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A Nanoparticle Platform to Evaluate Bioconjugation and Receptor-Mediated Cell Uptake using Crosslinked Polyion Complex Micelles Bearing Antibody Fragments Stelios Florinas, Marc Liu, Ryan Fleming, Lilian Van Vlerken-Ysla, Joanne Ayriss, Ryan Gilbreth, Nazzareno Dimasi, Changshou Gao, Herren Wu, Ze-Qi Xu, Shaoyi Chen, Anjaneyulu Dirisala, Kazunori Kataoka, Horacio Cabral, and R. James Christie Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b00239 • Publication Date (Web): 23 Mar 2016 Downloaded from http://pubs.acs.org on March 24, 2016

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A Nanoparticle Platform to Evaluate Bioconjugation and Receptor-Mediated Cell Uptake using Crosslinked Polyion Complex Micelles Bearing Antibody Fragments 1

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Stelios Florinas , Marc Liu , Ryan Fleming , Lilian Van Vlerken-Ysla , Joanne Ayriss , Ryan Gilbreth , Nazzareno 1

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Dimasi , Changshou Gao , Herren Wu , Ze-Qi Xu , Shaoyi Chen , Anjaneyulu Dirisala , Kazunori Kataoka 4

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Horacio Cabral , and R. James Christie 1

Antibody Discovery and Protein Engineering, MedImmune, Gaithersburg, MD 20878, USA, 2Oncology Research, MedImmune, Gaithersburg,

MD 20878, USA, 3SynChem, Inc., Elk Grove Village, IL 60007, USA, 4Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan, 5Department of Materials Engineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan. 6Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. 7The Innovation Center of Nanomedicine, 66-20 Horikawa-cho, Saiwai-ku, Kawasaki 212-0013, Japan.

ABSTRACT Targeted nanomedicines are a promising technology for treatment of disease, however, preparation and characterization of well-defined protein-nanoparticle systems remains challenging. Here, we describe a platform technology to prepare antibody binding fragment (Fab) -bearing nanoparticles and an accompanying real-time cell-based assay to determine their cellular uptake compared to monoclonal antibodies (mAbs) and Fabs. The nanoparticle platform comprised of corecrosslinked polyion complex (PIC) micelles prepared from azide-functionalized PEG-b-poly(amino acids), i.e. azido-PEG-b-poly (L-lysine) [N3-PEG-b-PLL] and azido-PEG-b-poly(aspartic acid) [N3-PEG-b-PAsp]. These PIC micelles were 30 nm in size and contained approximately 10 polymers per construct. Fabs were derived from an antibody binding the EphA2 receptor expressed on cancer cells, and further engineered to contain a reactive cysteine for site-specific attachment and a cleavable His tag for purification from cell culture expression systems. Azide-functionalized micelles and thiol-containing Fab were linked using a heterobifunctional crosslinker (FPM-PEG4-DBCO) that contained a fluorophenylmaleimide for stable conjugation to Fabs thiols and a strained alkyne (DBCO) group for coupling to micelle azide groups. Analysis of Fab-PIC micelle conjugates by fluorescence correlation spectroscopy, size exclusion chromatography, and UV-Vis absorbance determined that each nanoparticle contained 23 Fabs. Evaluation of cellular uptake in receptor positive cancer cells by real-time fluorescence microscopy revealed that targeted Fab-PIC micelles achieved higher cell uptake than mAbs and Fabs,

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demonstrating the utility of this approach to identify targeted nanoparticle constructs with unique cellular internalization properties. KEYWORDS: targeted nanoparticle, polymeric micelle, bioconjugation, cancer cell uptake, nanobiomaterials

INTRODUCTION Polymer-, lipid- and inorganic-based nanoparticles have shown promise for the treatment of disease by serving as diagnostic, imaging, drug and gene delivery vehicles

1-3

. Nanoparticles typically

exhibit less toxicity and better PK/PD profiles when compared to small molecule drugs and can also achieve higher drug loading compared to protein-based drug delivery vehicles such as antibody-drug conjugates (ADCs)

4-6

. To date, numerous nanoparticle formulations are in clinical trials phase 1-3, and

several have advanced to the market

7, 8

. Production of targeted nanoparticles could further improve

clinical outcomes by guiding medicines into the cells targeted for treatment. Preparation and evaluation of targeted nanoparticles (NPs) in vitro and in vivo is challenging and often requires use of a hydrophobic toxic drug for nanoparticle formation

9-11

. Thus, comparison of

results across different NP platforms is difficult as the drug can affect physical properties such as size and stability

12-14

. Furthermore, many antibody-based targeting ligands used for attachment to NPs are

not specifically designed for that purpose and typically utilize functionality present on native antibodies 15-17

. Development of a simple and practical platform for evaluating targeted NPs with respect to parent

antibodies and corresponding fragments is crucial and necessary to understand basic principles relating to the biological process of cell targeting. Polymeric micelles (PMs) are an example of well-characterized and advanced NPs that show promising results in vivo

18-20

. PMs have a prolonged blood circulation time due to their ability to

circumvent renal clearance (MWCO > 50,000 Dalton) and also avoid reticuloendothelial system (RES) uptake due to a PEG corona that imparts stealth properties 21, 22. Furthermore, their small size (20-100 nm) allows for passive accumulation in cancer tissue by the enhanced permeability and retention (EPR) effect, which is characterized by leaky vasculature and impaired lymphatic drainage surrounding a tumor 23, 24. However, the EPR effect can vary in patients and cancer types, therefore, cannot be relied on as a universal mechanism to target NPs to cancer tissue 11, 25-27. Attachment of targeting ligands onto


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NPs has provided further advantages for drug delivery, such as increased cellular uptake and therapeutic efficacy in vitro as well as in vivo 28-31. Numerous targeting moieties are available for attachment to NPs including small molecules, peptides, monoclonal antibodies (mAbs), engineered proteins and nucleic acid aptamers. Choosing the correct targeting ligand & conjugation chemistry is crucial and can impact therapeutic outcome 17, 32, 33. Antibody-based cell-targeting moieties such as mAbs, F(ab’)2s, Fabs, scFv’s etc., are attractive for use as the binding component in targeted NPs because antibodies can be generated against a wide variety of targets with controlled affinity and specificity, and can also be produced in high yield using cell-based expression systems. Of the above-mentioned antibody-based targeting moieties, antigen binding fragments (Fabs) offer advantages for attachment onto NPs due to their relative small size (50 kDa) compared to mAbs (150 kDa) and simple structure (lack of an Fc domain)

34, 35

. Research-scale Fab

generation typically requires multiple digestion, reduction and purification steps to be performed on the parent antibody to obtain Fabs in a form ready for conjugation, which is often achieved via thiolmaleimide coupling to cysteines following reduction of hinge disulfides

28, 36

. Alternatively, cysteine-

engineered Fabs can be produced directly from cell expression systems to contain a convenient sitespecific conjugation handle for attachment to NPs 37, 38. Use of cysteine engineered Fabs for conjugation onto NPs is attractive because controlled conjugation can be achieved without interfering with native functional groups (which could impact stability, i.e. hinge disulfides) and the process is scalable and mild. Herein, we describe a simple method to produce corona functionalized, stable, drugless NPs for use as a proof-of-concept platform to evaluate antibody-based targeted NPs. Crosslinked polyion complex (PIC) micelles served as the central building block for attachment of targeting moieties and were formed by self-assembly of the oppositely charged ionic block copolymers N3-PEG-b-PAsp (anion) and N3-PEG-b-PLL (cation) followed by PIC micelle core crosslinking via amide bond formation

39, 40

.

