Modulation of the Biophysical Properties of Bifunctional Antibodies as

Article Cite This: Biochemistry 2019, 58, 3116−3132

pubs.acs.org/biochemistry

Modulation of the Biophysical Properties of Bifunctional Antibodies as a Strategy for Mitigating Poor Pharmacokinetics Amita Datta-Mannan,*,† Robin M. Brown,‡ Jonathan Fitchett,∥ Aik Roy Heng,∥ Deepa Balasubramaniam,∥ Jennifer Pereira,§ and Johnny E. Croy*,§

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†

Department of Experimental Medicine and Pharmacology, ‡Departments of Drug Disposition and Development/Commercialization, and §Biotechnology Discovery Research, Lilly Technology Center North, 1223 W. Morris Street, Indianapolis, Indiana 46221, United States ∥ Biotechnology Discovery Research, Lilly Research Laboratories, Lilly Biotechnology Center, San Diego, California 92121, United States S Supporting Information *

ABSTRACT: Interest in the development of bi- or multispecific antibody (BsAbs)-based biotherapeutics is growing rapidly due to their inherent ability to interact with many targets simultaneously, thereby potentially protracting their functionality relative to monoclonal antibodies (mAbs). Biophysical property assays have been used to improve the probability of clinical success for various mAb therapeutics; however, there is a paucity of such data for BsAbs. This work evaluates a fusion of an IgG with an isolated protein domain (deemed ECD) and serves to understand how molecular architecture influences biophysical and biochemical properties and, in turn, how these relate to drug disposition. The biophysical characteristics of the molecules (charge, nonspecific binding, FcRn and Fcγ receptor interactions, thermal stability, structure−dynamics, and hydrophobic properties) indicated preferred orientations of ECD and IgG, which supported better pharmacokinetic outcomes. In certain instances, in which ECD−IgG configurations led to suboptimal biophysical behavior in the form of increased hydrophobicity and global ECD instability, drug clearance was found to be increased by ≥2-fold, driven by endothelial cell-based association/clearance mechanisms in the liver, kidneys, and spleen. Improvements in the pharmacokinetic properties were afforded by positional modulation of ECD that was able to bring the disposition characteristics in line with those of the parental mAb. The findings provide some pragmatic, broadly applicable strategies and guidance for the design considerations and evaluation of ECD−BsAb constructs. Additional studies, delineating the precise interactions involved in the clearance of the ECD−BsAb constructs, remain an opportunistic area for improving their in vivo kinetic properties.

B

mAb component.9−16 Some of these disposition observations have been ascribed to nonspecific binding driven by solventexposed charge patches or Fcγ interactions in vivo.4−8 However, there are several examples of aberrant BsAb clearance phenomena that cannot be explained or readily attributed to these more “typical” mechanisms that can influence mAb disposition.16 For example, we reported that the major clearance mechanism of several BsAbs was linked to binding/association with liver sinusoidal endothelial cells (LSECs) in vivo and cannot be attributed to target binding, weakened FcRn interactions, or poor molecular/biochemical properties of the BsAbs compared to those of their parental mAbs, which did not show any LSEC interaction.16 Because these inferior in vivo properties can limit the potential advantages offered by the pharmacological synergism of BsAbs, mitigation of poor disposition through the flexibility

ifunctional/bispecific antibodies (BsAbs) have the potential to affect several disease pathways because of their inherent ability to interact with multiple targets simultaneously. Hundreds of BsAbs have been evaluated in preclinical studies, and there are >30 currently under clinical investigation; however, there are only two approved for use in the U.S. and European markets.1 Like most mAb therapeutics, the causalities of clinical failure for BsAbs can be generally related to poor exposure−efficacy profiles, insufficient safety margins, and strategic industry decisions. As such, the focus of this report is on interrogating the key biochemical/biophysical factors that influence BsAb pharmacokinetics (PK) and disposition using preclinical model systems. It is reasonably well accepted that specific and nonspecific factors that influence mAb PK are also involved in the disposition of BsAbs.2−8 Notwithstanding these likenesses, an important concern that has been observed in the study and development of BsAbs is the fact that they can demonstrate aberrant or inferior in vivo PK properties (short half-life and rapid clearance) relative to a kinetically well-behaved parental © 2019 American Chemical Society

Received: January 25, 2019 Revised: June 17, 2019 Published: June 26, 2019 3116

DOI: 10.1021/acs.biochem.9b00074 Biochemistry 2019, 58, 3116−3132

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Biochemistry

Figure 1. Cartoon representation of molecules evaluated in this study. (A) The common antibody (G1) was altered among a humanized IgG1 (G11), IgG2 (G21), and IgG4PAA (G41) while maintaining the C-HC fusion position for the ECD on each isotype construct; these constructs are denoted G11-C-HC, G21-C-HC, and G41-C-HC, respectively. Alternatively, we prepared a second set of fusions that altered the fusion poisition of the D2 ECD from the C-terminus of the G41 heavy chain to either (B) the N-terminus of the G41 light chain (G41-N-LC) or (C) the N-terminus of the G41 heavy chain (G41-N-HC). In all variants, a common linker separating G1 and the D2 ECD consisting of a repeating G4Sx2 element was used.

afforded in the structural design of these molecules is critical to their successful development as medicinal agents. Protein engineering has facilitated the development of a number of different structural BsAb formats (described extensively elsewhere).17,18 Many of these formats involve the fusion of a mAb-derived domain unit or another protein/ peptide with inherently unique target binding activity to a mAb.17,18 The studies herein utilize the IgG−ECD BsAb structure as a model system. In this BsAb format, a pharmacologically active peptide/protein (deemed ECD) may be fused to an agonistic/antagonistic mAb to generate an IgG−ECD construct that provides additional biological activities. Relative to the other BsAb formats, the IgG−ECD constructs are unique in that these molecules offer alternate ligand binding activity via addition of a non-IgG target binding domain such as the extracellular domain of a cell surface receptor fused to the mAb structure. The IgG−ECD format offers structural tractability in being able to position the fusion unit at the N- or C-terminus of the IgG via a flexible linker region. The ability to generate different fusion architectures allows for the study of protein structure on the balance of multiple chemical and biological factors. Additional considerations include retaining mAb and/or ECD target binding activities and the metabolic stability/integrity of the parental mAb and ECD components prior to interrogation of the in vivo activity. While also critical, the position-dependent effect of fusing the IgG and ECD units on the in vivo PK of the BsAb is to a much lesser extent prospectively apparent through in vitro interrogation. Because few studies have shown the fusion

position of the ECD protein domain to the mAb can unpredictably affect the bispecific molecule’s blood clearance,7,15,16 the configuration of the fusion position for BsAbs adds another level of complexity in the consideration of the in vivo fate relative to a mAb. From a pragmatic perspective, changes in the configuration of the protein fusion position with the mAb have been used as an empirical strategy to mitigate aberrant PK findings with mixed success.7,15 A more systematic understanding that encompasses relating the influence of fusion position format on the biophysical properties of the BsAbs and the connection of these factors with the disposition of the molecules is an important aspect in developing a deeper and deliberate versus serendipitous determination of the appropriate BsAb configuration for further therapeutic development. In this study, we designed and evaluated several BsAb architectures with the D2 domain of vascular endothelial growth factor receptor 1 (VEGFR1, reported previously), an ∼11.5 kDa protein, as the ECD fusion partner, connected via a flexible linker to a common antibody platform (Figure 1).19 All but the humanized G41-N-HC BsAb architecture demonstrated inferior in vivo drug disposition properties in cynomolgus monkeys and mice relative to those of the parental G41 mAb. A biodistribution analysis of the molecules that had poor disposition character implicated an endothelial cell-based clearance mechanism(s) localized to the liver, kidney, and spleen as the root cause of their rapid elimination. Retrospective evaluation of biophysical characteristics of these molecules [charge, nonspecific binding, FcRn and Fcγ interactions, thermal stability, hydrogen−deuterium exchange 3117


