was employed to calculate the kinetic parameters, and a Langmuir one-site model was used for the analysis. Preparation of RICs. tmAbFab(DTPA)-SAv and tmAbFab-DTPA, as well as the two polymer-tmAbFab-SAv complexes (tmAbFab-SAvBi-PAm(EDA-DTPA)40 and tmAbFab-SAv-Bi-PAm(DET-DTPA)36) were radiolabeled by incubation of 10 μg of each in 100 mM sodium acetate (pH 6.0) with 111InCl3 at 0.04 MBq/μg for 1 h at RT. These RICs are denoted as tmAbFab-DTPA- 111 In, tmAbFab-SAvDTPA-111In, tmAbFab-SAv-Bi-PAm(EDA-DTPA)36-111In, and tmAbFab-SAv-Bi-PAm(DET-DTPA)36-111In, respectively. In addition to these RICs, the two polymer-containing RICs were also prepared with stable indium saturation of residual noncomplexed DTPA groups. They were prepared by treatment with excess stable indium (InCl3, 50fold molar excess to the DTPA groups of each conjugate) following the radiolabeling procedure described above. The indium-saturated polymers are denoted as tmAbFab-SAv-Bi-PAm(EDADTPA)40-111In/115In and tmAbFab-SAv-Bi-PAm(DETDTPA)36-111In/115In, respectively. HER2 Immunoreactivity. The immunoreactivity of 111In-labeled tmAbFab(DTPA), tmAbFab-SAv, and tmAbFab-SAv complexed to MCPs was examined as previously reported.7 HER2 binding was determined by incubating 1.5 × 105 HER2+ SKOV-3 human ovarian cancer cells overnight in 24-well plates with 10 nmol/L of the RICs (0.5−1 MBq/μg) in 0.5 mL of serum-free medium for 3 h at 4 °C. Nonspecific binding (NSB) was evaluated by repeating the assay in the presence of a 100-fold excess of tmAb IgG. The medium was removed, and the adherent cells were rinsed with PBS, pH 7.4, 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 γ-counter. Specific binding (SB) was calculated by subtracting NSB from total binding and expressed as a percentage of total binding of RICs per 1.5 × 105 SKOV-3 cells. In addition, the

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RESULTS AND DISCUSSION The main hypothesis that we test in the experiments described here is that zwitterionic repeat units in a polyacrylamide-based metal chelating polymer saturated with trivalent metal ions and conjugated to tmAbFab-SAv will lead to significantly less liver uptake in vivo in mice, allowing for improved tumor uptake, than a corresponding polymer with a negative charge associated with each repeat unit. The polymers of interest are BiPAm(DET-DTPA)36, with a Bi end group and a DET spacer connecting the DTPA chelator to the polymer backbone, and Bi-PAm(EDA-DTPA)40, a polymer of similar length with an EDA group connecting each DTPA to the polymer. We begin by describing the synthesis and characterization of BiPAm(DET-DTPA)36. Synthesis of Bi-PAm(DET-DTPA)36 (4). Samples of BiPAm(EDA-DTPA)40 examined in previous publications were obtained by RAFT polymerization of tBoc-EDA acrylamide. To D

DOI: 10.1021/acs.biomac.5b01066 Biomacromolecules XXXX, XXX, XXX−XXX

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Biomacromolecules Scheme 1. Synthesis of Bi-PAm(DET-DTPA)36

obtain polymers with the DET spacer group, we had to use a different monomer to construct the polymer backbone. For this purpose, we carried out RAFT polymerization of pentafluorophenyl acrylate (PFPA) with the same Bi-containing chain transfer agent used previously, which enabled us to synthesize structurally well-defined Bi-P(PFPA) samples with good control over chain length. The presence of the dithiobenzoate group in this polymer allowed the most precise determination of DPn through comparison of 1H NMR peak intensities of the aromatic ring at δ = 7.91, 7.57, and 7.39 ppm (Figure S1) to that of the polymer backbone protons in the range 2.0−3.3 ppm. For the experiments described here, we employed a BiP(PFPA) sample 1 with DPn of 36. The overall synthesis of BiPAm(DET-DTPA)36, including aminolysis of the PFP esters with DET and attachment of DTPA, is outlined in Scheme 1. The dithiobenzoate group of 1 was then removed by an AMBN-initiated free radical reaction to yield polymer 2. This step was carried out to avoid complications that might arise from thiol groups during the subsequent pendant group transformations. The DET groups were introduced by aminolysis of the pentafluorophenyl esters with DET in a solvent mixture of dioxane and DMF. Dioxane is a good solvent for P(PFPA), while DMF was added to maintain the solubility of the aminecontaining product polymer. A large excess of DET was