Furthermore, block copolymers were designed to contain an azide group on the PEG terminus to serve as the attachment site for Fabs to the NP exterior in a controlled manner by mixing non-functionalized block copolymers into the micelle formulation. Fab targeting the EphA2 receptor, which is overexpressed in several types of cancer, was engineered to contain a surface-exposed cysteine residue for site-specific conjugation to NPs. The site-specific conjugation strategy to attach targeting ligands to NPs used a fluorophenylmaleimide and DBCO containing heterobifunctional crosslinker (FPM-PEG4DBCO) for conversion of cysteine thiols to azide-reactive DBCO functional groups via a stable thioether


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linkage. Analysis of real-time cell-internalization in cultured receptor-positive PC3 cancer cells revealed that targeted PIC micelles showed significantly higher cell uptake and a different internalization profile compared to targeted antibodies, free Fab, and non-targeted PIC micelle constructs (mock Fab-PIC and PICs without Fab), demonstrating the utility of this platform to observe the effects of NP-mediated cell targeting.

MATERIALS AND METHODS Synthesis of dye labeled crosslinked polyion complexes (PICs) Azido-PEG-b-PLL and azido-PEG-b-PAsp block copolymers were prepared as previously described and characterized by 1H NMR (Supplementary Figures S1 and S2)

39-42

. Azido-PEG-b-PLL was labelled

with Alexa Fluor 555 NHS-ester (Life Technology Corporation, Grand Island, NY) by combining 10 mg polymer (23.5 mM amines) with 80 µL of dye (3.2 mM in DMAC) for 1 h in 0.1 M sodium phosphate dibasic (pH 8.6) to allow conjugation, followed by PD-10 column (GE, Marlborough, MA) purification using 500 mM NaCl as an eluent to remove free dye from the polymer. Polymer-conjugate was then dialyzed into Milli-Q water using 3.5k MWCO Slide-A-Lyzer dialysis cassettes (Life Technology Corporation, Grand Island, NY). The water was changed every 4-12 h over 2 days. Dye content was determined by UV absorbance of Alexa Fluor 555 dye using a dilution series and found to be 0.3 dyes per block copolymer. Optimal PIC micelle formation conditions were determined empirically by mixing different molar ratios of polycation:polyanion followed by DLS analysis (described below). Subsequently all PIC micelle constructs were prepared by mixing equimolar ratios (COOH:NH2) of azido-PEG-b-PLL and azido-PEG-bPAsp in 10 mM phosphate buffer pH 7.3. Crosslinked PIC micelles were obtained by adding EDC crosslinker (Life Technology Corporation, Grand Island, NY) at various ratios (10:1, 5:1, 1:1, 0.1:1 (EDC:COOH)) and incubated overnight. Excess EDC was removed from crosslinked PIC micelles by dialysis using a 3.5k MWCO Slide-A-Lyzer dialysis cassette against 10 mM phosphate buffer pH 7.3 overnight.


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Molecular weight analysis of PIC micelles Molecular weight values for PIC micelles were determined using static light scattering experiments. Dye-free non-crosslinked and crosslinked PIC micelles were prepared as described above, 0.22 µm filtered and samples were analyzed at a total polymer concentration of 2 mg/mL. Noncrosslinked PIC micelles were analyzed in 10 mM phosphate, pH 7.3 with and without 600 mM NaCl. Crosslinked PIC micelles were analyzed in PBS (5 mM phosphate, 150 mM NaCl, pH = 7.4). Light scattering measurements were made using a DAWN HELEOS II light scattering instrument (Wyatt Technology, Santa Barbara, CA) and data processing and analysis were conducted using software provided by the manufacturer. Briefly, excess Rayleigh ratios (R(θ)) were measured at seven angles spanning 57 to 117⁰. At each angle, the ratio Kc/R(θ) was calculated where c is the total polymer concentration in mg/mL and K is a constant equal to 4π2(dn/dc)2η02/Naλ04. In this expression, dn/dc is the change in refractive index per g/mL for the micellar polymer mixture. This value was measured using an Optilab T-rex instrument (Wyatt Technology, Santa Barbara, CA) and determined to be 0.162 for the PIC micelles as formulated. η0 is the refractive index of the solvent (estimated as 1.3308 for all samples), Na is Avogadro’s number (6.022 x 1023 mol-1) and λ0 is the wavelength of incident light (662 nm for the instrument used). Kc/R(θ) was plotted as a function of sin2(θ/2) as described in

43, 44

to assess any

angular dependence of scattering, but for all samples measured, scattering was found to be isotropic. Thus, Mw values were calculated independently at each angle using the relation R(θ)/Kc = Mw. As expected for isotropic scattering, Mw values calculated at all scattering angles agreed closely and an average value is reported in the text. To further ensure accuracy of Mw values, additional measurements were made for crosslinked micelles at 0.3, 1 and 2 mg/mL. No significant concentration effects were observed over this range with calculated Mw values remaining consistent.

Synthesis and fluorescence dye labeling of cysteine engineered monoclonal antibodies (mAbs) and antigen binding fragments (Fabs) Anti-EphA2 (1C1) and an isotype-matched non-binding control mAb (mock) contained an engineered cysteine at position 239 and were produced and purified at MedImmune using standard molecular biology methods as described by Dimasi et al.

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generated as described herein. The Fab heavy chain genes for the 1C1 control antibodies were obtained from GeneArt, Invitrogen (Carlsbad, CA), and cloned into an in-house antibody expression vector for


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expression in HEK293F cells using FreeStyle 293 Expression Media (Thermo Scientific, Waltham, MA) (1 L final volume). Briefly, the Fab heavy chain genes were designed with the appropriate 5’ and 3’ DNA restriction sites to facilitate cloning into the antibody expression vector. The Fab constructs contained an A114C (Kabat numbering) mutation in the immunoglobulin CH1 domain and a thrombin cleavage site followed by a glycine-serine (G4S) linker and a six His tag at the C-terminus of the construct prior to the 3’ DNA restriction site (Supplementary Figure S3). The constructs were digested with the corresponding restriction enzymes and the heavy chain fragment was cloned into a similarly digested antibody expression vector containing the appropriate light chain expression cassette following standard cloning techniques 46. Using 293fectin and manufacturer’s protocol, (Invitrogen, Carlsbad, CA), the Fabs were expressed in HEK293F cultured in Freestyle medium for ten days. Conditional media were harvested and Fabs were purified using a His-Hitrap column following the manufacturer's protocol (GE Healthcare, Piscataway, NJ). A typical expression experiment yielded ~100 mg of purified Fab. Fab-FPM-PEG4-DBCO and mAb-FPM-PEG4-DBCO were labeled with AlexaFluor 555 -azide via click chemistry attaching the dye to A114C engineered cysteine mutation site through the FPM- PEG4DBCO crosslinker. Proteins were passed through a PD-10 column and dialyzed against PBS for 48 h using 10k MWCO Slide-A-Lyzer dialysis cassettes to remove unconjugated dye. PBS was exchanged every 4-12 h and samples were analyzed by UV-Vis and mass spectrometry to confirm conjugation, which showed ~1 dye/Fab & ~2 dyes/mAb (Supplementary Figure S4).

Fluorophenyl maleimide crosslinker synthesis The heterobifunctional fluorophenyl maleimide crosslinker (FPM-PEG4-DBCO, Figure 3 (3)) was synthesized as described herein. First, the active ester intermediate (2-Fluoro-5-maleimido)benzoic acid N-hydroxysuccinimide) (Figure 3 (1)) was prepared according to the literature procedures 47. A DBCO linker containing a free amine N-(NH2-PEG4)-N'-DBCO-succinamide (Figure 3 (2)) was purchased from Click Chemistry Tools (Scottsdale, AZ). N-(NH2-PEG4)-N'-DBCO-succinamide (0.3 g, 0.573 mmol) was added to a stirred solution of 2-Fluoro-5-maleimido)benzoic acid N-hydroxysuccinimide (0.181 g, 0.545 mmol) in CH2Cl2 (5 mL) at ambient temperature under N2. The resulting mixture was stirred at ambient temperature overnight, then diluted with CH2Cl2 (5 mL) and thoroughly washed with H2O (3 x 5 mL). The organic phase was separated, dried over Na2SO4 and concentrated to afford the target compound FPMPEG4-DBCO (400 mg, 0.540 mmol, 99%) as a light-yellow solid. 1H NMR (CDCl3) δ 8.05 (br s, 1H), 7.66 (d,


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J= 7.2 Hz, 2H), 7.38 - 7.18 (m, 11H), 6.87 (s, 2H), 5.13 (d, J= 13.8 Hz, 2H), 3.70 - 3.50 (m, 21H), 3.55 - 3.43 (m, 2H), 1.97 - 1.82 (m, 2H); MS (ESI+) m/z 763 (M+Na), 741 (M+1, base peak).