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Biochemistry

Hydrophobic Interaction Chromatography Analysis. The gross hydrophobicity of the molecules evaluated in this study was assessed using hydrophobic interaction chromatography methods. For this analysis, the proteins of interest were first diluted to 1 mg/mL in 1× PBS and then subsequently diluted to 0.5 mg/mL with 100 mM potassium phosphate (pH 6.7) containing 2 M ammonium sulfate. These “salted” samples were then injected using an Agilent 1100 highperformance liquid chromatography (HPLC) instrument onto a NPR butyl HIC column (Tosoh Bioscience). Bound protein was then eluted using a linear gradient from 0 to 100% 50 mM potassium phosphate (pH 6.7), and the hydrophobicity of individual compounds was inferred on the basis of the observed column retention (measured in minutes) and the corresponding minimal concentration of ammonium sulfate required for column retention. FcRn Column Retention Assay Analysis. The binding and pH-dependent release properties of the molecules evaluated in this study were assessed using a column format originally described by Schlothauer et al.22 Briefly, speciesspecific soluble avi-tagged FcRn proteins were coupled to a 1 mL Hi-Trap streptavidin agarose support (GE Healthcare) by adding 1.2 mg of each respective soluble FcRn protein to a single column. Columns were washed extensively following coupling with 1× PBS to remove unbound FcRn materials. For the analysis, test articles were diluted to 1 mg/mL with 20 mM MES (pH 5.5) with 150 mM sodium chloride and confirmed to be pH 5.5 using a three-point calibrated pH meter. Samples were then loaded using an Agilent 1100 HPLC instrument onto each species-specific FcRn column at a flow rate of 1.0 mL/min. Bound proteins were eluted using a 0 to 100% linear gradient over 60 column volumes of 20 mM Tris (pH 8.8) containing 150 mM sodium chloride at a flow rate of 1.0 mL/ min at ambient temperature. FcRn binding properties were benchmarked using previously established antibodies Ustekinumab hIgG1 and Briakinumab hIgG1, and an increased time for elution from the FcRn column relative to that of the Ustekinumab hIgG1 standard was implied as altered FcRn engagement and release.22 Hydrogen−Deuterium Exchange (Don) Analysis. Hydrogen−deuterium exchange mass spectrometry (HDX-MS) experiments were performed on a Waters nanoACQUITY system with HDX technology, including a LEAP HDX robotic liquid handling system. The deuterium exchange experiment was initiated by adding 55 μL of D2O buffer containing 0.1× PBS to 5 μL (10 μg) each of the G41-D2 variant and the parental G41 mAb. Exchange was carried out at 15 °C for various amounts of time (0 s, 10 s, 2 min, 10 min, 60 min, and 240 min). The reaction was quenched using an equal volume of 0.32 M TCEP, 3 M Gdn HCl, and 0.1 M phosphate (pH 2.5) for 4 min at 1 °C; 50 μL of the quenched reaction mixture was digested online in an immobilized nepthensin II (Nep II) column, packed in house, using 0.2% formic acid in water as the mobile phase at a flow rate of 300 μL/min for 2 min. The resulting peptic peptides were then separated on a C18 column (Waters, Acquity UPLC BEH C18, 1.7 μm, 1.0 mm × 50 mm) fitted with a Vanguard trap column using a 3 to 85% acetonitrile (containing 0.2% formic acid) gradient over 10 min at a flow rate of 50 μL/min. The peptides generated were directed into a Waters Xevo G2-Tof mass spectrometer for mass analysis (MSE mode). The Xevo G2 instrument was calibrated using Glu-fibrinopeptide fragment ions, and mass spectra were recorded over the range of m/z 255−1950 in ESI

mass spectrometry (HDX-MS) stability, and hydrophobic properties] showed a connection between overall increases in global hydrophobicity, driven largely by a destabilized ECD structure, and poor drug disposition character. Modulation of the ECD fusion position on the antibody led to a profound decrease in hydrophobicity and improved ECD stability, as measured by differential scanning calorimetry (DSC) thermal melt and decreased deuterium uptake in HDX-MS experiments. These improvements ultimately manifested in drug disposition properties that were more closely related with the parental antibody. Taken together, these findings highlight the importance of exploring the site of fusion on an antibody for selecting BsAb formats with optimal biophysical and in vivo PK properties. Linking these findings to the precise mechanisms/ molecular interactions involved in the clearance of BsAbs remains an opportunistic area for improving the in vivo properties and potency of these molecules.

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MATERIALS AND METHODS Expression and Purification of Recombinant Monoclonal and Bispecific Antibody Proteins. The mAbs and BsAbs described within this report were expressed in either transient HEK-293 or stably transfected Chinese hamster ovary cells that were generated at Eli Lilly and Company (Indianapolis, IN). Purification of these compounds from concentrated cell culture supernatants proceeded via a two-step purification method. In a first step, crude conditioned cell culture medium containing the antibody was captured on Protein A MabSelect chromatography resin (GE Healthcare, Waukesha, WI), and following column washing, the bound antibody was eluted using a single-step gradient of 10 mM citrate (pH 3.0) containing 150 mM sodium chloride; pooled fractions were then concentrated using an Amicon Ultra-15 concentrator (Millipore, Billerica, MA). A second purification step that utilized Superdex G200 preparative gel filtration (GE Healthcare) facilitated the final polishing of the Protein Acaptured materials. In this step, the gel filtration column was first pre-equilibrated in 1× phosphate-buffered saline (PBS) (pH 7.4) running buffer, after which proteins of interest were injected and eluted using an isocratic gradient of 1× PBS (pH 7.4). The protein was pooled on the basis of the size properties, and purity was assessed using both simply blue stained (Life Technologies, Carlsbad, CA) sodium dodecyl sulfate−polyacrylamide gel electrophoresis gel analysis and analytical gel filtration on a TSKG3000SWXL column (Tosho Bioscience, Toyko, Japan) and was generally found to be >95%. Recombinant, soluble cynomolgus monkey FcRn was expressed in 293EBNA cells transfected with the plasmids encoding the soluble portion FcRn and β2m, and the protein was purified as previously described.20,21 Differential Scanning Calorimetry Analysis. Thermal melting properties of mAbs and BsAbs evaluated in this work were assessed using DSC using a NanoDSC instrument equipped with an autosampler (TA Instruments, New Castle, DE). Samples were first dialyzed into 1× PBS (pH 7.4) overnight at 4 °C, diluted to a final concentration of 1 mg/mL, and thoroughly degassed under vacuum for 15 min. Samples and corresponding buffer controls were then subjected to increased temperature gradients ranging from 20 to 110 °C with a temperature ramp of 1 °C/min under a constant cell pressure of 45 psi. Resultant data obtained from sample scans were buffer scan subtracted, converted to molar heat capacity, and fit to a two-state scaled model to obtain Tm. 3118


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Biochemistry + mode. All acquired data were mass corrected using a 2 μg/ mL solution of LeuEnk in 50% ACN, 50% H2O, and 0.1% FA at a flow rate of 5 μL/min every 30 s (m/z 556.2771). The peptides were initially identified by Waters Protein Lynx Global Server 3.02. The resulting peptide list was imported into Waters DynamX 3.0 software, where the relative deuterium incorporation for each peptide was determined by processing the MS data for deuterated samples along with the nondeuterated control. Pharmacokinetic Studies. Four pharmacokinetic studies were conducted in accordance with Standard Operating Procedures (SOPs) and the protocol as approved by Eli Lilly and Company and in compliance with the requirements of Covance Laboratories. Cynomolgus Monkey Pharmacokinetic Study. The cynomolgus monkey pharmacokinetic study was performed with male cynomolgus monkeys (2.3−3.2 kg). Two monkeys were assigned to each study group, and all animals received a single intravenous (iv) bolus dose of either G41, G41-C-HC, G41-N-LC, or G41-N-HC dissolved in PBS (pH ∼7.4) at a concentration of 2.0 mg/kg. Each animal had blood samples collected via a femoral vein. Blood samples were collected predose and 0.25, 1, 3, 6, 12, 24, 48, 72, 120, 168, 240, 336, 432, 504, and 600 h after administration of the dose. The blood samples were collected into tubes containing sodium citrate anticoagulant maintained in chilled cyroracks and centrifuged to obtain plasma. CD-1 Mouse Pharmacokinetic Study. The CD-1 mouse pharmacokinetic study was performed with male mice (20−22 g). Three mice per time point were assigned to each study group, and all animals received a single intravenous (iv) bolus dose of either G41, G41-C-HC, G41-N-LC, or G41-N-HC dissolved in PBS (pH ∼7.4) at a concentration of 2.0 mg/kg. Each animal had blood samples collected via cardiac puncture. Blood samples were collected predose and 1, 8, 24, 48, 72, 120, 168, 240, and 336 h after administration of the dose. The blood samples were collected into tubes containing sodium citrate anticoagulant maintained in chilled cyroracks and centrifuged to obtain plasma. CD-1 Mouse Pharmacokinetic Study with Clodronate Liposome Treatment. The kinetics of the G41 mAb and G41-C-HC were evaluated in CD-1 mice pretreated with liposomes containing clodronate to deplete macrophage cells in vivo. The pretreatment material, liposomal clodronate (Clodrosome), was received from Encapsula NanoSciences as a preformulated suspension in PBS (pH ∼7.4). Approximately 24 and 48 h prior to administration of G41 mAb or G41-C-HC, each animal received a single 0.1 mL iv injection of either liposomal clodronate at a concentration of ∼20−23 mg/ kg via a saphenous vein and once daily thereafter for the entire duration of the study. Each animal received an iv bolus dose of G41 mAb or G41-C-HC dissolved in PBS (pH ∼7.4) at a concentration of 2.0 mg/kg. Blood samples were collected from each animal via a femoral vein prior to dosing and 1, 8, 24, 48, 72, 120, 168, 240, and 336 h after administration of the dose into tubes containing sodium citrate anticoagulant. The blood samples were maintained in chilled cryoracks and centrifuged to obtain plasma. FcγRIIb Knockout Mouse Pharmacokinetic Study. The kinetics of G41 mAb and G41-C-HC were evaluated in FcγRIIb knockout mice (B6 mouse background strain) (Jackson Laboratory). Each animal received an iv bolus dose of G41 mAb or G41-C-HC dissolved in PBS (pH ∼7.4) at a