employed (ca. 65 equiv per PFP ester) to minimize possible cross-linking of the polymer. The product polymer 3 was examined by 1H NMR and seen to exhibit three distinct peaks from the pendant DET groups (δ = 3.45, 3.30, 3.10 ppm; Figure S2). DTPA chelators were then attached as described previously.12 Briefly, we treated a large excess of DTPA with a limiting amount of DMTMM in water at pH 8.5 to activate one of the carboxylic acid groups of DTPA. After 5 min, a solution of the polymer 3 was added and allowed to react for 30 min to form the monoamide. The polymer 4 was purified by spin filtration and washed multiple times with DI water. The characteristic signals of the DTPA groups in the 1H NMR spectrum (in the range from 1.0 to 2.5 ppm, Figure S3) allowed us to calculate the mean number of DTPA groups per polymer. In this way we determined that there was on average one DTPA molecule per pendant group. The SEC (Figure S4) shows a symmetric peak with a relatively narrow size distribution (Đ = 1.2), providing evidence that no significant coupling or cross-linking of polymers occurred in the reaction of excess DET with the Bi−P(PFP) polymer. The solution of Bi-PAm(DET-DTPA)36 (4) was freeze-dried to yield a white powder. To determine its effective molar mass, accounting for the sodium ions that partially neutralized the DTPA groups as well as residual moisture, we carried out TGA measurements (Figure S5) as described previously in ref 12, and found an E

DOI: 10.1021/acs.biomac.5b01066 Biomacromolecules XXXX, XXX, XXX−XXX

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Biomacromolecules average of 2.9 Na+ ions and 2.1 water molecules per repeat unit, corresponding to Mn = 23 000 Da. A further comment is about the detailed structure of polymer 4. As drawn in Scheme 1, we presume that each DTPA is attached to the primary amine of the DET spacer. It is not possible by NMR to know whether some DTPA amides are attached to the secondary amine or whether there might be two DTPA groups on one DET pendant. We tend to discount either possibility based on many experimental results that show that, even in the presence of very large excesses of activated DTPA, we never obtained more than one DTPA per DET pendant.9 For the antigen binding studies described below, as well as for in vitro and in vivo experiments, Bi-PAm(DETDTPA)36 was complexed to a SAv covalently linked to tmAbFab via a PEG24 spacer (tmAbFab-SAv).7 This complex was prepared by incubating a 1:1 molar mixture of the polymer and tmAbFab-SAv in PBS at pH 7.4 at RT for 1 h. The complex of Bi-PAm(EDA-DTPA)40 with tmAbFab-SAv was prepared in a similar manner. Antigen Recognition. SPR Measurements. The ability of the tmAbFab-SAv-Bi-PAm(DET-DTPA)36 complex to recognize its target HER2 antigen was first tested by SPR measurements. This technique provides a useful way to examine the on-rates and off-rates for the binding of tmAbFab derivatives with the ECD of HER2 immobilized on an SPR gold chip. In a previous report, we showed that metal-free tmAbFab-SAv-Bi-PAm(EDA-DTPA)50, with a longer polymer chain, had comparable on- and off-rates, as well as a similar equilibrium dissociation constant KD as tmAbFab-SAv.8 Here we extend these measurements to tmAbFab-SAv-Bi-PAm(DET-DTPA)36 and include both tmAbFab and tmAbFabSAv in our comparison. Experiments were run with samples in PBS buffer (pH = 7.4) containing 0.05% Tween 20. Recall that both the tmAbFab and tmAbFab-SAv derivatives employed in these measurements, and in the RIC experiments described below, are covalently derivatized with DTPA. Metal-free conjugates were examined in the SPR measurements. The sensorgrams are presented in Figure 2. After the injection of samples, analyte binding to immobilized HER2 ECD occurs, resulting in a significant signal increase. The subsequent rinsing with buffer led to a slow decrease in signal. By analyzing the association and dissociation phases of binding with ProteOn manager software, we determined the association (ka) and dissociation (kd) rate constants of the different samples and calculated the equilibrium dissociation constants Kd = kd/ka. These data are presented in Table 1. For completeness, we also include in this table data from ref 7 for the values reported for tmAbFab-SAv and tmAbFab-SAv-BiPAm(EDA-DTPA)40. The most important information in Table 1 is that tmAbFab, tmAbFab-SAv, and both MCP complexes have similar binding affinities to the ECD of HER2, characterized by KD values in the low nM range. The on-rates and off-rates are similar for all of the samples. We note that the on-rate determined here for tmAbFab-SAv was somewhat higher than that reported in ref 8, but this may just reflect small differences in sample preparation. The main conclusion, however, is that neither the presence of SAv nor the ligation of the MCPs hinders the interaction between tmAbFab and the ECD of HER2. SB to HER2 on SKOV-3 Cells. Antigen Recognition on SKOV-3 Cells. The ability of radiolabeled tmAbFab derivatives to recognize cell surface HER2 antigens was assessed in binding experiments with SKOV-3 cells, a HER2+ human ovarian