Preparation and analysis of Fab-crosslinker conjugates FPM-PEG4-DBCO and N-alkyl maleimide crosslinker (AM-PEG4-DBCO) (Sigma Aldrich, St Louis, MO) were conjugated to the A114C cysteine of mutated Fabs in multiple steps. First, Fab (50 mg, 3.5mg/mL) was incubated with thrombin (500 units dissolved in 500 µL PBS) (GE, Marlborough, MA) for 2 h on a rotator to cleave the HIS-tag. Fabs were then reduced with 50 mM TCEP (moles Fab:moles TCEP, 1:40) in conjugation buffer (CB) (1xPBS, 1mM EDTA, pH 7.2) to generate free sulfhydryls. The reduction reaction was carried out for 90 min at 37 °C followed by dialysis using 10k MWCO Slide-ALyzer dialysis cassettes against CB at 4 °C. The buffer was changed every 4-12 h for 24 h to remove all TCEP. Dehydroascorbic acid (dhAA) (50mM stock in DMSO) was added (Fab:dhAA, 1:20 molar ratio) to Fabs followed by incubation for 4 h in CB to reform inter-chain disulfides. Crosslinkers (FPM-PEG4-DBCO or AM-PEG4-DBCO, 10mM stock in DMAc) were added to Fab solution containing 10% v/v DMSO at the Fab:crosslinker molar ratio of 1:5 and incubated at room temperature for 1 h. The reaction was terminated by the addition of N-acetyl-L-cysteine (NAC) (100mM stock in water, Fab:NAC, 1:50 molar ratio) and incubation for 15 min at room temperature. Fab-crosslinker product was purified using a type II ceramic hydroxyapatite (CHT) column chromatography (Biorad, Hercules, CA) to remove excess crosslinker. Fab-crosslinker solutions were loaded onto CHT column with 10mM sodium phosphate buffer, pH 7 and washed with 5 column volumes. The Fab-crosslinker was eluted using 10mM sodium phosphate buffer, 2 M NaCl, pH 7 and final product was obtained by dialysis of the Fab-crosslinker against PBS overnight in a 10k MWCO Slide-A-Lyzer dialysis cassettes. Typical yield: 38-45 mg. Fab crosslinker conjugates were characterized as described previously

45, 48

. Briefly, Fab

conjugates were analyzed by mass spectrometry (LC/MS) and reduced reverse phase HPLC (rRP-HPLC) to determine conjugation efficacy and confirm site-specific conjugation on the Fab heavy chain and size exclusion chromatography (SEC) to determine aggregate content. For LC/MS analysis, Fab solutions (5 uL of 0.2 mg/mL) were injected and analyzed using an Agilent 6520B Q-TOF mass spectrometer connected to a RP-HPLC column (Agilent Poroshell 300SB-C3; 5 um, 2.1 mm x 75mm). HPLC was carried out at 0.4 mL/min using mobile phase A (0.1% (v/v) formic acid in HPLC-grade water) and mobile phase B (0.1%


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(v/v) formic acid in acetonitrile). Mass spectra were deconvoluted and analyzed as previously described using Agilent v.B.04.00 MassHunter Qualitative Analysis software 45. For rRP-HPLC analysis, Fab solutions (1 mg/mL) were reduced at 37 °C for 15 minutes in the presence of 42 mM dithiothreitol (DTT) in PBS pH 7.2. 10 µg of reduced Fab was loaded onto a PLRP-S, 1000A column (2.1 x 50 mm, Agilent) and eluted using mobile phase A (0.1% Trifluoroacetic acid in water, JT Baker) against a gradient of mobile phase B (0.1% Trifluoroacetic acid in acetonitrile) at 80 °C and a flow rate of 1 mL/min. Eluted protein was detected by absorbance at 280 nm. SEC analysis was performed using an Agilent 1100 Capillary LC System equipped with a triple detector array (Viscotek 301, Viscotek, Houson, TX), the wavelength was set to 280 nm and samples were run on a TSK-GEL G3000SWXL column (Toso Bioscience LLC, Montgomeryville, PA) using 100 mM sodium phosphate buffer, pH 6.8 at a flow rate of 1 ml/min.

Conjugation of Fabs to PIC micelles Dye labeled, crosslinked Fab-PIC micelles were prepared as described in Scheme 1. Fab-FPMPEG4-DBCO conjugate was incubated with crosslinked PIC micelles at an azide:Fab ratio of 1:2 to form Fab targeted PIC micelles and “click” reaction was allowed overnight. Excess of Fabs were removed by ultrafiltration using a Vivaspin®6 centrifugal tube (MWCO 100 kDa). Fab-PIC micelles with a 2mg/mL final polymer concentration (500 µL) were diluted with 1x PBS (5 mL) and spun down at 1000 rpm to the final volume of 100 µL. The last step was repeated for a total of five times. After the last purification step the Fab-PIC micelles were passes through a 0.2-micron filter to obtain the final Fab-PIC micelles.

Quantification of Fab-PIC micelle conjugation efficiency Fab content in PIC micelle conjugates was determined using three different analytical methods: i) SEC, ii) FCS and iii) UV absorbance. SEC was used for qualitative assessment of FAB conjugation onto PICs following the protocol as described above for Fab analysis using a Superdex 75 column (GE, Marlborough, MA) and PBS (flow rate 1 ml/min) as eluent. Fab-FPM-PEG4-DBCO was incubated with


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excess amount of 100%-azide PIC micelle at a molar ratio 1:3 (Fab:azide) to confirm reactivity of azide groups. Free Fab and Fab incubated with 0%-azide PIC micelles were used as appropriate controls. FCS analysis was performed as previously described using a confocal fluorescence laser scanning microscope (Carl Zeiss MicroImage Co., Ltd., LSM 510 META/Confocor 3) equipped with Ar lasers (458, 488 and 514 nm) 49. Briefly, laser scanning mode was selected to analyze samples using the appropriate wavelength and filter set to obtain high signal to noise micrographs. A 100 µL droplet of each sample was added into an eight-well chamber (NUNC, USA) and intensity fluctuation information was collected in terms of an autocorrelation function. Samples were prepared for FCS measurement in multiple steps. First, crosslinked PIC micelles were formulated with different azide concentrations (100, 50, 25, 10, 0% azide groups) by mixing azideand methoxy-terminated- PEG-b-PAsp and –PEG-b-PLL polymers. A set of samples was prepared to determine the number of available azide groups for “click” chemistry conjugation to various PIC micelles (100, 50, 25, 10, 0% azide groups). Azido-PIC micelles were reacted with Alexa Fluor 647 DBCO and passed through a PD-10 column using 500 mM NaCl as a mobile phase and dialyzed against PBS for 48 h in a 10k MWCO Slide-A-Lyzer dialysis cassette to remove residual unbound dye. In the second set of samples, Fab-FPM-PEG4-DBCO was conjugated to various PIC micelles (100, 50, 25, 10, 0% azide groups) and purified as described above. Alexa Fluor 647 DBCO (Life Technology Corporation, Grand Island, NY) was added (10 µL, 5000 uM) to Fab-PIC micelle conjugates (200 µL, 2 mg/mL) to label unreacted azide groups. Dye labeled Fab-PIC micelles were purified as described above and used as a control. FCS analysis was used to determine number of dyes per PIC micelle for both Fab-PIC-Alexa Fluor 647 and PIC-Alexa Fluor 647 micelle constructs. Total number of Fabs per PIC micelle was calculated by Equation 1. Free dye was used as control to calibrate the FCS instrument. = − (Equation 1) : (/) + : (/) + + UV absorbance analysis was used to calculate the amount of Fabs per PIC micelles by comparing the ratio of protein absorbance (280 nm) Alexa Fluor 555 absorbance (555 nm). Absorbance was measured on a Nanodrop ND-1000 (Thermo Scientific, Waltham, MA). Final protein concentration was calculated using a dye corrected A280 value calculated using the Alexa Fluor 555 correction factor (0.08)


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provided by the manufacturer. The absorbance spectra shown in Figure 6C were obtained using a SpectraMax M3 plate reader (Molecular Devices, Sunnyvale, CA).