concentration of 2.0 mg/kg. Blood samples were collected from each animal via a femoral vein prior to dosing and 1, 8, 24, 48, 72, 120, 168, 240, and 336 h after administration of the dose into tubes containing sodium citrate anticoagulant. The blood samples were maintained in chilled cryoracks and centrifuged to obtain plasma. Bioanalytical Assays and Pharmacokinetic Data Analysis. Concentrations of the G41, G41-C-HC, G41-N-LC, or G41-N-HC molecules in cynomolgus monkey or mouse plasma were determined using anti-human IgG enzyme-linked immunosorbent assays (ELISAs) for each of the molecules. In brief, each well of an Immulon 4HBX microtiter plate (Thermo Scientific, Waltham, MA) was coated with either goat anti-human IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) at 4 °C overnight. After washing and blocking, all other standards, control samples, and study samples were added to the plates and then incubated for 1 h at room temperature. After washing, the bound molecules were detected with a horseradish peroxidase-conjugated mouse antihuman IgG (Fc) antibody (Southern Biotechnology Associates, Birmingham, AL) by the TMB Microwell Peroxidase Substrate System (KPL, Gaithersburg, MD) for a colorimetric response. Plates were read at 450−493 nm with a reference of 630 nm. Concentrations from plasma or serum samples were determined from a standard curve prepared with known amounts of G41, G41-C-HC, G41-N-LC, or G41-N-HC in the appropriate cynomolgus monkey matrix using a four- or fiveparameter algorithm. The standard curve range for G41, G41C-HC, G41-N-LC, and G41-N-HC was from 3.91 to 500 ng/ mL, and the lower limit of quantitation (LLOQ) was defined as 25 ng/mL. Plasma concentration−time data following iv administration were described using a model-independent method according to the statistical moment theory using the either WinNonlin Professional 5.0 or Phoenix WinNonlin software package (Pharsight, A Certara Company, St. Louis, MO). The calculated parameters included the maximum serum concentration (Cmax), the area under the curve (AUC0−∞), the clearance (CL), and the elimination half-life (t1/2). Murine Tissue Distribution Study. The murine tissue distribution study was conducted in accordance with SOPs and the protocol as approved by Eli Lilly and Company and in compliance with the requirements contained in the MPI Research Radioactive Materials License Number 21-11315-02 and all applicable regulations issued by the Nuclear Regulatory Commission (NRC). Briefly, nonradiolabeled G41, G41-C-HC, and G41-N-HC were conjugated to diethylenetriaminepentaacetic acid (DTPA) and radiolabeled with 111In at MPI Research, Inc., to target a low specific activity. To prepare the dosing formulations, the appropriate amount of the radiolabeled test article was combined with the required volume of a vehicle, phosphate-buffered saline (PBS), and mixed. The radiolabeled compounds were compared to the appropriate nonlabeled compounds by the total human IgG ELISA (described in Bioanalytical Assays and Pharmacokinetic Data Analysis) to determine the effect of the label on the linear range in the ELISA. The dosing formulations were prepared on the day prior to administration. The dosing formulations were administered once via iv injection into the tail vein. The actual amount administered was determined by weighing the dose syringe before and after administration of the dose. Each dosing syringe was also counted in the dose calibrator prior to and after dosing. Doses 3119


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Biochemistry were labeled to a target dose level of 2 mg/kg (15 μCi/ animal). The final dose was based on the A280 concentration, and effort was made to administer approximately the same mass and volume of dosing formulation to each group. Individual doses were based on body weights collected on day −1. A dose wipe of the injection site was collected into a plastic container and was retained at room temperature prior to analysis by LSC to determine residual radioactivity. The recovered radioactivity was subtracted from the administered amount to give the actual radioactive dose administered. Blood samples (∼200−300 μL) were collected from all animals via the maxillary vein or cardiac puncture after carbon dioxide inhalation (terminal samples only) into tubes containing sodium citrate. All samples were analyzed using the gamma counter to determine residual radioactivity. Blood samples were collected 0.083, 24, 96, 120, and 168 h after the dose (cohorts of three animals per group). The total weight of each blood sample was recorded and analyzed for radioactivity using the gamma counter. The %ID/g values were corrected for radioactive decay over time. Following evaluation of the samples for hematocrit values, the samples were processed to plasma, and the total weight of each packed cell fraction and plasma sample was recorded. Packed cell fraction and plasma samples were analyzed for total radioactivity via the gamma counter. Individual gamma radioactivity counts were utilized for image reconstruction and analysis. Following the final scan, all surviving animals were euthanized by carbon dioxide inhalation followed by cervical dislocation, and the entire carcass was placed into the dose calibrator for measurement of the total remaining radioactivity. The adrenal gland, bladder (urinary), bone (femur), bone marrow (femur), brain, muscle (gastrocnemius, both quadriceps, and scapular region), heart, kidney, large intestine/ cecum with contents, liver, lung, lymph nodes (mesenteric), pancreas, skin (ventral and upper and lower dorsal), small intestine with contents, spleen, stomach with contents, testes, thymus, thyroid, and fat pad were collected from all three animals per time point (24, 96, 120, and 168 h after the dose). The tissues were rinsed with 0.9% saline and blotted dry as appropriate. All tissues were weighed and collected into a plastic container at room temperature and maintained under this condition until they were analyzed for total radioactivity via the gamma counter. Like the blood samples, the %ID/g values for the tissues were corrected for radioactive decay over time. An independent t test was conducted to compare the tissue:blood ratio data between pairs of compounds. Following analysis, tissues were discarded. Following tissue collection, the residual carcass of all animals was analyzed for total radioactivity using the gamma counter. The remaining carcasses were stored at approximately −10 to −30 °C until they decayed and were then discarded. Individual gamma and dose calibrator radioactivity counts were utilized for image reconstruction and analysis. A subset of the animals was analyzed by SPECT/CT scans. Animals were anesthetized with 2lpm oxygen/1.5−2% isoflurane, and eye lube was placed on the animals prior to scanning. Static SPECT scans were performed on all surviving animals 24, 120, and 168 h after the dose for 60 min. Each static scan was followed by a CT scan for anatomical reference. After each scan time point, each animal was placed in the dose calibrator and the total remaining reactivity was recorded. SPECT/CT data were transferred to inviCRO, LLC, for image analysis.

Immunohistochemistry Detection of G41 and G41-CHC in CD-1 Mouse Tissue. The study was conducted in accordance with SOPs and the protocol as approved by Eli Lilly and Company and in compliance with the requirements of Covance Laboratories. Male CD-1 mice (20−22 g; one mouse per time point) received a single iv bolus dose of either G41 or G41-C-HC dissolved in PBS (pH ∼7.4) at a concentration of 2.0 mg/kg. Tissues were collected 1, 6, 24, and 72 h after administration. Liver biopsy samples were collected from the left lobe of each animal. The left kidney and the entire spleen were collected from each animal. Tissue samples from animals treated with G41, G41-N-HC, or G41-CHC were embedded in optimal cutting temperature (OCT) medium and then frozen on a dry ice/2-methylbutane bath. OCT frozen tissue blocks were sectioned at a thickness of 8 μm. The slides were fixed in acetone for 5 min, air-dried, and then stored frozen at approximately −70 °C prior to use. Tissue samples from animals were transferred to 10% NBF (neutral buffered formalin solution) for up to 72 h under ambient conditions. After fixation in 10% NBF, the tissue samples were transferred to 70% ethanol. The tissues were then embedded in paraffin within 9 days of transfer to 70% ethanol. Following the embedding procedure, the paraffin blocks were sectioned at a thickness of 5 μm and placed on slides using standard procedures. Prior to staining, the formalin-fixed paraffin embedded (FFPE) slides were processed and antigen retrieval was conducted. Briefly, each FFPE slide was baked at 60 °C (Boekel Incubater Shaker II, Feasterville, PA) for approximately 60 min to adhere the tissue to the glass slide. Tissues were then deparaffinized and rehydrated using the following steps. Warmed slides were moved from the incubator to the first xylene (Fisher, Pittsburgh, PA) bath, for 5 min, and then moved into a second bath of xylene for an additional 5 min. The next steps describe movement through the graded alcohols to water, 10 dips and 2 min each into 100% ethanol (Decon, King of Prussia, PA), two times into 95% ethanol (Decon), and one time into 70% ethanol (Decon), before being transferred to doubly distilled H2O (ddH2O) (Millipore, Billerica, MA). Antigen retrieval unmasking of formalin-fixed cross-linked epitopes was conducted using a 1× heat-induced epitope retrieval solution, Diva Decloaker (BioCare Medical, Concord, CA), in a Decloaking Chamber (BioCare Medical) for 30 s at 125 °C under pressure. Upon removal from the decloaking chamber and following a ddH2O rinse, each tissue section was outlined with a PAP pen (BioGenex, Freemont, CA) to control solution placement prior to being placed on the Dako (Carpinteria, CA) Autostainer. A Dako Autostainer was programmed to stain all slides for the detection of human IgG with standard hematoxylin and eosin staining using the following steps. Briefly, slides were prerinsed with 1× Dako Wash Buffer (Dako) prior to blocking with 10% horse serum (Lampire Biologics, Pipersville, PA) in 1× PBS (Hyclone, Logan, UT). The blocking reagent was replaced with goat anti-human IgG (Bethyl Laboratories, Montgomery, TX) at a concentration of 10 μg/mL. A control IgG for the detection of mouse IgG was obtained from Jackson ImmunoResearch (West Grove, PA) or R&D Systems (Minneapolis, MN) and used at concentrations equivalent to those of the primary antibodies as a control to determine the specificity of detection. Following a 1 h incubation with the primary antibody or control IgGs, slides were rinsed a minimum of four times with 1× wash buffer followed by 3120