Figure 2. SPR sensorgrams of the interaction of (A) tmAbFab, (B) tmAbFab-SAv, and (C) tmAbFab-SAv-Bi-PAm(DET-DTPA)36 with the extracellular domain of HER2. All three groups of measurements initiated with a concentration of 200 nM and were diluted 2-fold in serial dilutions to produce 5 decreasing concentrations.

Table 1. Binding Kinetics Data from SPR Measurements sample tmAbFab tmAbFab-SAv tmAbFab-SAv- Bi-PAm(DETDTPA)36 tmAbFab-SAvd tmAbFab-SAv- Bi-PAm(EDADTPA)50d

10−4 ka (M−1 s−1)a

104 kd (s−1)b

KD (nM)c

8.8 ± 0.1 6.7 ± 0.1 7.5 ± 0.1

0.93 ± 0.04 1.6 ± 0.1 0.62 ± 0.09

1.1 2.4 0.83

1.2 ± 0.1 1.9 ± 0.1

1.8 ± 0.1 1.7 ± 0.1

1.5 0.88

All the binding assays were performed at 25 °C with a flow rate of 100 μL/min. bThe dissociation was monitored for 10 min. cAnalysis of kinetic parameters employed a Langmuir model, with the ProteOn manager software. dData reported in ref 8. a

cancer cell line. Here we compare the RIC of tmAbFab-SAv-BiPAm(DET-DTPA)36-111In, with the DET spacer, with the RIC of tmAbFab-SAv-Bi-PAm(EDA-DTPA)40-111In, with the EDA pendant group spacer. In these experiments, neither polymer was saturated with stable In3+. These experiments were carried F

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Figure 3. Binding of RICs to HER2+ SKOV-3 cell. Panel A depicts the total RIC bound (pmol), and panel B shows the percent SB to HER2+ SKOV-3 human ovarian cancer cells of (A) 111In-labeled-tmAbFab(DTPA), (B) 111In-labeled-tmAbFab(DTPA)-SAv, (C) tmAbFab-SAv-BiPAm(DET-DTPA)36-111In, (D) tmAbFab-SAv-Bi-PAm(EDA-DTPA)40-111In, (E) Bi-PAm(EDA-DTPA)40-111In, and (F) Bi-PAm(DETDTPA)36-111In. Error bars represent the mean ± SEM (n = 3). Where error bars are not visible, they are smaller in magnitude than the width of the line at the top of the bar. Significant differences of interest are indicated by an asterisk (*) for both the total bound (pmol) and % SB, as indicated by comparison bars (p < 0.05). Since panel A shows the total pmoles bound was significantly lower for the unconjugated polymers in comparison to any of the RICs (p < 0.05), they were not considered further in panel B in terms of their SB as a percent of their total binding. In panel A, all bars are significantly different from each other except for (E) and (F), which are not significantly different (n.s.) from each other.