Characterization of PIC micelles by transmission electron microscopy (TEM) and dynamic light scattering (DLS) The core morphology of PIC micelles was directly observed by TEM using an H-700 TEM (Hitachi Ltd., Tokyo, Japan) according to a previously reported method

50

. Briefly, carbon-coated collodion

membrane containing copper TEM grids (Nisshin EM Co., Ltd., Japan) were treated by glow-discharge using an Eiko IB-3 ion coater (Eiko Corp., Tokyo, Japan). The hydrophilized TEM grids were then immersed into PIC micelle solutions (0.02 mg/mL polymer) pre-treated with uranyl acetate (UA) (2% (w/v)) for 30 s to achieve effective deposition of the samples on the grids. The PIC micelle-deposited grids were blotted onto a piece of filter paper to remove excess solution, air-dried at room temperature for 30 min and then transferred to the TEM instrument for imaging. Images were captured at an acceleration voltage of 75 kV. PIC micelle core size distributions were determined from TEM images using ImageJ 1.48v software (National Institutes of Health, USA). A total count of more than 100 spherical structures—144 counts for PIC micelles and 126 counts for Fab-PIC micelles—was measured to obtain micelle core size distributions. Z-average particle size, polydispersity and kcps were measured by DLS using a ZetasizerNano ZS (Malvern Instruments, Worcesterhire, UK). PIC and crosslinked PIC stability were evaluated by DLS (counts) in the presence and absence of 600 mM NaCl.

Fab-FPM-PEG4-DBCO conjugate stability in buffer Fab-crosslinker conjugates were evaluated for their propensity to undergo retroMichael deconjugation as described previously by Christie et al.

47

. Briefly, Fab

crosslinkers (1.33 µM) were incubated at 37 °C in PBS and PBS containing 1% v/v (143 mM) β-mercaptoethanol (BME) and evaluated by M/S overtime. Fresh BME (1% v/v) was added 24 h prior to the final measurement on day 7 to account for potentially oxidized BME after prolonged incubation. For each time point samples were sterile filtered and reduced


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using DTT (50mM) followed by LC/MS analysis as described above for characterization of Fab conjugates. Deconjugation of crosslinkers was calculated from peak heights of mass spectra using Equation 2. = ∗ 100/( + + ) (Equation 2) a= Fab heavy chain with unhydrolyzed crosslinker* b= Fab heavy chain with hydrolyzed crosslinker* c= Fab without crosslinker *included crosslinker reacted with BME

Cell culture and generation of GFP expressing cell line PC3 (human prostate cancer cell line) and GFP-PC3 (GFP sorted PC3 cells) were cultured in DMEM containing 10% FBS at 37 °C in a humidified incubator and an atmosphere of 5% (v/v) CO2. PC3 cells were seeded at a subconfluent density (approximately 2-5x104 cells/cm2) and infected with lentiviral vector containing GFP construct and puromycin-resistance cassette at an MOI of 3. Cells were transduced using a spin transduction protocol by centrifuging cells containing virus and 8 µg/mL polybrene (Sigma Aldrich, St Louis, MO) at 2500 rpm for 90 min at 32oC. Following overnight incubation, media was refreshed and cells were cultured undisturbed for an additional 72 h before selecting for transduced cells under 5 µg/mL puromycin (Sigma Aldrich, St Louis, MO). GFP-positive cells were further enriched by sorting for uniform GFP expression on a BD FACS AriaII. Cell binding studies EphA2 binding was evaluated in PC3 cells using flow cytometry. Fabs, mAbs and PIC micelles were labeled with Alexa Fluor 750 (Life Technology Corporation, Grand Island, NY) as described above. Cells were incubated with DPBS containing 10 mM EDTA for 10 min to detach cells, centrifuged at 1000 rpm for 3 min, resuspended in 2% FBS/DPBS and seeded into a U bottom plate at a density 2 x 105 cells per well. Samples were added to cells in the amount of 4 µg total protein concentration per well and incubated for 15 min on ice to allow cell binding. Cells were washed with 2% FBS/PBS and spun down at 1000 rpm to remove unbound samples and stored on ice. Samples were analyzed by a flow cytometer


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(LSRFortessa Cell Analyzer, Becton-Dickinson, Mountain View, CA) using a 640 nm laser (100 mW) and 780/60 filter settings. Forward scatter and side scatter was used to identify viable, single cell events with an Alexa Fluor 750 positive signal. Dead cells (DAPI positive) were excluded from analysis.

Real-time cell uptake Internalization of mAbs, Fabs and PIC micelles was measured by high content screening using a Cellomics Arrayscan VTI instrument (ThermoFisher Scientific, Rockville, MD) equipped with a live cell chamber set to humidified incubation at 37oC with 5% CO2. Live cells were imaged kinetically every 20 minutes for a minimum of 5 h at 20x magnification. Fluorescence images were captured at an excitation of 485 nm (GFP-positive cells) and excitation of 549 nm (Alexa Fluor555 labeled constructs Fab-PIC, mAb and Fab) with an appropriate emission filter for each dye. Images were analyzed under the colocalization bioapplication in HCS Studio V2 software, quantitating the degree of co-localization of Alexa Fluor 555 set as target, to individual cells ROIs identified by the GFP signal. Data was captured as the mean fluorescence intensity of Alexa Fluor 555 that was co-localized within individual cell ROIs, and from there further normalized to control. To initiate the assay, GFP-labeled PC3 cells were plated (0.75 x 105 cells/well) into black-walled 96-well plates (Corning) and allowed to adhere overnight. Cells were kept on ice while dosing with 0.1 µg/mL protein per well. Once treatments were administered, the plate was loaded into the live cell chamber and scanning was immediately initiated, with t=0 scan occurring within 3 min of plate transfer into the incubated chamber. Non-targeted PIC micelles, which did not have conjugated Fab, were administered at the same polymer concentration as targeted 1C1 PIC micelles. Two independent assays were performed with N=200 individual cell measurements per time point (Supplementary Figure S5).

RESULTS AND DISCUSSION Crosslinked PIC micelle formation and characterization A stable nanoparticle functionalized with reactive groups is necessary for ligand conjugation, purification and subsequent biological assays. For these reasons, we chose core crosslinked PIC micelles containing surface azide groups as the nanoparticle platform for conjugation of cell-targeting Fabs, with


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each component depicted in detail below (Scheme 1).

Scheme 1. Preparation of dye labeled, crosslinked Fab-PIC micelles.