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Biochemistry Table 1. Biophysical and Biochemical Summarized Results for GX1-D2 BsAbsa FcRN column elution (min)

Tm (°C)

molecule

mouse

cyno

heparin binding ([NaCl] at main peak elution apex, mM)

HIC retention (min)

CH 2

CH3/FAB

ECD

G41 G11-C-HC G21-C-HC G41-C-HC G41-N-LC G41-N-HC Ustekinumab Briakinumab

41.5 40.7 41.5 41.5 N/A N/A 42.6 45.2

39.2 39.1 39.4 39.4 N/A N/A 39.7 41.6

none none none none none none N/A N/A

10.8 15.2 14.9 15.2 12.7 10.6 N/A N/A

70.4 67.9 73.6 71.2 69.6 69 N/A N/A

75.9 75.4 80.7 81.1 73.9 74.5 N/A N/A

N/A 51.9 50.4 49.4 54.9 ND N/A N/A

a

N/A, data not available; ND, not detected; none, no column retention observed.

impacted molecular biophysical behavior. Specifically, molecular interactions governed by hydrophobic and charge-based (heparin) mechanisms as well as interactions with FcRn were evaluated using established chromatographic methods. In addition, molecules were also assessed for overall stability using two orthogonal approaches, DSC and HDX-MS. Table 1 summarizes the biophysical profiling of these BsAbs in a battery of analyses aimed to understand molecular attributes associated with pharmacokinetics character. Alterations in FcRn binding and engagement were conducted using a column-based method as outlined in Schlothauer et al.22 In this format, biotinylated cynomolgous monkey and mouse FcRn proteins were indivually captured on a solid phase streptavidin agarose support and BsAb molecules were captured under the permisive FcRn binding conditions of low pH (∼5.5). Captured BsAbs were eluted using a linear pH gradient to pH 7.0, and elution times were monitored and compared aganist two control antibodies, Ustekinumab and Briakinumab, which have been previously used to establish connectivity between well-studied antibodies that exhibit good and poor pharmacokinetic behavior linked to FcRn interaction modifications.22 In this assay, when either mouse or cynomologus monkey FcRn was evaluated, none of the Cterminally D2 ECD fused variants exhibited any measurable changes in FcRn elution, relative to the paretntal G41 mAb, and they were found to elute well before that of strong FcRn interacting, Briakinmab control (Table 1). Given that these molecules, independent of isotype, did not show any altered FcRn engagement properties, G41-N-LC and G41-N-HC were not evaluated using this method. Measurements of global molecule hydrophobicity were measured using a chromatographic HIC-based method previously described.16 Evaluation of the elution times of G11-, G21, and G41-C-HC family of molecules showed that HIC retention was found to be increased relative to the parental G41 mAb and was not dependent on the isotype of the G1 mAb (Table 1). Positional modulation of the D2 ECD from the C-terminus to the N-terminus of the LC and HC of G41 served to reduce the hydrophobic character of the molecules to differing degress. Specifically, the movement of the D2 ECD fusion position from the C-terminus of the HC to the N-terminus of the light chain in G41 resulted in a modest but meaninigful reduction in HIC retention. Surprisingly, a full recapitulation of parental G41 mAb HIC retention was afforded when the D2 ECD was fused to the N-terminus of the HC, indicating that the overall molecule hydrophobicity was governed by the specific placement of the D2 ECD. To further explore the causes for these differences in hydro-

detection in combination with DAPI at a 1:500 dilution for 30 min prior to being water rinsed. Stained slides were coverslipped using Dako fluorescent mounting medium. Reagent volumes ranged between 150 and 200 μL/slide, depending on the tissue area to be covered. All steps were performed at ambient temperature having slides and reagents protected from light.

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RESULTS Design and Biophysical Characterization of the G1ECD BsAb Molecules. The studies described herein were performed using a series of related compounds built upon a well-characterized antibody targeting an undisclosed soluble low-abundance antigen fused with a potent inhibitory ∼11.5 kDa domain isolated from the extracellular domain of human VEGFR1/FLT1 receptor (deemed D2).19 The D2 ECD utilized in this study was altered from that described by Shen et al. to remove two N-linked glycosylation sites (N164 and N196) that resulted in poor disposition due to improper sialylation and rapid ASPGR-mediated clearance (data not shown) and mutations designed to improve upon the chemical stability of the D2 domain (Y139, M148, N212, and N227).19 For these studies, several related molecules were expressed and purified and are diagrammed in Figure 1A−C. D2 was fused to three different human antibody isotypes [IgG1 (G1), IgG4PAA (G4), and IgG2 (G2)]. Additionally, the location of the fusion of the D2 ECD was placed at the N- and C-termini of the heavy and light chains of G4mAb (N-HC, C-HC, N-LC, and C-LC, respectively). Attempts to generate a C-terminal light fusion of D2 on G41 (G41-C-LC) were unsuccessful as the molecule behaved poorly during protein expression/purification, and insufficient quantities were procured (data not shown) and will not be further discussed in the context of this report. Purified samples were assessed by analytical gel filtration and found to be >95% pure with similar levels of soluble aggregate, and LC−MS analysis confirmed the expected mass and absence of glycosylation, except for the expected IgG CH2 glycans (data not shown). As a second step, the purified G41-C-HC, G41-N-HC, and G41-N-LC molecules were assessed for the binding of the target to human VEGFA121 (D2 ECD) and the cognate binding partner for G41 mAb using Biacore SPR. These studies did not show any major differences in the calculated equilibrium binding affinities or kinetics for either ligand tested (data not shown). Given that there were no major differences in molecular behavior noticed during purification and binding affinities were unperturbed with the positional modulation of the D2 ECD, we set forth to determine if these architectural modifications 3121


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Figure 2. Differential scanning calorimetry thermograms for (A) G41 mAb, (B) G41-C-HC, (C) G41-N-LC, and (D) G41-N-HC molecules in 1× PBS (pH 7.4) at 1 mg/mL. Plots represent the obtained thermal transitions as a function of the increase in temperature (black lines) and associated fits to a two-state fitting model (red lines).

HC, a dramatic improvement in its apex melting temperature was observed, which became indistinguishable from that of the G41 CH2/FAB domain. Given these differences in the thermal stability of the D2 ECD as a function of positional fusion on G41, we assessed the global structure changes in both elements using HDX-MS Don experiments. The peptide coverages for the D2 ECD domain and G41 mAb regions obtained following pepsin digestion are shown in Figures S1−S3. Good peptide coverage was obtained for the D2 ECD and heavy chain across the three G41 variants that were evaluated [>95% and 79%, respectively (Figures S1 and S2)]. The light chain peptide coverage was not as robust comparatively (60%); notably absent was consistent coverage within the vL domain of the G41 mAb, while good coverage of the CL domain was obtained (figure S3). A pairwise comparative analysis of the changes in deuterium uptake in the G41 mAb regions of the three BsAbs as compared to the parental mAb is presented in Figure S4A−C. This analysis shows minimal changes in the mAb portions of these BsAb variants, suggestive of the fact that the structure and dynamics of the mAb region were conserved in these BsAbs. Moving our focus to the D2 ECD region among the three positional fusions evaluated, we observed a decreased level of deuterium exchange that was dependent on the fusion position of the D2

phobicity, we evaluated the overall stability of the G41 series of molecules using DSC and HDX-MS methods. Results from DSC thermal signatures indicated that regardless of the molecule tested the mAb regions of each fusion, independent of fusion position, were comparable (Figure 2A−D and Table 1). While mAb stability appeared to be unaltered with different D2 ECD fusion positions , the thermal stability properties of the D2 ECD were not. In the context of the G41-C-HC molecule (the most hydrophobic molecule evaluated), the D2 ECD was found to have a broad, thermal melting profile with an apex melting temperature of ∼50 °C (Figure 2B). In comparison to that of the D2 ECD alone, the apex melting temperature for the D2 ECD when fused to G41 appears to be slightly reduced by ∼5 °C relative to that measured for the FcD2 fusion measured in our previous report.16 Modulation of the D2 ECD position from the C-terminus of the HC to the LC of G41 led to a subtle but meaningful increase in the apex melting temperature of the D2 ECD (from 49.4 to 54.9 °C), but a broad flattened melting profile was oberved. This positional alteration and observed change in Tm was found to also bring the D2 ECD thermal stability back to that reported in our mAbs report when fused to Fc alone.16 Interestingly, when the D2 ECD was fused to the N-terminus of the G41 3122


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Figure 3. Heat maps showing the relative fractional uptake differences for (A) the D2 ECD on G41-C-HC compared to G41-N-HC and (B) the D2 ECD on G41-C-LC compared to G41-N-LC. Regions with no bars represent the absence of peptide coverage. This figure was generated using DynamX (Waters Corp.).