Figure 4. Biodistribution of tmAbFab-SAv-PAm-Bi-PAm(EDA-DTPA)40-111In/115In or tmAbFab-SAv-Bi-PAm(DET-DTPA)36-111In/115In at 24 h p.i. in Balb/c mice. Error bars represent the mean %ID/g ± SEM (n = 4). The organs examined include Bl (blood), H (heart), Lu (lungs), L (liver), Sp (spleen), St (stomach), Int (intestines), K (kidneys, M (muscle). Significant differences in the blood, liver, and intestinal accumulation of radioactivity are noted by the asterisks (p < 0.05).

out by incubating 1.5 × 105 cells overnight in 24-well plates with 10 nmol/L of the various RICs (0.5−1 MBq/μg) in 0.5 mL of serum-free medium for 3 h at 4 °C, following the procedure described in ref 7. NSB was evaluated by repeating the assay in the presence of a 100-fold excess of tmAb IgG. SB was calculated as (total binding) − NSB. The results are presented in Figure 3. The SPR experiments and the experiments with SKOV-3 cells provide two different and complementary measures of the immunoreactivity of tmAbFab-SAv and its MCP complexes toward HER2. In the SPR experiments with the ECD of HER2, we found that the polymer conjugates had similar on-rates and off-rates as tmAbFab and tmAbFab-SAv, and thus similar values of the dissociation constants KD, in the low nanomolar range. From these results, one learns that the polymer does not interfere with the ability of the Fab fragment to interact with the ECD of HER2. In contrast, the experiments with HER2overexpressing SKOV-3 cells examines the fraction of binding of tmAbFab, tmAbFab-SAv, and the tmAbFab-SAv-Bi-MCP complex that can be blocked by presaturation of the HER2 binding sites on the SKOV-3 cells with excess tmAb IgG. In these in vitro HER2 binding assays we see some important differences between the ability of the RICs to bind specifically to HER2+ SKOV-3 cells. Almost all of the binding of 111In-

tmAbFab(DTPA) and 111 In-tmAbFab(DTPA)-SAv was blocked in the presence of excess tmAb IgG, resulting in a high percentage of SB (87.4 ± 2.0% and 92.2 ± 0.04%, respectively). Both MCPs complexed to tmAbFab-SAv showed persistent binding in the presence of tmAb IgG. The proportion of total binding that was blocked by tmAb IgG and considered HER2 specific for tmAbFab-SAv-Bi-PAm(DETDTPA)36 (57.9 ± 12.2%) was in fact found not to be significant according to the unpaired t test in comparison to tmAbFab(DTPA) (87.4 ± 2.0%; p = 0.076) though, significantly lower than tmAbFab-SAv (92.2 ± 0.04%, p < 0.05). In contrast, we found a very significant (p < 0.005), 4-fold, reduction in the proportion of total binding that was blocked by tmAb IgG and considered HER2 specific for tmAbFab-SAv conjugated to BiPAm(EDA-DTPA)40 compared with tmAbFab(DTPA) (87.4 ± 2.0%; p < 0.005) as well with tmAb-SAv (23.7 ± 9.8%, vs 92.2 ± 0.04%), Figure 3, panel B. In previous experiments in cell culture with several other Bi-MCPs complexed to tmAbFab-SAv, we found significant reductions in HER2 SB, as determined by competition with excess tmAb.7 In all of these in vitro experiments, the MCPs were trace-labeled with 111In but not saturated with stable In3+. Thus, the polymers in each of these complexes were polyanionic at the pH of the medium (7.4). In light of the SPR results, we conclude that although G