Formation of PIC micelles occurs by electrostatically driven self-assembly of oppositely charged polymers, which in this study were N3-PEG-b-PLL (positive charged amines) and N3-PEG-b-PAsp (negative charged carboxylates). PIC micelles formed in this manner may be fragile under physiological conditions, disassociate, and fall apart due to charge shielding by salts and interactions with charged proteins or naturally occurring polyelectrolytes. Therefore, PIC micelles must be further stabilized in order to make them useful for studies under biological conditions. PIC micelles were stabilized in this work by chemical crosslinking using EDC, a carbodiimide reagent, to chemically link polymer chains through amine-carboxy coupling (amide bonds) in the micelle core (Figure 1A). PIC micelle formation was optimized by mixing different ratios of PEG-b-PLL (NH3+) and PEG-bPAsp (COO-) followed by DLS measurements to determine size, PDI and kcps (Figure 1B). PIC micelles prepared at the charge neutral ratio of 1:1 (NH3+:COO-) showed the lowest PDI and highest kcps value indicating that micelle formation was optimal at this condition. This PIC micelle formation behavior suggests that excess polyion interferes with the self-assembly process, which is consistent with previous


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reports 39, 40, 51, 52. Next, PIC micelle crosslinking was optimized by using different ratios of EDC crosslinker to carboxyl groups in the PEG-b-PAsp polymer backbone (ratios [EDC]/ [COOH]: 0, 0.1, 1, 5, 10) to determine the amount of crosslinking required to produce stable PIC micelles. Crosslinked PIC micelles stability was evaluated by exposing micelles to increased ionic strength conditions (NaCl) and analyzing their diameter, PDI and kcps, and then comparing those values to non-crosslinked PIC micelles. NaCl interferes with electrostatic interactions, resulting in dissociation of non-crosslinked PIC micelles and subsequent loss of scattered light intensity. As seen in Figure 1C, crosslinking increased PIC micelle stability, as crosslinked PIC micelles remained intact under high salt conditions when a ratio of [EDC]/ [COOH] > 5 was used for crosslinking, evidenced by their low PDI and high kcps when compared to noncrosslinked controls. Based on this result, an EDC/[COOH] ratio of 10 was used to produce crosslinked PIC micelles for all experiments. To further assess the PIC micelle structure and estimate the polymer association number (NA) in each particle, we determined the apparent molecular weight (Mw) of PIC micelles in both crosslinked and non-crosslinked forms using static light scattering. For non-crosslinked PIC micelles in phosphate buffer, measurements indicated an Mw of 281,800 Da. We also measured Mw of the non-crosslinked PIC micelles after addition of 600 mM NaCl, to determine the apparent Mw value for the monomeric polymer units after dissociation of PIC micelle structures

39, 40, 53

. Under high salt conditions, light

scattering measurements gave a Mw value of 24,140 Da, somewhat larger than the average formula weight of the polyanion and polycation polymers (18,865 Da). This difference might be explained by associated counterions that add to the coherent scattering mass or incomplete dissociation of the polymer units, or incomplete dissociation of pairs of oppositely charged block ionomers (termed unit PICs) 54. Assuming that the actual mass of the monomeric polymer units in the PIC micelles assembly lies somewhere between the measured and calculated values, a range of NA of 11.7 – 14.9 polymers/PIC micelle can be estimated for the non-crosslinked state. After crosslinking, light scattering gave a Mw value of 232,900 Da under high salt conditions, corresponding to a NA of 9.7 – 12.3 polymers/PIC micelle, which is similar to the NA value calculated for non-crosslinked PIC micelles under low salt conditions. Maintenance of NA at high salt concentration confirms that EDC-mediated crosslinking stabilized PIC micelle structures. Altogether polymer association number analysis revealed that each PIC micelle structure comprised about 10 polymers/micelle, which is in good agreement with polymer association number estimates from FCS experiments that determined the number of azide groups/PIC micelle (described below).


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Figure 1. Crosslinked PIC micelle formation, characterization and stability. (A) Overview of PIC micelle formation by electrostatic interaction and subsequent crosslinking via amide bond formation in the micelle core. (B) Optimization of PIC micelle formation by varying ratios of polycation and polyanion, and subsequent DLS analysis. (C) Effect of crosslinking on PIC micelle stability. PIC micelles were incubated with increasing amount of EDC crosslinker and analyzed by DLS. Stability was assessed by comparison of size, PDI and kcps of crosslinked PIC micelles to non-crosslinked PIC micelles under low (0 mM) and high (600 mM) NaCl conditions.

Site-specific and stable incorporation of DBCO functionality into Fabs Generation of antibody-based targeting ligands in a ready-to-conjugate format can be challenging, often involve processing of full-length antibodies to produce desired fragments for conjugation. Furthermore, Fabs produced by mAb processing have two potential drawbacks relating to the type of chemistry available for conjugation; i) amine-based coupling results in random conjugation


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with little control over the degree and position of Fab modification, ii) thiol-based coupling consumes one or both of the native hinge disulfides, which could weaken the heavy-light chain interface and impact Fab stability. Thus, we designed a cysteine-engineered Fab to allow precise and site-specific conjugation and also utilized a cell-based expression system to generate the Fab material. Cysteine engineered Fabs comprised an A114C mutation site on the heavy chain for site specific attachment of a nanoparticle-reactive heterobifunctional crosslinker to the cysteine thiol using maleimide chemistry (Figure 2A and B). This mutation site was chosen based on a previous report which showed that this surface-exposed position exhibits high conjugation efficiency without affecting antigen binding after conjugation 55. In addition to the cysteine mutation, a thrombin cleavage site was included between the HIS-tag and Fab to allowed HIS-tag removal after Fab purification. HIS-tags are positively charged entities that allow Fab purification through chelating columns and could interfere with crosslinker and/or nanoparticle conjugation and potentially impact biological assays. Removal of the Histag was achieved by incubation with thrombin for 2 h (Figure 2D). While cysteine-engineering provides a chemical handle for micelle attachment, thiols can’t directly couple to azide groups contained on micelles and thus a crosslinker was used to link the two components together. It should be noted that maleimide-terminated polymers could also be used to couple directly to cysteine-engineered Fabs. However, the hydrolytic instability of maleimides (T1/2 ~30300 min, pH 7.4, 22 oC) would require prompt conjugation after micelle preparation 47. Thus, we chose to utilize the azide-alkyne reaction due to the hydrolytic stability of those reactive groups. The crosslinker used (FPM-PEG4-DBCO) comprised a fluorophenyl maleimide functional group for reaction with Fab thiols and a DBCO group for reaction with micelle azides, with both groups separated by a short PEG (4-units) spacer. The rationale for selecting a fluorophenyl maleimide is to improve conjugate stability and is explained in more detail below. Overall, use of this crosslinker enabled simple conversion of thiols to DBCO groups in high yield and also allowed purification and characterization of the Fabcrosslinker product prior to micelle conjugation. The conjugation/purification protocol developed for crosslinker attachment to Fabs showed high conjugation efficiency, no overconjugation, and did not cause aggregation (Figure 2C). First, the HIS-tag was removed by incubation with thrombin for 120 min at room temperature. Following HIS-tag removal, Fabs were reduced to activate the engineered cysteine for crosslinker conjugation, followed by mild oxidation to reform interchain disulfides. This activation step is necessary because the engineered cysteine in isolated Fab exists as a disulfide with a free cysteine amino acid, which must be reduced to


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generate a reactive sulfhydryl. Maleimide crosslinkers were reacted with activated Fabs to convert the A114C cysteine thiol into DBCO functionality. The Fab conjugation reaction was very efficient with >95% conjugation determined by mass spectrometry (peak height) and rRP-HPLC (AUC) as seen in Figure 2D and 2E respectively. Site specific conjugation was demonstrated by rRP-HPLC as shown in Figure 2E, where the FPM-PEG4-DBCO crosslinker was conjugated onto the Fab heavy chain as expected. Additionally, no over-conjugation of the FPM-PEG4-DBCO crosslinker was observed at 5:1 (crosslinker:Fab) molar ratio. Aggregation of Fabs before and after crosslinker conjugation was evaluated by SEC. As seen in Figure 2F aggregation of Fabs was significantly low with 1.2% and 2.1% before and after conjugation respectively. A slight increase in retention time was noted after crosslinker conjugation, which could be attributed to hydrophobic DBCO groups interacting with the column matrix. Overall, this process was very efficient to produce pure Fab in a format ready for micelle conjugation. It has been reported that thiol conjugates formed with N-alkyl maleimides (AMs) are unstable due to a retro-Michael reaction which regenerates the free thiol and maleimide. This process especially effects conjugate stability in biological media where free thiols can serve as a sink for liberated maleimide groups, which prevents simple reattachment of maleimides to the thiol, and ultimately results in loss of the maleimide-containing component in the original conjugate 56-59. Thus, we utilized a fluorophenylmaleimide (FPM-PEG4-DBCO) crosslinker to overcome the aforementioned disadvantages, based on findings described in our previous work (Figure 3)