Figure 4. Mean pharmacokinetic profiles of G41, G41-C-HC, G41-N-LC, and G41-N-HC following a single 2 mg/kg iv administration in (A) cynomolgus monkeys and (B) CD-1 mice. Data are the means for two animals per time point for all molecules.

particularly interesting that both of the N-terminal G41-D2 fusions resulted in such different biophysical behavior, which rules out the simple explanation of attaching the D2 ECD via the N- or C-terminal end to G41 mAb as being the driving principle for these observations. Additional studies outside the scope of this report are needed to fully understand the molecular architecture subtleties that drive these differences and will be the focus of a separate report. Pharmacokinetics of the G41-ECD Bispecific mAbs in Cynomolgus Monkeys and Mice. Because the isotype changes had little influence on the ECD BsAb biophysical properties, we focused our in vivo characterizations on the G41ECD BsAbs with modulations in the fusion position of the ECD given the observed differences in hydrophobicity, selfassociation, and Tm across these constructs. The kinetics of the three G41-ECD constructs (G41-C-HC, G41-N-HC, and G41N-LC) were assessed relative to the G41 mAb following a

ECD. Specifically, pairwise comparison of G41-C-HC and G41N-LC shows no significant differences in deuterium exchange indicating a conserved structure (Figure 3A). However, this same conservation was not present when G41N-HC was compared with G41-C-HC as a decrease in the level of deuterium exchange was observed when the D2 ECD domain was attached to the N-HC of G41 mAb (Figure 3B). These changes in the HDX signature are also in agreement with the thermal stability data obtained for these variants (Table 1 and Figure 2A−D). Taken together, these data suggest that the overall biophysical behavior of the G41-D2 variants is largely dictated by the fusion position of the D2 ECD domain. Given that the linker elements that connect the D2 ECD to the G41 mAb were conserved in each of the fusion positions, these data imply that the interplay between the G41 mAb local environment and/or structural constraints invoked by the BsAb architecture drive these differences. It is 3123


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Table 2. Mean Cynomolgus Monkey Pharmacokinetic Parameters of the G41 Bispecific Molecules following a Single 2 mg/kg iv Administrationa molecule G41 G41-C-HC G41-N-LC G41-N-HC

dose (mg/kg) 2 2 2 2

(n (n (n (n

= = = =

2) 2) 2) 2)

Cmax (μg/mL)

AUC0−∞ (h μg−1 mL−1)

Vss (mL/kg)

clearance (mL h−1 kg−1)

T1/2 (h)

69.7 51.4 44.9 66.4

6615 606 1533 4273

40.9 60.9 35.1 34.2

0.3 3.32 1.39 0.47

37.6 12.5 18.1 51.2

a

Cmax, maximal observed plasma concentration; AUC0−∞, area under the plasma concentration curve from zero to infinity; Vss, volume of distribution at steady state; CL, clearance; T1/2, elimination half-life determined from the last three measurable concentration vs time points; n, number of animals per group.

Table 3. Mean Mouse Pharmacokinetic Parameters of the G41 Bispecific Molecules following a Single 2 mg/kg iv Administrationa molecule G41 G41-C-HC G41-N-LC G41-N-HC

dose (mg/kg) 2 2 2 2

(n (n (n (n

= = = =

2) 2) 2) 2)

Cmax (μg/mL)


Vss (mL/kg)


T1/2 (h)

21.9 25.5 28.6 33.9

1451 873 1157 2193

76.9 70.7 104 52.1

1.38 2.29 1.73 0.91

38.6 21.4 41.7 39.6

a

Cmax, maximal observed plasma concentration; AUC0−∞, area under the plasma concentration curve from zero to infinity; Vss, volume of distribution at steady state; CL, clearance; T1/2, elimination half-life determined from the last three measurable concentration vs time points; n, number of animals per time point (nonserial sampling was used in the mouse PK study).

Figure 5. Mean (± SD) concentrations (%ID/g) of G41 mAb, G41-C-HC, and G41-N-HC in male CD-1 mice following a single iv administration of ∼2 mg/kg (∼0.015 mCi/animal) in (A) plasma, (B) liver, (C) kidneys, (D) muscle (gastrocnemius), (E) skin, and (F) spleen. Tissue and plasma data are n = 3 per time point. The legend indicates the time point of each measurement in hours for panels B−F.

comparisons of the concentration versus time profiles in the context of the same studies. Following administration to cynomolgus monkeys or mice, the G41 mAb and G41-N-HC constructs showed similar pharmacokinetics (panels A and B of Figure 4 and Tables 2 and 3, respectively). The clearance of G41 and G41-N-HC was 1.38 and 0.91 mL h−1 kg−1, respectively, in mice and 0.30 and 0.47 mL h−1 kg−1, respectively, in cynomolgus monkeys. The half-lives of G41 and

single 2 mg/kg iv administration to cynomolgus monkeys or CD-1 mice. This dose level was selected because in previous studies it had been established that G41 displayed nearly dose proportional increases in exposure over a broad dose range (1−10 mg/kg).16 The pharmacokinetics of G41 and G41-CHC were reported previously in cynomolgus monkeys;16 however, in the context of the studies herein, the kinetics of both molecules were re-evaluated to allow for head-to-head 3124


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Biochemistry G41-N-HC were 38.6 and 39.6 h, respectively, in mice and 37.6 and 51.2 h, respectively, in cynomolgus monkeys. In contrast, G41-N-LC and G41-C-HC were characterized by faster elimination and shorter half-lives in both species relative to those of G41. In mice, G41-N-LC and G41-C-HC showed ∼1.3- and ∼1.7-fold faster clearance and shorter half-lives, respectively, relative to those of G41. G41-C-HC had an ∼11fold more rapid clearance and an ∼3-fold shorter half-life than G41 in cynomolgus monkeys, consistent with our previous findings.16 G41-N-LC showed better pharmacokinetics than G41-C-HC in cynomolgus monkeys, but the clearance and half-life of the G41-N-LC molecule were ∼4.6- and ∼2-fold poorer, respectively, relative to the parental G41. Antidrug antibodies were observed for all of the molecules in both species >336 h after the dose apart from G41-C-HC in cynomolgus monkeys (ADA titer data not shown). Because ADA could be measured only >336 h after administration, there were sufficient concentration versus time data to calculate the pharmacokinetic parameters of the molecules, and thus, the ADA did not influence the interpretation of the data. In addition, concentrations of the ECD-based BsAbs measured using a VEGF-based antigen capture and Fc (i.e., mAB) detection ELISA also showed the BsAbs had rapid clearance relative to their parental mAbs, indicating that functional ECD is present on the BsAbs and not clipped from the mAb in vivo (data not shown). The data suggested there were factors specifically related to the context of ECD fusion position with the G41 mAb that influenced the in vivo pharmacokinetics of the BsAb constructs differentially. Tissue Distribution of the G41-ECD Bispecific mAbs in Mice. The biodistribution and tissue accumulation kinetics of the G41-ECD BsAbs were studied in mice to gain additional mechanistic insight into the differential peripheral clearance observations. G41-N-HC and G41-C-HC were selected for these murine studies because these molecules were potential drug candidates intended for further development, whereas G41-N-LC was not. Following a single iv injection of 111Inlabeled DTPA-conjugated versions of the two BsAb molecules and the G41 mAb into CD-1 mice, the concentrations of five major highly vascularized organs and/or tissue (liver, kidney, spleen, skin, and muscle) were measured for each molecule over the course of 168 h after administration (Figure 5A−F). Plasma concentrations of the 111In-labeled DTPA-conjugated versions of the two BsAb molecules and the G41 mAb were measured over the course of 168 h after the dose for each of the molecules. In addition, animals were also imaged using SPECT/CT to ensure that the biodistribution was monitored for organs and/or tissues that were not being collected for quantitative analyses up to 168 h after compound dosing (Figure S3). Radiometrically derived and ELISA-derived total antibody plasma kinetic data were largely comparable, showing the exposures of G41 > G41-N-HC > G41-C-HC (Figure 5A). The exposure profiles were also similar to those of the unlabeled counterpart of each construct (Figure 4B), suggesting labeling the molecules did not impact the disposition. The comparison of the blood:plasma partitioning ratio data of the three molecules shows the findings are also mainly alike (Table 4), indicating blood cell binding is not involved in the differential clearance observations for the G41-ECD BsAbs. SPECT/CT images show the BsAbs and G41 mAb are distributed to similar tissues over time following administration (Figure S5). Three-dimensional reconstruction of the imaging

Table 4. Mean (±SD) Blood:Plasma Partitioning Ratios following a Single iv Administration of ∼2 mg/kg (∼0.015 mCi/animal) to CD-1 Mice for G41, G41-C-HC, and G41-NHC over Timea compound G41

G41-C-HC

G41-N-HC

time (h after dose) 0.083 24 96 120 168 0.083 24 96 120 168 0.083 24 96 120 168

mean (SD) 0.61 0.83 0.83 0.82 0.90 0.55 0.88 0.88 1.22 1.48 0.54 0.87 0.87 0.96 0.93

(0.12) (0.06) (0.11) (0.02) (ND) (0.11) (0.04) (0.08) (0.24) (ND) (0.11) (0.01) (0.07) (0.10) (0.10)

a Data are n = 3 per time point except when noted otherwise. The standard deviation (SD) is reported only for time points with n = 3 per animals. Mean and SD (standard deviation) values were determined from the individual animal data (%ID/g blood divided by %ID/g plasma) ND, not determined due to N = 2 data point for the time point.