DOI: 10.1021/acs.biomac.5b01066 Biomacromolecules XXXX, XXX, XXX−XXX

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Biomacromolecules tmAbFab’s affinity for HER2 is not affected by conjugation to MCPs, a difference in its ability to bind HER2+ SKOV-3 cells in an in vitro environment is observed when conjugated to the polyanionic MCP species. The nature of the NSB to SKOV-3 cells is not understood at this time, nor do we have an explanation for the differences in NSB observed for the tmAbFab-SAv-Bi-PAm(DET-DTPA)36-111In and tmAbFabSAv-Bi-PAm(EDA-DTPA)40-111In RICs. Tissue Biodistribution and Imaging Studies. Biodistribution and imaging studies of the tmAbFab-SAv-Bi-MCP conjugates were carried out in Balb/c mice with 111In-labeled/ In3+-saturated polymers. In Figure 4 we present the normal tissue distribution as percent injected dose/g (%ID/g) at 24 h p.i. for tmAbFab-SAv-Bi-PAm(EDA-DTPA)40-111In/115In and tmAbFab-SAv-Bi-PAm(DET-DTPA)36-111In/115In. These data showed significant differences in elimination from the blood, liver accumulation, and intestinal accumulation. Liver uptake of tmAbFab-SAv-Bi-PAm(EDA-DTPA)40-111In/115In was 4-fold greater than tmAbFab-SAv-Bi-PAm(DET-DTPA)36-111In/115In; 53.1 ± 5.5% ID/g vs 13.7 ± 0.8% ID/g, respectively (p < 0.005). Radioactivity circulating in the blood for 111IntmAbFab-SAv-Bi-PAm(EDA-DTPA)40 was 4.5-fold lower than for tmAbFab-SAv-Bi-PAm(DET-DTPA)36-111In/115In; 5.2 ± 0.1% ID/g versus 23.2 ± 0.9, respectively (p < 0.001). There were no major differences in the radioactivity concentrations in other normal organs between these two RICs. We also calculated the total percent of the injected dose of 111In (% ID) recovered from all sampled organs. These values are shown in Figure 5, where a significantly higher recovery of tmAbFabSAv-Bi-PAm(EDA-DTPA)40-111In/115In was found compared to tmAbFab-SAv-Bi-PAm(DET-DTPA)36-111In/115In.

Figure 6. Maximum intensity projection whole body posterior microSPECT/CT images at 24 h p.i. in Balb/c mice of (A) tmAbFab-SAv-Bi-PAm(EDA-DTPA)40-111In/115In or (B) tmAbFabSAv-Bi-PAm(DET-DTPA)36-111In/115In. Panel A is reprinted with permission from ref 7. Copyright 2013 AAPS. Both images were adjusted to the same intensity for comparison. There is lower liver uptake (large arrowhead) and higher circulating radioactivity in the heart (small arrowhead) in the image in panel B compared to panel A.

circulating radioactivity as noted by the counts found in the mediastinum which includes the heart (small arrowhead). We next turn our attention to tumor uptake in athymic mice with s.c. HER2-overexpressing SKOV-3 ovarian cancer xenografts. Here we compare 111In-labeled tmAbFab-SAv-BiPAm(DET-DTPA)36 without indium saturation, with the polymer in the form of an anionic polyelectrolyte, with 111Inlabeled and stable indium-saturated tmAbFab-SAv-Bi-PAm(DET-DTPA)36-111In/115In with the polymer in its zwitterionic form. The tumor and normal tissue distribution of the radioactivity was determined at 48 h p.i., with the results presented in Table 2. Tumor uptake of tmAbFab-SAv-BiPAm(DET-DTPA) 36 - 111 In was 1.9 ± 0.1% ID/g and significantly increased to 2.8 ± 0.07 ID/g when the DTPA groups were saturated with stable In3+ (p < 0.001). The blood

Figure 5. Total relative 111In recovery [percent injected dose (%ID)] from all sampled organs shown in Figure 4 for tmAbFab-SAv-BiPAm(EDA-DTPA)40-111In/115In or tmAbFab-SAv-Bi-PAm(DETDTPA)36-111In/115In in Balb/c mice at 24 h p.i. Error bars represent the mean %ID ± SEM (n = 4). The difference between the two RICs was significant (p < 0.05).

Table 2. Tumor and Normal Tissue Biodistribution at 48 h p.i. of tmAbFab-SAv-Bi-PAm(DET-DTPA)36-111In and tmAbFab-SAv-Bi-PAm(DET-DTPA)36-111In/115In percent injected dose/g (% ID/g) tissue blood lungs liver spleen stomach intestines kidneys muscle tumor

MicroSPECT/CT images of representative Balb/c mice injected i.v. (tail vein) with tmAbFab-SAv-Bi-PAm(DETDTPA)36-111In/115In were obtained at 24 h p.i. and compared with similar images acquired previously for tmAbFab-SAv-BiPAm(EDA-DTPA)40-111In/115In in Balb/c mice.7 These images show results consistent with the biodistribution studies presented in Figure 4. As seen in Figure 6, the polyanionic polymer complex tmAbFab-SAv-Bi-PAm(EDADTPA)40-111In/115In shows a very high concentration in the liver, whereas the complex involving the zwitterionic polymer tmAbFab-SAv-Bi-PAm(DET-DTPA)36-111In/115In exhibited much lower liver accumulation (large arrowhead) and more