47

. The fluorophenyl maleimide-thiol

reaction product, a fluorophenyl thiosuccinimide, rapidly undergoes ring-opening hydrolysis after thiol conjugation and prevents the retro-Michael reaction from occurring, thus stabilizing the conjugate. Stability of the fluorophenyl maleimide crosslinker Fab-conjugate in the current work was evaluated in buffer containing free thiols, which react with deconjugated crosslinker generated by retro-Michael reaction as reported previously

60-62

. Furthermore, a traditional AM-PEG4-DBCO crosslinker Fab

conjugate was evaluated simultaneously with the Fab-FPM-PEG4-DBCO conjugate as a control. Conjugates were incubated at 37 °C in PBS, pH 7.2 containing BME and analyzed by mass spectrometry over time. Analysis by mass spectrometry allows quantitative analysis by comparison of peak intensities of conjugated and non-conjugated species

63

. These conditions yielded similar results to serum

incubation when maleimide deconjugation


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Figure 2. Fab conjugation, derivatization and characterization. (A) Overview of the cysteine engineered Fab anti-EphA2 with A114C mutation site. (B) Site specific conjugation scheme using Fab anti-EphA2 and crosslinkers FPM-PEG4-DBCO and AM-PEG4-DBCO. (C) Step by step overview of the crosslinker conjugation process. (D) Mass spectrometry analysis of Fab intermediates and the final conjugate. (E) Confirmation of site-specific heavy chain (HC) conjugation by reduced reverse phase HPLC (rRP-HPLC).


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Fabs were reduced prior to analysis. (F) Aggregation analysis of Fab-crosslinker conjugates before and after conjugation by size exclusion chromatography (SEC) (zoomed in area shows aggregates).

from mAbs was determined in our previous work 47. It should also be noted that BME reacted with the alkyne group of DBCO, but that did not interfere with crosslinker deconjugation analysis. As seen in Figure 4A2 peak e, the AM-PEG4-DBCO conjugate showed increased free Fab content over the 7 day incubation time both with and without BME in the buffer. However, linker deconjugation increased when incubated in BME compared to the PBS control lacking BME (Figure 4A1; peak e) which is attributed to reaction of BME with the maleimide in free AM-PEG4-DBCO crosslinker. Free Fab content did not increase for the FPM-PEG4-DBCO Fab conjugate in PBS buffer and only slightly increased (~8%) upon incubation in PBS containing BME (Figure 4A3 and 4A4), demonstrating that the FPM-PEG4-DBCO crosslinker indeed stabilized the thiol-maleimide conjugate.

Figure 3. Synthesis of FPM-PEG4-DBCO crosslinker (3).


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Figure 4. Stability of FPM-PEG4-DBCO and AM-PEG4-DBCO-crosslinker Fab conjugates. (A) Mass spectrometry analysis of Fab-crosslinker after 0, 3 and 7 days incubation in PBS (2), 4)) and βmercaptoethanol (BME) (1), 3)). (B) Kinetics of crosslinker deconjugation from Fabs over 7 days in PBS and BME. (C) One-way-ANOVA statistical analysis at day 7 of FPM-PEG4-DBCO and AM-PEG4-DBCOcrosslinker Fab conjugates in PBS and BME (P < 0.001, N=3). Analysis of the time dependence of crosslinker deconjugation revealed that in general, the FPMPEG4-DBCO Fab conjugate showed higher stability over the timeframe of 7 days at 37 °C when compared to AM-PEG4-DBCO Fab conjugate with > 90% of conjugated Fab observed (Figure 4B). Specifically, in the presence of BME the FPM-PEG4-DBCO Fab conjugate showed significantly higher (P < 0.001) Fab conjugate stability (91%) when compared to the AM-PEG4-DBCO Fab conjugate (77.42%) (Figure 4C). Hydrolysis of the thiosuccinimide in the FPM-PEG4-DBCO conjugate was confirmed by mass spectrometry (Figure 4A3; peak g) which corroborates well with the observed stability of the conjugate. These results highlight the importance of choosing appropriate chemistry for preparation of stable bioconjugates. In the context of this work, use of the traditional N-alkyl maleimide crosslinker would


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result in loss of Fabs from the surface of micelles over time and thus could impact their targeting ability. Thus, we chose to use the FPM-PEG4-DBCO crosslinker for preparation of functional nanoparticles in this work in order to eliminate uncertainty associated with conjugate stability.

Conjugation of Fabs to PIC micelles Fabs were linked to PIC micelles via a copper-free click reaction between the strained alkyne DBCO group on Fabs, and azides contained on the surface of PIC micelles 64. Conjugation of Fabs onto PIC micelles was evaluated using three characterization methods; SEC, FCS and absorbance at 280 nm. First, crosslinked PIC micelles were prepared with various azide content (0%, 10%, 25%, 50%, 100%) by combining different ratios of azide functionalized polymers (N3-PEG-b-PLL, N3-PEG-b-PAsp) with methoxy-terminated polymers (MeO-PEG-b-PLL, MeO-PEG-b-PAsp) to evaluate conjugation efficacy as a function of azide content on the surface of PIC micelles. Initial experiments aimed to confirm the feasibility of this reaction involved reaction of Fab-FPM-PEG4-DBCO with a molar excess of micelle azide groups. Qualitative analysis by SEC allowed the conjugation reaction to be monitored, as micelle and Fab peaks exhibited different retention times. As seen in Figure 5A Fab-FPM-PEG4-DBCO incubated with 0% azide PIC micelles (molar ratio N3:Fab, 3:1) showed 0% conjugation as confirmed by AUC values of free Fab (44.31) when compared to 0% azide PIC + Fab (44.82). Further, these results indicate that there is no non-specific interaction between PIC micelles and free Fab-FPM-PEG4-DBCO crosslinker which would make them appear conjugated in the SEC trace. AUC values of free Fab decreased when reacted with increasing amounts of azide modified PIC micelles (Figure 5A), indicating that the azide groups on the PIC micelles are functional and the conjugation reaction correlates with azide content. We then evaluated the number of Fabs per PIC micelle using fluorescence correlation spectroscopy (FCS). This technique allows quantification of fluorescent dyes by counting photons emitted from materials as they pass through the focal point of a confocal microscope 49. This technique allows quantification of Fabs on individual PIC micelles rather than measuring an average value of all PIC micelles in solution, as in the case of simple absorbance measurements. For this work, the fluorescence of an azide-reactive dye was monitored after conjugation to PIC micelles, which allowed quantification of reactive polymer chains, as each polymer contains only one azide group. First, we analyzed the absolute number of reactive azide groups per PIC micelle by reaction with DBCO-Alexa Fluor 647. PIC micelles were prepared with 0%, 10%, 25%, 50%, and 100% theoretical azide groups by simply mixing


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azide-terminated block copolymers with methoxy-terminated block copolymers. As seen in Figure 5B 100% azide PIC micelles contained 9.58 ± 1.27 dyes per micelle, which correlates to about 10 available azide groups for “click” chemistry. This result is in good agreement with light scattering results, which also determined the polymer association number (NA) to be ~10 polymers/micelle. Furthermore, this result confirms that azide groups are functional and that EDC crosslinking did not affect azide reactivity.

Figure 5. Evaluation of Fab-PIC micelle conjugation by SEC, FCS and UV-Vis absorbance. (A) Fab conjugation onto various PIC micelles bearing different azide contents analyzed by SEC. Numbers represent the AUC of Fab peaks. (B) FCS measurements for determination of Fabs on PIC micelles prepared with different azide content (0%, 10%, 25%, 50% and 100%). PIC micelles were analyzed after; i) reaction with Cy5-DBCO only (black bars) and ii) reaction with Fab-FPM-PEG4-DBCO followed by Cy5DBCO (grey bars). (C) UV-Vis absorption spectra naked (no Fab) and Fab conjugated PIC micelles. Fabconjugated PIC micelles were prepared from 100% azide PIC micelles.