data 24 h after the dose shows both the BsAbs and the G41 mAb molecule display an increased disposition to highly vascularized tissues with high cardiac output, including the liver, kidneys, and spleen. Quantitative analyses of five major highly vascularized tissues (liver, spleen, kidney, skin, and muscle) over time show the majority of the %ID/g (percent injected dose per gram of tissue) for the molecules is within these five tissues (Figure 5B−F). Normalization of the tissue concentrations by the amount in the blood across groups was performed to determine differences in tissue concentrations (Figure 6). Higher concentrations of G41-C-HC are evident in many of the tissues relative to those of G41 and G41-N-HC. There were statistically significant differences (P < 0.05) noted within a time point between the molecules within each tissue (Figure 6). These results suggest that there is a meaningful difference in the mechanism of clearance between the molecules. Notably, liver and kidney show the highest level of accumulation of G41-C-HC. The data indicate the differences in the peripheral clearance of G41-C-HC relative to G41-N-HC and G41 are mainly due to accumulation of G41-C-HC in these two organs. Like G41-C-HC, we also noted the preponderance of the G41-N-HC data show evidence of increased disposition to the kidney and liver tissues relative to the parental G41 mAb. The extent of tissue accumulation of G41-N-HC is much lower than that of G41-C-HC, which is consistent with the similar peripheral pharmacokinetic findings of G41-N-HC with the G41 mAb. The %ID/g (or concentration) values of the three molecules were relatively low in the muscle, skin, and spleen compared to those in the kidney and liver tissues (Figure 6). With respect to the skin, lower concentrations of G41-C-HC were observed at almost each of the time points relative to the G41-N-HC BsAb and G1 mAb (Figure 6D). Like the spleen concentrations, the skin, kidney, and muscle concentrations of each of the molecules are lower than the liver concentrations, 3125


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Figure 6. Average tissue:blood ratios (mean %ID/g tissue:mean %ID/g blood) of G41 mAb, G41-C-HC, and G41-N-HC in male CD-1 mice following a single iv administration of ∼2 mg/kg (∼0.015 mCi/animal) in (A) liver, (B) kidneys, (C) muscle (gastrocnemius), (D) skin, and (E) spleen. Data are for N = 3 per time point. The asterisk indicates statistically significant (P < 0.05) differences between G41 mAb and G41-C-HC or G41-N-HC. The carrot symbol indicates statistically significant (P < 0.05) differences between G41-C-HC and G41-N-HC.

pharmacokinetic profiles displaying a reasonable divergence in the peripheral exposure of the compounds (Figure 4B). At each of the time points analyzed, neither the G41 mAb nor the two BsAbs were detected by IHC in the skin or gastrocnemius muscle tissue (data not shown). With regard to the liver, kidney, and spleen tissues, only the G41-C-HC was detected in each of the tissues (panels A−C of Figure 7 show representative images 1 h after the dose). Under the anti-human IgG staining conditions that were used, the qualitative intensity of the staining for G41-C-HC was much greater than that of either G41-N-HC or G41 in each of the three tissues at each time point tested. In addition, the level of detection of G41-C-HC in terms of intensity and frequency was also higher at earlier time points and progressively diminished at later time points in all of the groups (data not shown). In terms of the cellular disposition in the liver, the IHC detection of the molecules was located multifocally mainly in small segments of the sinusoids (likely endothelial cells) and in the cytoplasm of scattered Kupffer cells (liver macrophages) throughout the parenchyma (Figure 7A). The spleen tissue also showed positive IHC labeling occurred in the sinuses (presumably endothelial cells) and within the cytoplasm of mononuclear cells (presumably macrophages) multifocally scattered throughout the red pulp (Figure 7C). Like the liver and spleen, IHC evaluation of kidney tissue showed G41-C-HC was located multifocally in variably sized segments of the interstitial vasculature (mainly endothelial cells) in the cortex and medulla and in the cytoplasm of scattered mononuclear cells (presumably macrophages) throughout the parenchyma (Figure 7B).

suggesting the mechanisms of clearance for the molecules in these organs make a smaller contribution than those in the liver. Cellular Distribution of G41, G41-N-HC, and G41-C-HC Constructs in Normal Mouse Tissues by Immunohistochemistry. In a previous report, we demonstrated that the aberrant clearance of G41-C-HC in cynomolgus monkeys was linked to a mechanism(s) involving the association with liver sinusoidal endothelial cells (LSECs).16 In the context of our previous report, we did not fully interrogate our BsAb cellular disposition in other organs.16 While current murine radiolabel biodistribution studies (results reported above) clearly show that liver is mechanistically linked to the rapid clearance of G41-C-HC, additional organs of clearance, including the kidney and spleen, were identified. Thus, as means to further extend our understanding of the types of cells involved in the disposition of our BsAbs and interrogate the connectivity of our findings across species, we chose to conduct immunohistochemistry (IHC) studies on liver, kidney, and spleen sections derived from normal mice administered either G41, G41-NHC, or G41-C-HC. Although the radiolabel biodistribution study in mice indicated low concentrations of our molecules accumulated in skin and muscle (specifically gastrocnemius), these tissues were also evaluated by IHC for G41 and the BsAbs cellular disposition profiles as an orthogonal approach to verify these findings. The distribution within each of the three tissues mentioned above was evaluated 1, 6, 24, and 72 h following a single 2 mg/ kg iv administration of the G41, G41-N-HC, or G41-C-HC construct. The time points were chosen on the basis of the previously described murine biodistribution study and 3126


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Figure 7. Immunohistochemistry (IHC) detection of G41-C-HC in CD-1 mouse (A) liver, (B) kidney, and (C) spleen following a single 2 mg/kg iv administration 1 h after administration. Data are from representative tissue sections from a single mouse for each compound. The scale bars represent 50 μm.

Figure 8. Mean pharmacokinetic profiles of G41 and G41-C-HC following a single 2 mg/kg iv administration to (A) CD-1 mice pretreated with clodronate once daily for 2 days and once daily thereafter until the end of the study and (B) FcγRIIb knockout mice (B6 mouse background strain). Data are the means for three animals per time point for all molecules.

HC.16 We chose to extend this interrogation to mice to build a better understanding of the connectivity of our previously reported findings across species and apply these approaches to the other BsAb configuration, G41-C-HC, and parental G41 mAb studied herein. Immunohistochemistry showed pretreatment (i.e., prior to administration of G41 or G41-C-HC) of mice along with continued once daily administration (i.e., after administration of G41 or G41-C-HC) with the chlodronate liposomes completely depleted liver macrophages and circulating monocytes in the blood (macrophage precursors); in contrast, pretreatment with control (empty) liposomes in conjunction with once daily administration for the duration of the study

Characterization of the Pharmacokinetics of G41-CHC and G41-N-HC following Macrophage Depletion in Normal Mice. Macrophages can function as a high-capacity nonspecific clearance mechanism(s) and are known to engulf and catabolize circulating species nonspecifically.23 The IHC findings in liver, kidney, and spleen indicated some association of G41-C-HC with macrophages. Given the qualitative nature of the IHC findings, we chose to interrogate the role of macrophages in the clearance of our molecules in a more deliberately quantitative manner via depletion of these cells in vivo. Previously, we showed liposomes containing chlodronate can be used to deplete macrophages in cynomolgus monkeys to study the role of macrophages in the clearance of G41-C3127


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Table 5. Mean Pharmacokinetic Parameters of the G41 mAb and G41-C-HC Molecules following a Single 2 mg/kg iv Administration in Mice Pretreated with Clodronate Once Daily for 2 Days and Once Daily Thereafter until the End of the Studya molecule

dose (mg/kg)

Cmax (μg/mL)


Vss (mL/kg)


G41 G41-C-HC

2 (n = 3 per time point) 2 (n = 3 per time point)

26.2 25.9

8376 2168

151.4 113.4

0.2 0.9

a

Cmax, maximal observed plasma concentration; AUC0−∞, area under the plasma concentration curve from zero to infinity; Vss, volume of distribution at steady state; CL, clearance; n, number of animals per time point (nonserial sampling was used in the mouse PK study).