tmAbFab-SAv-BiPAm(DET- DTPA)36-111Ina 0.2 0.7 12.3 1.5 0.6 0.5 12.3 0.3 1.9

± ± ± ± ± ± ± ± ±

0.03 0.07 0.9 0.3 0.1 0.04 1.0 0.05 0.05

tmAbFab-SAv-Bi-PAm(DETDTPA)36-111In/115Inb 1.0 1.5 4.2 2.8 0.4 0.4 1.5 0.3 2.8

± ± ± ± ± ± ± ± ±

0.2c 0.5 0.7d 0.7 0.1 0.03 0.2d 0.02 0.07e

Mean ± SEM (n = 4). bMean ± SEM (n = 3). cSignificantly different (p < 0.05). dSignificantly different (p < 0.005). eSignificantly different (p < 0.0001). a

H

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bulky pendant group (PNIPAm, PHPMA) makes a much bigger contribution to the Kuhn length (a measure of chain stiffness) and to the magnitude of Rg. It is interesting to note, for this chain length, that the values of Rg are comparable to (PNIPAm) or smaller (PHPMA) than the Kuhn length. One can conclude that polymers with this type of bulky pendant group are stiff and worm-like rather than flexible random coils. While we do not have a direct measure of the mean polymer dimensions and chain stiffness for indium-saturated PAm(EDADTPA)40 and PAm(DET-DTPA)36, we assume that, with their bulky pendant groups, they are not too different from that of PNIPAm of comparable length, with Rg values on the order of 2 to 3 nm. One can also ask how the charges along the backbone of PAm(DET-DTPA)36-111In/115In affect the polymer dimensions in solution, since electrostatic interactions in a polyelectrolyte can lead to expansion of the polymer coil. Charge repulsion, however, is screened at high salt concentrations. Under typical physiological conditions (i.e., PBS buffer: 150 mM NaCl, 25 mM phosphate, pH 7.4), the calculated Debye screening length (rD = 0.7 nm) is smaller than the Kuhn length (ca. 2 nm) for these polymers. This result shows that electrostatic interactions do not contribute to the polymer dimensions under physiological conditions. We next examine the evidence that charge can affect biodistribution of polymers in vivo. In studies in rats of HPMA copolymers with 3- or 8 mol % carboxyl pendant groups as well as two oligo peptides, all of the copolymers exhibited more rapid renal clearance than their neutral counterparts.24 Very different results were found for an antibody (humanized anti-Tac IgG) derivatized with a second generation polyamidoamine dendrimer with its exterior primary amines covalently modified with the DTPA derivative 2-(pisothiocyanatobenzyl)-6-methyl-DTPA acid (IB4M).25 The dendrimer-derivatized antibody trace-labeled with 111In showed much higher liver, kidney, and spleen accumulations than the 111 In-labeled 1B4M-derivatized antibody. The authors attributed this to the large number of negative charges introduced by the DTPA groups on the dendrimer. Charged functional groups also play a critical role in the biodistribution of inorganic NPs such as gold NPs (AuNPs) and quantum dots (QDs). For example, Arvizo et al.,26 reported the biodistribution profiles of AuNPs bearing different surface charges in a tumor-implanted mouse model. They found that the AuNPs carrying zwitterionic groups on their surface had a long plasma half-life similar to that of nonchargebearing AuNPs, and much longer than either negatively charged or the positively charged AuNPs. Furthermore, due to their longer blood circulation time, the zwitterionic AuNPs and neutral AuNPs exhibited a much higher tumor uptake than the AuNPs with negative charges or with positive charges. Choi et al.23 reported that, in a mouse model, surface charge strongly influenced the biodistribution and renal clearance of QDs coated with small solubilizing ligands. Both cationic and anionic NPs interacted with serum proteins, increasing their dh, leading to reduced body clearance and high levels of liver uptake. Corresponding QDs (dh = 4.6 nm) with a zwitterionic surface coating were rapidly excreted through the bladder. One can surmise from these experiments that having a small number (3 or 8 mol %) of negative charges along the backbone of an HPMA polymer small enough to be excreted is not sufficient to promote interactions with serum proteins. In contrast, a high density of negative or positive charges, as on