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FCS analysis of PIC micelles (after reaction with DBCO-Alexa647) prepared with calculated azide contents of 50%, 25%, 10% and 0% were found to contain 5.84 ± 0.28, 4.19 ± 0.3, 2.33 ± 0.32 and 1.12 ± 0.08 dyes per PIC micelle respectively, demonstrating that azide content can be controlled fairly well simply by incorporating methoxy-terminated block copolymers into the micelle formulation. Fab content in PIC micelle conjugates was determined indirectly by measuring the amount of free azides remaining after reacting PIC micelles with Fab-FPM-PEG4-DBCO (molar ratio of 2:1) using the DBCO-Alexa647 dye method as described above. As seen in Figure 5B, 100% and 50% azide PIC micelles showed 2.5 ± 1.2 and 1.1 ± 0.5 Fabs per PIC respectively. Thus, 100% azide PIC micelles could conjugate ~1.5-3.5 Fabs & 78 dye molecules. PIC micelles with 25%, 10% and 0% azide groups showed no Fabs per PIC micelle. Conjugated Fab content in micelles was also determined by measuring protein absorbance (A280) and polymer absorbance (A555) of Fab-conjugated to fluorescent-labelled PIC micelles. This approach exploited the fact that all Fab-micelle conjugates were prepared from the same Alexa555labeled crosslinked PIC micelle stock. Thus, all micelle solutions could be normalized by A555 values to determine the A280 at the same polymer concentrations for all constructs. Using Equation 3 we accounted for the Alexa-555 contribution to A280 absorbance and calculated the final protein concentration per PIC micelle. 1C1- and mock- Fab PIC micelle conjugates showed 2.1 and 1.9 Fabs per PIC micelle, respectively, when compared to non-targeted PICs with no Fabs (Figure 5C). The results confirmed our findings by FCS where 1-3 Fabs/PIC were determined for the 100% azide formulation. Overall, Fab density on PIC micelles determined by FCS and UV-Vis measurements were in excellent agreement, but showed incomplete consumption of available azide groups. PIC micelle azide content could be effectively tuned between 0-10 reactive groups per PIC micelle, whereas Fab density was limited to a maximum of ~3 Fabs per PIC micelle. We tried to “push” the reaction further under extreme conditions, such as concentrating the reaction mixture 10 times by ultrafiltration and using a freeze-thaw protocol as described by Takemoto et. al.

65

, however, we were not able to observe an

improvement in conjugation. It should also be noted that both Fab-DBCO and PIC micelle azide reactivity was confirmed with small-molecule reactions. DBCO reactivity was confirmed (>90%, mass spectrometry) by conjugating azido-Alexa555 onto Fab-FPM-PEG4-DBCO constructs (Supplementary Figure S4), while FCS results demonstrated that PIC micelle azide groups were readily available for conjugation of small dye molecules. However, only 20-30% of PIC micelle azide groups were available for Fab conjugation. Dye molecules (MW: ~1250) are around 40 times smaller than the Fabs (MW: ~50,000), suggesting that steric hindrance might contribute to low Fab conjugation efficiency. Furthermore, FPM-


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PEG4-DBCO crosslinker length may also impact conjugation. The FPM-PEG4-DBCO crosslinker used in this work is fairly short (~2 nm), which may not sufficiently extend the linker beyond the protein surface to allow for efficient reaction. Further experiments are necessary to evaluate and optimize this proteinmicelle macromolecular reaction to achieve higher Fab densities via azide-DBCO coupling. Determination of Particle Size and Morphology of PIC Micelle constructs DLS measurements revealed that PIC micelles with and without conjugated Fabs were narrowly distributed with unimodal size distribution and moderate polydispersity indices (Table 1). PIC micelles with Fabs (32 d.nm) were slightly larger than PIC micelles without Fabs (25 d.nm). The increase in size (~7 nm) for PIC micelles with Fabs is reasonable considering the size of a single Fab (~5 nm). It is worth noting that the diameter distribution of PIC micelles conjugated with Fabs was relatively broader and showed a higher PDI than that of PIC micelles without Fabs (Figure 6 Ai and Bi), which could suggest a distribution of Fab densities on the PIC micelle surface. TEM images revealed that the core of the PIC micelles were nearly spherical and narrowly dispersed structures with an average diameter less than 15 nm for both Fab-conjugated and unconjugated PIC micelles (Figure 6Aii, Bii and Table 1). PIC micelle core diameter distribution after Fab conjugation was more broadly distributed than that of PIC micelles without Fabs (Figure 6Aii and Bii). Note that the PEG shell of uranyl acetate stained micelles cannot be observed due to the low affinity of PEG for uranyl cations compared to carboxyl groups in the core of the PIC micelles. In the case of Fabconjugates, some staining of protein is expected which could contribute to size measurement.


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Figure 6. Particle size measurements of 100% azide PIC micelles with and without conjugated Fab determined by DLS (A-i, B-i) and TEM (A-ii, Bii).

Table 1. Diameters and polydispersity indices (PDI) of PIC micelles conjugated with and without Fabs observed by DLS and TEM measurements. Construct

z-average (d.nm)

PDI

PIC (DLS)

25

0.123

Fab-PIC (DLS)

32

0.254

PIC (TEM)

11

ND

Fab-PIC (TEM)

15

ND

Cell-Surface Receptor Binding and Real-time Cellular Uptake of Targeted PIC Micelles, mAbs and Fabs Our underlying objective for generating cell-targeted micelles was to produce a material with biological activity, i.e. effective cell binding and internalization. Therefore, receptor binding of PIC micelles was evaluated by flow cytometry in EphA2 receptor (+) PC3 cells. All targeted 1C1 constructs (Fab-PIC micelle, mAb and Fab) showed high cell binding properties with > 90% binding compared to non-targeted controls, and similar geo mean values were observed amongst the different constructs (Figure 7). Thus, Fab-PIC micelle conjugates were functional with respect to binding the EphA2 receptor, and receptor binding was apparently not affected by micelle attachment. Furthermore, we included mock controls (isotype-matched non-binding Fab) for each construct (Fab-PIC, mAb and Fab) and they


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all showed low binding efficacy (< 1%) which confirms that EphA2 receptor binding in targeted constructs occurred through specific interactions and not non-specific protein adsorption. Non-targeted PIC micelles themselves did not bind to cells at the concentrations tested in this work, which is expected due to their hydrophilic and “stealth” PEG corona.

Figure 7. Cell binding analysis of targeted and non-targeted mock constructs in receptor positive PC3 cancer cells by flow cytometry. (A) Targeted 1C1-Fab versus mock-Fab. (B) Targeted 1C1-mAb versus mock-mAb. (C) Targeted 1C1-Fab-PIC micelle versus mock-Fab-PIC micelle versus non-targeted PIC micelle (no Fab). (D) Geo mean values of measurements from (A), (B) and (C). After cell binding of targeted constructs was confirmed, we then turned to cell-uptake assays to understand the biological impacts of nanoparticle-mediated EphA2 targeting. Therefore, we investigated cell uptake of our constructs using a Cellomics ArrayScan instrument, a real-time fluorescence microscope imager capable of high density automated image analysis. PIC micelle cell uptake was