Table 6. Mean Pharmacokinetic Parameters of the G41 mAb and G41-C-HC Molecules in FcγRIIb Knockout Mice (B6 mouse background strain) following a Single 2 mg/kg iv Administrationa molecule

dose (mg/kg)

Cmax (μg/mL)


Vss (mL/kg)


G41 G41-C-HC

2 (n = 3 per time point) 2 (n = 3 per time point)

25 28.6

7105 3075

92.3 98.1

0.3 0.7

a Cmax, maximal observed plasma concentration; AUC0−∞, area under the plasma concentration curve from zero to infinity; Vss, volume of distribution at steady state; CL, clearance; n, number of animals per time point (nonserial sampling was used in the mouse PK study).

behaved BsAbs could be rescued.16 In this study, we focused on one of the antibodies from this previous study (G1) to determine if modulation of the antibody isotype and/or D2 ECD fusion position could reduce and/or remove these biophyscical deteriments and in turn improve the pharmacokineitc behavior of the BsAb. Changing the antibody isotype (i.e., IgG4 vs an IgG1) in the presence of a fixed C-terminal heavy chain orientation D2 ECD fusion did little to alter the poor biophysical character of the molecules (Table 1), and as a result, PK studies were not performed as we expected these to succumb to a rapid clearance disposition. However, placement of the D2 ECD on the various termini of the G41 antibody (LC and HC) did have meaningful and positive impacts on its biophyscial behavior, with a clear preference for a particular orientation. Specifically, fusions of the D2 ECD to the Nterminus of the HC of G41 led to a more preferred biophysical character that was mediated by a dramatic and global structural stabilization and reduced hydrophobicity. It should be noted that simple alteration of the D2 ECD fusion position (Nterminal vs C-terminal) on G41 was insufficient to explain the observed improvements in PK and biophyscial character, as fusions made to the N-terminus of the LC of G41 were met with only modest improvements in HIC retention and DSC thermal stability, relative to the G41-C-HC variant. These modest improvements were also complemented by HDX-MS data that indicated little difference in D2 ECD structure and/ or dynamics when G41-N-LC and G41-C-HC variants were compared. These findings provide an interesting perspective on the effects of positional modification for BsAbs, as the data cannot be simply explained by a change in BsAb fusion position, as as both N-terminal fusions led to dramatically different outcomes with a defined preference for N-terminal HC fusion. The poor biophysical character exhibited by the D2 ECD in these fusions (as well as in other fusions described in our last report16) was slightly unexpected, as data presented in Shen et al. described the D2 ECD as a relatively well behaved fusion partner.19 It is worth noting that while we maintained a consistent linker length and chemical composition described in Shen et al. for attaching the D2 ECD to G1 mAb in all of these positional variants, mutations were introduced to improve the chemical stability of the D2 ECD. Furthermore, two additional mutations were made to remove two N-linked glycosylation sites to reduce drug disposition risks associated with poorly

had no effect on either of these parameters in mice (data not shown). The pharmacokinetics of G41 and G41-C-HC showed no difference following a single 2 mg/kg iv administration to mice treated with clodronate-containing liposomes relative to those treated with empty liposomes (Figure 8A and Table 5). Furthermore, the pharmacokinetic differences between the G41 mAb and G41-C-HC were still maintained under the macrophage depletion conditions (0.2 and 0.9 mL h−1 kg−1, respectively). While ADA titers were not measured in this study, the shape of the kinetic profiles does not suggest the formation of any ADA. These findings indicate macrophages are not a predominant mechanism involved in the rapid clearance of the G41-C-HC BsAb. Characterization of the Pharmacokinetics of G41-CHC in FcγRIIb Knockout Mice. Previous reports have suggested that BsAbs may be cleared through binding to FcγRIIb, which is expressed on LSECs.15,16 Given that our findings suggest LSECs are involved in the clearance of our molecules, we chose to study the pharmacokinetics of G41 mAb and G41-C-HC in FcγRIIb knockout mice to characterize the role of this specific mechanism in our clearance observations. G41-N-HC was not included as the kinetics of this molecule were comparable to those of G41 (see results above). The kinetics of G41-C-HC were assessed relative to those of the G41 mAb following a single 2 mg/kg iv administration to FcγRIIb knockout mice. The findings show G41-C-HC has a clearance that is more rapid than that of the parental G41 mAb in FcγRIIb knockout mice as observed in normal mice kinetics reported above. The clearance values of G41 and G41-C-HC were 0.3 and 0.7 mL h−1 kg−1, respectively, in the FcγRIIb knockout mice (Figure 8B and Table 6). The shape of the kinetic profiles did not suggest the formation of any antidrug antibodies (ADA); however, ADA titers were not performed. The findings indicate FcγRIIb is not a predominant mechanism involved in the rapid clearance of the G41-C-HC BsAb.

■

DISCUSSION In a previous assessment, we evaluated an array of different IgGs fused to the D2 ECD of VEGFR1 and their repective biophysical properties and pharamacokinetics.16 However, no attempts were made to eliminate these undesired biophysical properties or to test whether the disposition of the poorly 3128


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Figure 9. Comparisons of spatial aggregation prone sites in wild-type D2 ECD vs D2Mutant ECD. SAP calculations were performed in MOE 2018 (Chemical Computing Group) following the algorithms described in ref 25 with an R of 10 Å. (A) Raw values obtained for SAP on wild-type D2 ECD (orange bars) and mutated D2 ECD (blue bars) on a per residue basis. Positive values indicate residues that exhibit increased hydrophobic character, while negative values denote positions that are more hydrophilic. Mapping of SAP values onto (B) D2 mut ECD and (C) D2 ECD wildtype structures. Bound PLGF is represented by ribbons.

sialylated carbohydrate in CHO-expressed materials and clearence via liver ASPGR.24 While these modifications were found to be compatible with retained D2 ECD biological and biochemial activity (data not shown), a comparison of the DSC data for the G41-C-HC molecule and those reported for the unaltered D2 ECD fused to C-terminus of the heavy chain in IGF1R in Shen et al. reveals a profound decrease in D2 ECD thermal stability (Tm values 49 and 64 °C, respectively). Clearly, these modifications in their totality impart a poor thermodynamic character to the fused D2 ECD, ultimately resulting in the observed increased HIC retention and poor structural and/or dynamic stability. To better understand the mechanisms driving increased HIC retention for these molecules, we peformed an in silico assessment of surface hydrophobicity using a well-established spatial aggregation propensity (SAP) method.25 For this analysis, we obtained the public domain structure of the D2 ECD bound to PLGF (Protein Data Bank entry 1RV6),26 removed the PLGF ligand, and performed SAP calculations using MOE on both the unaltered D2 ECD domain and the same D2 ECD modeled with our set of mutations (Figure 9A−C). This analysis showed a clustered region of residues within the D2 ECD (164−175) exhibiting an aggregation prone surface (i.e., positive SAP scores) in the wild-type D2 ECD (Figure 9A) that was unassociated with PLGF binding. Interestingly, of the several mutations used in our D2 ECD, only one was found to make any meaningful increase in positive SAP values (Figure 4A). Specifically, alteration of asparagine to aspartic acid at position 164 to remove one of the two glycans led to to an extension of the hydrophobic surface in this region (Figure 8B,C) when compared with that of the wild-type D2 ECD. The exacerbation of this hydrophobic patch coupled with the loss of the masking and protective glycan at position 164 may have led to thermodynamic instability in the D2 ECD in our BsAbs. Indeed, such effects of addition of engineered glycans to stabilize protein structure have been well documented.27,28

Interestingly, through positional modulation of the D2 ECD on G41, these deficits could be overcome without additional engineering to reduce the unmasked patch of hydrophobicity and instabilities arising from it. The HDX-MS data collected revealed some potential insights into how this may have occurred. We did not see any major differences in the deuterium uptake across the peptides present in the G41 mAb heavy and light chains when the D2 ECD was positionally modulated, despite the fact that D2 ECD HDX-MS stability could be altered. These data suggest that D2−mAb interactions are not important for mediating the stability improvements; rather, they appear to be governed by the D2 ECD domain itself through putative self-protective D2−D2 intramolecular interactions. The preference for D2 at the Nterminus of the HC of the G41 mAb may reflect an optimal orientation and geometry of D2 to faciliate these interactions with a minimal thermodynamic penalty in stability. While this speculation is intriguing to consider, additional complementary structural and dynamic studies, including nuclear magnetic resonance, X-ray crystallography, and/or HDX, are required to further resolve the structural basis for these observations and establish a mechanistic model for these empirical observations and will provide additional data about how to approach protein engineering strategies to further mitigate future findings wtihin this class of molecules. The disparity in the PK findings across the various hIgG4 BsAb configurations facilitated the second aim of the study involving the interrogation of the physiological mechanism(s) for in vivo disposition of the BsAbs. Because both the D2 domain and mAb used for these studies interact with soluble ligands that have low or negligible peripheral concentrations in normal animals, this eliminated specific TMD factors as potential mechanisms for the observed differential disposition/ exposure across the BsAb formats. In a previous report of studies limited to liver tissue, we linked the aberrant clearance of various BsAb C-HC fusion formats with an ECD or scFv to a liver sinusoidal endothelial cell (LSEC) mechanism.16 Herein, a more complete examination of whole body- and 3129