radioactivity concentration of tmAbFab-SAv-Bi-PAm(DETDTPA)36-111In/115In was 1.0 ± 0.2% ID/g, 4-fold higher than tmAbFab-SAv-Bi-PAm(DET-DTPA)36-111In (p < 0.005; Table 2). In contrast, the localization of tmAbFab-SAv-Bi-PAm(DETDTPA)36-111In in normal tissues, particularly in the liver and kidneys, was substantially elevated compared to the complex containing the zwitterionic polymer. For example, the liver uptake of tmAbFab-SAv-Bi-PAm(DET-DTPA)36-111In was 12.3 ± 0.9% ID/g, 3-fold higher than tmAbFab-SAv-Bi-PAm(DETDTPA)36-111In/115In (p < 0.005; Table 2), and the kidney uptake of tmAbFab-SAv-Bi-PAm(DET- DTPA)36-111In is 12.3 ± 1.0% ID/g, 10-fold higher than tmAbFab-SAv-Bi-PAm(DETDTPA)36-111In/115In (p < 0.005; Table 2). In summary, the polyanionic RIC tmAbFab-SAv-Bi-PAm(EDA-DTPA)40-111In/115In exhibited a poor biodistribution profile, particularly low retention in the blood and high liver sequestration, both of which are undesirable for maximizing tumor uptake. This problem was to a large extent overcome with the RIC tmAbFab-SAv-Bi-PAm(DETDTPA)36-111In/115In with the zwitterionic MCP. Origin of the Differences in Biodistribution of the Polymer Complexes. In designing these experiments, our working hypothesis was that negative charges along the polymer backbone of tmAbFab-SAv-Bi-PAm(EDADTPA)40-111In/115In were responsible for the rapid blood clearance and high liver uptake of this complex in mice. In contrast, tmAbFab-SAv-Bi-PAm(DET-DTPA)36-111In/115In, with zwitterionic pendant groups, exhibited much higher blood levels at 24 h p.i. and much lower levels of normal tissue accumulation. Here we first consider the possibility that other factors, such as the size and shape of the MCP might play a role in its biodistribution. Studies of soluble polymers carrying drugs, targeting moieties, and imaging modalities have shown that size, shape, and charge can influence in vivo biodistribution.20 Most of these studies have focused on dendrimers and linear polymers, e.g., hydroxypropylmethacrylamide (HPMA) copolymers with hydrodynamic sizes close to the renal cutoff (equivalent to a globular protein with Mr < 60 kDa).21 For example, attachment of drugs and other substituents to HPMA in this molecular weight range lead to a more folded structure, and this reduction in hydrodynamic size decreased circulation half-life and tumor accumulation.22 To confirm that our tmAbFab-SAv RICs are much larger than the renal cutoff, we used dynamic light scattering to measure the z-average hydrodynamic diameter (dh) of the tmAbFab-SAv conjugate (SI and Figure S8) and found a value of 14 nm. This is well above the limiting size for renal excretion of 7−8 nm determined for inorganic nanoparticles (NPs) and for globular proteins.23 Attachment of the MCP will increase the size of the RIC. To estimate the magnitude of this contribution, we next consider the size of the PAm(EDA-DTPA) and PAm(DET-DTPA) polymers in solution, and for the purposes of comparison examine the case of 40 repeat units in the polymer. We begin by examining the mean coil dimensions of various polyacrylamides and polymethacrylamides. These data are collected in Table S1 for polyacrylamide (PAm), polymethacrylamide (PMAm), poly(N-isopropylacrylamide (PNIPAm) and PHPMA, where we also tabulate calculated values of the root-mean-squared radius of gyration (Rg) of these polymer with DPn = 40. These data show that there are relatively small effects associated with introducing the backbone methyl group in the repeat unit (acrylamides vs methacrylamides), whereas a I

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uncomplexed DTPA sites with stable 115In, which in turn allowed for significantly increased tumor uptake in SKOV-3 xenograft mouse models. Although the Bi−SAv system has enabled us to obtain systematic insights into the correlation between the structure of MCPs and tissue localization of RICs, these complexes are not suitable for therapeutic applications. The presence of SAv will elicit immunogenicity.29 In the future, it will be important to attach polymers covalently to antibodies or antibody fragments.10 We anticipate that zwitterionic MCPs will play an important role in these studies.