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compared to corresponding mAbs and Fabs to determine if uptake was impacted by the type of targeting construct. In order to compare cell uptake across different formats, the fluorescence intensity of each construct was controlled (Table 2) to yield similar values across all targeting systems investigated. Dye content in Fabs and mAbs was controlled by site-specific conjugation to yield 1 and 2 dyes/protein, respectively, which is similar to the dye content in PIC micelles (1.5 dyes/micelle). An overview of targeted and non-targeted constructs binding valency, size (d.nm) and fluorescence intensity (RFU/mg protein) is shown in Table 2 66, 67. Cell internalization was measured over 12 h in GFP-expressing PC3 cells (green) using Alexa Fluor 555-labeled (red) constructs. First, the internalization behavior of all targeted 1C1 constructs was evaluated (Fab-PIC micelle, mAb and Fab), as seen in Figure 8A. 1C1-Fab-PIC micelles initially bound onto the cell surface, indicated by the red fluorescent ring surrounding green PC3 cells at t = 0. This ring rapidly disappeared (minutes) to yield punctate staining visible within cells. At 12 h (Figure 8B) 1C1-FabPIC micelles were mostly observed within punctate structures associated with cells. It should be noted that the images obtained and analyzed were not confocal images, however, it seems reasonable to assume that punctate staining represents subcellular endosome and/or lysosome compartments. Additionally, cell-surface bound fluorescent constructs were only visible on the cell edge (rings) at early time points, and did not contribute to the amount of construct calculated inside the cell, as fluorescence intensity measurements at early time points were very low. Targeted 1C1-Fab-PIC micelles showed significantly higher cellular uptake (P < 0.001) than targeted mAbs, with the measured intensity ultimately reaching a 5-fold higher value (Figure 8D). Fabs showed very little internalization, even though cell-binding was confirmed by flow cytometry. Lack of Fab internalization is consistent with the mechanism of EphA2 mediated endocytosis, as the EphA2 receptor must dimerize to trigger internalization

68, 69

. Fabs are not able to dimerize the receptors

because they have only one antigen binding arm, and thus, efficient cell uptake is not possible. Fab-PICs and mAbs have at least 2 antigen binding arms, capable of dimerizing the EphA2 target, which leads to receptor mediated endocytosis. Furthermore, it is well known that EphA2 receptors are degraded and expression is down-regulated upon internalization triggered by mAbs 68, 69. Thus, the plateau observed for mAbs after ~3 h is likely due to depletion of cell-surface receptors. However, Fab-PIC micelles did not follow the same plateau behavior and higher uptake was achieved. It can be argued that the observed increase in fluorescence intensity for PIC micelles was caused by dequenching or dye released from the PIC micelle core. Therefore, we performed a pulse-chase assay to determine the stability of the


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Alexa555 fluorescence signal. In this experiment, Fab-PIC micelles were incubated with cells for 5 h, then the culture media was exchanged with sample-free media, followed by analysis for 12 h to observe any increase/decrease of the fluorescence intensity. Since no further internalization is possible after Fab-PIC micelles are removed the signal is expected to remain constant. As seen in Figure 8F the fluorescence intensity slowly decreased over time, demonstrating that there is no release of the dye and that the previously observed increase of Fab-PIC micelle fluorescence signal is truly caused by enhanced cellular uptake.

Figure 8. Real-time cellular uptake of targeted and mock constructs in receptor positive PC3 cancer cells. (A) GFP expressing cells (green) and Alexa Fluor 555 labeled 1C1-Fab-PIC micelles (red ring surrounding cells) at the starting time point. (B) 1C1-Fab-PIC micelles after 12 h incubation. (C) ROI overview of Cellomics ArrayScan analysis. (D) Analysis of 1C1-targeted constructs (Fab-PIC, mAb, Fab) (P < 0.001). (E) Comparison between targeted 1C1-Fab-PIC micelle, mock-Fab-PIC micelle and non-targeted PIC micelle (no Fab). (F) Evaluation of fluorescence dye quenching by pulse-chase assay using Fab-PIC micelle constructs.


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Table 2. Binding valency, size (d.nm), fluorescence intensity (RFU/mg protein) of Fab-PIC, PIC, mAb and Fab constructs analyzed in the cell uptake assay. (a) and (b) are reference values from (64) and (65) respectively. (*) Value was calculated using same polymer concentration as Fab-PIC micelle construct. (c) Range reported from FCS measurement. Construct

Fab

Binding Valency

2

PIC

0

Fab-PIC

1.3-3.7

Mock

Non-targeted

126865

247535

N/A

177881

274422

N/A

25

N/A

N/A

147764

32

159551

147273

N/A

5

10-15

c

RFU/mg protein 1C1

a

1

mAb

Size (d.nm)

b

Finally, specificity of the receptor mediated endocytosis pathway was confirmed by evaluating Fab-PIC micelles comprising an isotype-matched non-binding control Fab (Mock Fab-PIC micelle) and a non-targeted PIC micelle without conjugated Fab. Often, this type of analysis is not included in the evaluation of targeted NPs, which usually compare targeted constructs with the corresponding NP lacking the targeting ligand. As seen in Figure 8E, mock Fab-PIC micelle and non-targeted PIC micelle showed almost no uptake when compared to 1C1-Fab-PIC micelle, thus demonstrating that cellular uptake was facilitated by specific interaction of 1C1-Fab on the micelle with EphA2 receptors. There are several possible explanations for increased uptake of PIC micelles compared to mAbs: i) Fab-PIC micelles have a longer distance between their antigen binding arms, which could allow more efficient scavenging of receptors on the cell surface that can’t be accessed by short mAb arms, ii) Fabs may be more flexible at the end of a linear PEG, which could allow more conformational freedom to bind epitopes on EphA2 receptors, and iii) Fab-PIC micelles likely have species with greater binding valency than a mAb (i.e. higher than 2) and receptor clustering at higher valency could impact internalization and trafficking of the EphA2 receptor. More detailed biological experiments are needed to elucidate the exact mechanism of increased targeted micelle uptake observed here. Altogether, the real-time cell uptake assay allowed distinct differences between the different targeting platforms to be observed, and that NPs demonstrated different uptake behavior than analogous mAbs and Fabs.


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CONCLUSION

We developed a targeted nanoparticle platform based on stable, crosslinked PIC micelles to evaluate protein conjugation and subsequent biological activity in vitro. Block copolymer materials allowed flexibility in micelle construction, enabling covalent crosslinking, fluorescent dye labeling, and Fab conjugation by click chemistry. Azide functionality contained on the micelle corona was easily tuned to contain up to 10 reactive groups per NP by mixing azide-terminated- and methoxy-terminated-PEG block copolymers, however, Fab density was limited to 1-3.7 Fabs per NP. Cysteine-engineering of the Fab targeting moiety allowed precise conjugation to micelles without disrupting the protein core structure (i.e. disulfide bridges), and also allowed generation of Fab material by direct expression in cell culture without need for manipulation of the parent antibody. The FPM-PEG4-DBCO crosslinker utilized in this work enabled efficient conversion of engineered cysteines into azide reactive DBCO groups and provided the benefit of improved conjugate stability resulting from the self-hydrolyzing thiosuccinimide group. Real-time evaluation of cell uptake allowed an interesting and somewhat unexpected behavior of targeted micelles to be observed. Not only was higher overall cell uptake achieved for EphA2 targeting micelles compared to mAbs, the internalization profile was also different - whereas mAb uptake saturated after a few hours and micelle uptake did not. Thus, real-time evaluation of cell uptake is a valuable tool to elucidate the effects of NP-mediated targeting compared to simple mAbs and Fabs. The technology and methods described in this work could be applied to evaluate the biological behavior of micellar NPs bearing a variety of Fabs directed towards potential cell- or tissue- based targets, enabling development of advanced targeted nanomedicines.

SUPPORTING INFORMATION Supplemental text contains 1H-NMR’s of block copolymers, sequence of heavy/light chain of Fab, mass spectrometry data of dye labeled mAbs and Fabs, and cellomics array scan analysis. This material is available free of charge via the Internet at http://pubs.acs.org.


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ACKNOWLEDGMENTS We thank the Institute of Bioscience and Biotechnology Research, University of Maryland, Hannah Mbatia and Puranik Purushottamachar for the use and assistance in NMR measurements. Anand Ravindran, Jacob Gorman assisted with flow cytometry measurements and Jooyeon Ahn assisted with FCS measurements. This work was financially supported by MedImmune, a member of the AstraZeneca group. Additional support was provided to S.F. by the Japan Society for the Promotion of Science postdoctoral fellowship program.


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TOC FIGURE


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A Nanoparticle Platform To Evaluate ... - ACS Publications

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