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unintended interaction mechanism, as the PK of the molecule shows the same pattern in FcγRIIb knockout mice for G41-CHC compared to G41 that was observed in wild-type mice (∼2-fold more rapid clearance). Moreover, the rapid clearance of G41-C-HC does not appear to be related to recognition by scavenger receptors (SRs). SRs strongly expressed on the surface of LSECs can effectively facilitate endocytosis and subsequent degradation of macromolecules. Previously, we speculated that aberrant BsAb clearance could potentially be linked to SRs but did not assess this parameter in our earlier studies.16 The similarly low level of binding of G41-C-HC and parental G41 mAb to nondifferentiated LSECs in vitro indicates SRs are likely not involved in the differential PK observations. Additionally, despite the increased HIC retention of G41-CHC and G41-N-LC, no enhanced cell surface binding was observed in vitro to HEK cells or various matrices compared to G41-N-HC or the G41 parental mAb. Together, in vitro and in vivo data suggest the differential clearance of the ECD BsAbs is not related to direct interactions via specific membrane receptor or nonspecific interactions with cellular membranes and/or their components. Despite the number of interrogations reported herein, the precise nature of the mechanism(s) involved in the differential ECD BsAbs remains a mystery. Given the preponderance of data suggesting the lack of involvement of cell surface interactions in the PK findings, it seems the biophysical liabilities within the BsAbs (i.e., increased HIC association and decreased Tm) may lead to intracellular trafficking and/or recycling deficiencies and subsequent intracellular degradation. Many laboratories, including ours, have shown altered or aberrant FcRn interactions can change mAb PK, resulting in a decreased level of partitioning of molecules to tissues that are known to have relatively higher levels of FcRn expression, including skin and muscle.33,34 Combined, the whole body distribution and in vitro data equivocally support an altered FcRn-based interaction mechanism contributing to the differential disposition findings between the G41-N-HC and G41-CHC BsAb constructs. Our studies do show G41-C-HC has lower level of accumulation in these tissues, which may be ascribed by some as in part being due to altered FcRn interactions when considered in isolation. While it is possible that the fusion configuration could affect the salvage of the molecule at the cellular level, it does not seem likely given the in vitro FcRn column interaction data are inconsistent with this finding (Table 1). It is possible that the lower concentration of G41-C-HC in the skin and muscle relative to those of G41-NHC and the parental mAb simply reflects the fact that less G41C-HC is available to partition to these tissues due to the intense accumulation and/or sequestration of G41-C-HC in the liver. Thus, while it remains difficult to conclusively attribute the differential disposition observations for the BsAbs to skewed FcRn interactions, there remains some thread connecting the structural perturbations noted in the C-HC and N-LC BsAb configurations to some type of hydrophobic interaction(s) that facilitates shunting these constructs to the lysosomes for degradation. Additional cellular trafficking and/ or electron microscopy studies may provide insight into the potential mechanism(s) affecting the BsAbs reported herein. The increased speed of therapeutic antibody and various antibody structure (i.e., BsAbs, Fc fusion, and ADCs)-based development has spurred a growing need for the establishment of generalized sets of biophysical characteristics with predictably favorable disposition and developability properties

tissue-specific distribution data implicates the clearance of G41C-HC (clearance ∼2-fold more rapid than that of the parental mAb) through an increased generalized endothelial cell binding/association mechanism detected in three major organs in mice, including the liver, kidneys, and spleen, relative to the parental mAb. Although endothelial cell association was more broadly observed in other tissues, the higher tissue:blood ratio of liver relative to those of the other sites still implicates it as the major clearance route for the rapidly cleared ECD BsAbs. In contrast, G41-N-HC, which showed PK comparable to that of the parental mAb, lacked the extent of increased tissue association observed for G41-C-LC. G41-N-HC did show the same distribution to tissues as G41-C-HC, but like the parental mAb, G41-C-HC did not preferentially associate with endothelia in the liver, kidneys, or spleen. The lack of substantial tissue deposition for the G41-N-HC BsAb, which has a clearance similar to that of the parental G41 mAb, indicates that there are some foundational structural differences between the molecules stimulated by the position of the ECD fusion (as hypothesized above) because both BsAb constructs contain the same G41 mAb and ECD components. While the biophysical characterization data support the contention that structural or conformational differences between the various BsAb configurations are connected to their differential PK, the data raise the question of whether the in vivo observations are a result of specific unintended and/or nonspecific cell surface interactions. Fcγ receptors expressed on the surface of blood and liver cells have been reported by others to facilitate rapid removal of mAb from circulation.29−31 The Fcγ receptor mechanism on blood cells has been shown to be particularly relevant to the IgG1 isotype molecules as compared to other isotypes, such as IgG2 or IgG4, given the interactions with the Fc region of IgG1 molecules and the receptors are part of an innate immune regulation function in higher species.30,31 Because the BsAbs studied in vivo herein are all IgG4-based structures and showed no inherent binding to a panel of Fcγ receptors expressed on blood cell surfaces in vitro (data not shown), we anticipated a low probability of clearance via interactions with Fcγ receptors. However, a few reports have shown that unidentified blood cell-mediated clearance could not be attributed to Fcγ receptor interactions even for IgG4s.2,14 The similarly low blood:plasma partitioning ratio data for the G41-N-HC, G41-C-HC, and parental mAb demonstrated direct binding with blood cells via specific interactions with Fcγ receptors was indeed not a viable clearance mechanism, consistent with the behavior of minor and/or marginal contributions of the effector function activity influencing the PK of IgG4 molecules. In addition, the similar blood:plasma ratio data for the molecules also do not support nonspecific or unintended interactions with blood cells as a potential clearance mechanism. These findings do support the fact that the endothelial cell association in tissues is not related to residual blood in the organs. This is a positive finding given the residual blood matrix can lead to potential artifacts related to incomplete tissue perfusion techniques. Similarly, our in vitro and in vivo data do not suggest a FcγRIIb (strongly expressed on the surface of LSECs)-based interaction mechanism as observed in a recent report by Kasturirangan et al.32 In this report, Kasturirangan and co-workers suggested that certain BsAb configurations could be mistaken as “antigenloaded” immune complex-like structures imparting some “specific” unintended nonspecific binding to FcγRIIb.32 For G41-C-HC, FcγRIIb does not appear to be a causative 3130


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for clinical development. Given the number of degrees of freedom present in these ≳150000 Da macromolecules (compared to small molecule drugs of ∼10−50 Da), it is interesting to speculate if indeed there can be a contiguous set of rules that applies to this class of biotherapeutics. This would likely require additional interrogation beyond the limited number of constructs presented herein. Indeed, for approximately the past 15 years (since ∼2003), investigators in the field of antibody drug development have been reporting the connectivity between the biophysical properties of antibodies and in vivo PK performance with mixed success in retrospective analyses of molecules with “poor PK”. In even perhaps the most comprehensive retrospective study in the field of 137 antibodies (including 48 approved for therapeutic use) in a dozen biophysical property assays, Jain and co-workers reported there appeared to be no single unfavorable assay that definitively predicted the failure to advance molecules to clinical trials and supported a more holistic approach of multiple biophysical properties.35 Interestingly, when we benchmarked the biophysical property assessments of the BsAbs herein to those reported by Jain et al., our BsAbs were stratified in a mixed manner but none were considered as unfavorable.35 This may suggest that while the same biophysical characterization tools used to assess mAbs can be applied to BsAbs, parametrization of “favorable” and “unfavorable” biophysical property bookends for BsAb disposition may be different from that of mAbs. Our findings indicate that as more of these structures advance into clinical studies, case-by-case biophysical property assessments and the connectivity of these to the in vivo PK and/or disposition of the molecules will be important for their continued success as therapeutic modalities.

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AUTHOR INFORMATION

Corresponding Authors

*Biotechnology Discovery Research, Lilly Technology Center North, 1223 W. Morris Street, Indianapolis, IN 46221. E-mail: [email protected]. Telephone: (317) 755-6003. *Eli Lilly and Company, Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN 46225. E-mail: datta_ [email protected]. Telephone: (317) 651-3577. ORCID

Johnny E. Croy: 0000-0002-5913-9408 Notes

The authors declare the following competing financial interest(s): This research was funded by Eli Lilly and Company. The funder provided support in the form of salaries for all authors. There are no patents, products in development, or marketed products to declare.

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ACKNOWLEDGMENTS The authors thank the following individuals for their thoughtful discussions: Linda Schirtzinger, Beth Strifler, Ming Ye, Stacy Torgerson, Bernice Ellis, and Victor Wroblewski.

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ABBREVIATIONS BsAbs, bi- or multifunction antibody; mAb, monoclonal antibody; LC−MS, liquid chromatography−mass spectrometry; HDX, hydrogen−deuterium exchange; HIC, hydrophobic interaction chromatography; ECD, extracellular domain; PK, pharmacokinetics; PD, pharmacodynamics; ELISA, enzymelinked immunosorbent assay; LSEC, liver sinusoidal endothelial cell.

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REFERENCES

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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.biochem.9b00074. Composite sequence map coverage for the peptide fragments obtained from the D2 ECD only following pepsin digestion of each D2 ECD fusion with the G41 mAb (Figure S1), composite sequence map coverage for the peptide fragments obtained from the heavy chain only following pepsin digestion of the parental G41 mAb and the three D2 ECD/G41 fusions that were tested (Figure S2), composite map sequence coverage for the peptide fragments obtained from the light chain only following pepsin digestion of the parental G41 mAb and the three D2 ECD/G41 fusions that were tested (Figure S3), heat maps showing the pairwise comparison of the relative fractional uptake differences in the heavy chain (top) and light chain (bottom) regions of parental G41 mAb with each of the three D2 ECD variants that were evaluated (Figure S4), and representative SPECT/CT images of G41 mAb, G41-C-HC, and G41-N-HC in male CD-1 mice following a single iv administration 24 h after administration of ∼2 mg/kg (∼0.015 mCi/animal) to the subgroup C animals (Figure S5) (PDF) 3131


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Modulation of the Biophysical Properties of Bifunctional Antibodies as

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