the surface of a NP or on a dendrimer, promotes interaction with serum proteins, reduces renal clearance, and leads to high liver uptake. We conclude that in spite of the short Debye screening length associated with the high ionic strength under physiological conditions, the high density of negative charges along the MCP backbone in tmAbFab-SAv-Bi-PAm(EDADTPA)40-111In/115In is responsible for the rapid blood clearance and high liver uptake. tmAbFab-SAv-Bi-PAm(DETDTPA)36-111In/115In, with zwitterionic pendant groups, is much less susceptible to interaction with the mononuclear phagocyte system that would lead to liver and spleen uptake. This result and the experiments described above with AuNPs and QDs are consistent with reports in the literature that zwitterionic species are able to suppress nonspecific protein absorption,27 and that zwitterionic polymers can serve as an alternative to PEG to provide a stealth effect and prolong the circulation time of nanomedicines and therapeutic proteins.17,28

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.5b01066. Additional experimental details including the synthesis and characterization of pentafluorophenyl acrylate (PFPA), RAFT polymerization of PFPA, removal of the dithiobenzoate end group of polymer 1, and 1H NMR of Bi-(PFPA)36 in CDCl3, of Bi-PAm(DET)36 in D2O, of Bi-PAm(DET-DTPA)36 in D2O, the size exclusion chromatography (SEC) trace of Bi-PAm(DET-DTPA)36, thermogravimetric analysis (TGA) of Bi-PAm(DET-DTPA)36, DLS measurements of SAv, tmAbFab and tmAbFab-SAv, and a table of mean dimensions of a series polyacrylamides and polymethacrylamides(PDF).

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SUMMARY AND CONCLUSIONS In the experiments reported here, we compared tmAbFab-SAv complexes with two metal chelating polymers with a polyacrylamide backbone, a Bi end group, and similar degrees of polymerization. The polymer component of tmAbFab-SAvBi-PAm(DET-DTPA)36 had a DET spacer separating the DTPA-monoamide chelator from the polymer backbone. Secondary amines such as the middle nitrogen in DET are protonated at physiological pH (∼7.4). Thus, when the DTPA monoamide of the DET-DTPA pendant groups are saturated with trivalent metal ions, the polymer is zwitterionic. By contrast, the polymer component of tmAbFab-SAv-Bi-PAm(EDA-DTPA)40 has an uncharged EDA spacer separating the DTPA-monoamide chelator from the polymer backbone. Even when the DTPA groups are saturated with trivalent metal ions, the repeat units carry a negative charge. Metal-free complexes of the tmAbFab-SAv-MCPs were examined for antigen recognition by SPR measurements using a chip coated with the ECD of HER2. Immunoconjugate complexes of both polymers showed similar and low nM KD values, comparable to that of tmAbFab itself. This result indicates that the presence of the polymer does not interfere with the affinity of binding of tmAbFab toward the HER2 ECD. In immunoreactivity experiments with HER2-overexpressing SKOV-3 ovarian cancer cells, both polymer immunoconjugate complexes, trace-labeled with 111In, showed reduced proportions of SB, that is, binding that could not be competed by excess tmAb IgG, compared to 111In-labeled tmAbFab(DTPA)SAv, suggesting moderate levels of non-HER2 mediated binding for the polymer complexes to these cells. The fact that these nonspecific interactions were not observed with binding to HER2 ECD on the SPR chip suggests interactions with cell-surface proteins on SKOV-3 cells other than HER2, likely mediated by the polymers, contributed to the total measured binding to the cells. We note, however, that the binding of uncomplexed polymers to these cells was very low. This result suggests that nonspecific cell binding requires the presence of both the tmAbFab bioaffinity moiety as well as the MCP. The biggest differences were seen in biodistribution studies, both in Balb/c mice and in athymic mice with s.c. SKOV-3 ovarian cancer xenografts. Here anionic charges along the polymer backbone led to high levels of uptake of the immunoconjugates by normal tissue, particularly by the kidneys and the liver. This was overcome by saturating the

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Author Contributions #

These two authors contributed equally to this paper.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge the financial support from the Canadian Institutes of Health Research (CIHR)/Natural Sciences and Engineering Research Council (NSERC) Collaborative Health Research Program (Grant No. CHRPJ 365423-09; CPG-95268) and from the CIHR Open Operating Grant Program (Grant No. MOP130322, and from DVS Sciences, Inc (now Fluidigm Canada). We also thank Mr. Nick Jarvik for his help with the SPR experiments.

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REFERENCES

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DOI: 10.1021/acs.biomac.5b01066 Biomacromolecules XXXX, XXX, XXX−XXX