Cellular assays to quantify target independent clearance of therapeutic molecules

Abstract

The present disclosure relates to in vitro cell-based methods that can be used to predict in vivo pharmacokinetic characteristics of candidate therapeutic proteins. In particular, the cell-based assays described herein can be used to predict in vivo non-target dependent clearance and subcutaneous bioavailability of candidate therapeutic proteins.

Description

[0001] This document is being filed in accordance with 35 U.S.C. 119(e) The disclosures of U.S. Provisional Application No. 63 / 523,868, filed June 28, 2023, and U.S. Provisional Application No. 63 / 547,184, filed November 3, 2023, are hereby incorporated by reference into this document, and their disclosures are hereby incorporated by reference herein in their entireties. TECHNICAL FIELD

[0002] The present disclosure relates to cell-based methods for predicting in vivo target-independent clearance of biomolecules. INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

[0003] INCORPORATION BY REFERENCE The sequence listing submitted electronically as an XML file named “10587-WO01-SEC_SequenceListing.xml” created on June 27, 2024, 8 kilobytes, is hereby incorporated by reference in its entirety. BACKGROUND

[0004] Pharmacokinetics (PK) of monoclonal antibodies (mAbs) and multispecific antibodies are determined by target-dependent clearance pathways and target-independent clearance (CL) pathways. Target-independent CL associated with mAbs (CL ind ) is mathematically described by first-order linear kinetics (i.e., (dA / dt) / [drug] = CL ind ). Mechanistically, CL indThe rate of endocytosis is caused by the interaction of at least two competing processes, in which nonspecific endocytosis is balanced by intracellular rescue / recirculation via neonatal Fc receptors (FcRn), which return the drug to systemic circulation (Ovacik, M. and Lin, K. (2018) Tutorial on Monoclonal Antibody Pharmacokinetics and Its Considerations in Early Development. ClinTransl Sci 11, 540-552; Ryman, JT and Meibohm, B. (2017) Pharmacokinetics of Monoclonal Antibodies. CPTPharmacometrics Syst Pharmacol 6, 576-588; Huisinga et al., Target-Driven Pharmacokinetics of Biotherapeutics). In *Pharmaceutical Sciences Encyclopedia*, pp. 1-15; and Meno-Tetang, GML, *Target-Driven Pharmacokinetics of Biotherapeutics*, pp. 1-12. Nonspecific endocytosis of therapeutic proteins can be driven by fluid-phase uptake and nonspecific adsorption internalization. Fluid-phase uptake is a mechanism by which solutes are internalized along with the extracellular fluid to a degree proportional to solute concentration (Steinman et al. (1983), *Endocytosis and therecycling of plasma membrane*, *J Cell Biol*, 96, 1-27; and Besterman, JM and Low, RB (1983), *Endocytosis: a review of mechanisms and plasma membrane dynamics*).Biochem J [Biological Chemistry Journal] 210, 1-13). When proteins interact with the cell membrane non-specifically (e.g., charge-based attraction), non-specific adsorptive pinocytosis occurs, which results in the internalization of the protein. The fluid phase is constitutive and can be cell type specific, where non-specific adsorption is protein dependent and can be influenced by factors including local charge (Lloyd and Williams (1984) Non-specific adsorptive pinocytosis. Biochem. Soc. Trans. [Biochemical Society Transactions] 12(3): 527-28). FcRn is the alpha chain of a non-covalent heterodimeric complex with beta2-microglobulin (β2m), which is primarily located within the endosomal membrane under basal conditions (Praetor, A. and Hunziker, W. (2002) beta(2)-Microglobulin is important for cell surface expression and pH-dependent IgG binding of human FcRn. J Cell Sci [Journal of Cell Science] 115, 2389-2397; Simister, N. E. and Mostov, K. E. (1989) An Fc receptor structurally related to MHC class I antigens. Nature [Nature] 337, 184-187; Antohe et al. (2001) Expression of functionally active FcRn and the differentiated bidirectional transport of IgG in human placental endothelial cells. Hum Immunol [Human Immunology] 62, 93-105; Roberts et al. (1990) Isolation and characterization of the Fc receptor from the fetal yolk sac of the rat.J Cell Biol 111, 1867-1876; Dickinson et al. (1999) Bidirectional FcRn-dependent IgG transport in a polarized human intestinal epithelial cell line. J Clin Invest 104, 903-911; D'Hooghe et al. (2017) Cell surface dynamics and cellular distribution of endogenous FcRn. PLoS One 12, e0182695). FcRn exhibits increased affinity for its endogenous ligands albumin and immunoglobulin G (IgG) at acidic pH (Chaudhury et al. (2003) The major histocompatibility complex-related Fc receptor for IgG (FcRn) binds albumin and prolongs its lifespan. J Exp Med 197, 315-322; Raghavan et al. (1995) Analysis of the pH dependence of the neonatal Fc receptor / immunoglobulin G interaction using antibody and receptor variants. Biochemistry 34, 14649-14657; Andersen et al. (2012) Structure-based mutagenesis reveals the albumin-binding site of the neonatal Fc receptor.Nat Commun [Nature Communications] 3, 610; Ober et al. (2001) Differences in promiscuity for antibody-FcRn interactions across species: implications for therapeutic antibodies. Int Immunol [International Immunology] 13, 1551-1559; Martin et al. (2001) Crystal structure at 2.8 A of an FcRn / heterodimeric Fc complex: mechanism of pH-dependent binding. Mol Cell [Molecular Cell] 7, 867-877), thus selectively retaining these proteins by intracellular trafficking away from lysosomal degradation and toward the plasma membrane to facilitate active recycling (Dickinson et al. (1999) Bidirectional FcRn-dependent IgG transport in a polarized human intestinal epithelial cell line. J Clin Invest [Journal of Clinical Investigation] 104, 903-911; Ober et al. (2004) Visualizing the site and dynamics of IgG salvage by the MHC class I-related receptor, FcRn. J Immunol [Journal of Immunology] 172, 2021-2029; Ober et al. (2004) Exocytosis of IgG as mediated by the receptor, FcRn: an analysis at the single-molecule level.Proc Natl Acad Sci U S A 101, 11076-11081; Kim et al. (2004) Net absorption of IgG via FcRn-mediated transcytosis across rat alveolar epithelial cell monolayers. Am J Physiol Lung Cell Mol Physiol 287, L616-622; Schmidt et al. (2017) Direct demonstration of a neonatal Fc receptor (FcRn)-driven endosomal sorting pathway for cellular recycling of albumin. J Biol Chem 292, 13312-13322; Bern et al. (2020) An engineered human albumin enhances half-life and transmucosal delivery when fused to protein-based biologics. Sci Transl Med 12; Strohl, W. R. (2015) Fusion Proteins for Half-Life Extension of Biologics as a Strategy to Make Biobetters.BioDrugs 29, 215-239). This salvage mechanism prolongs the serum half-life of FcRn ligands relative to similarly sized proteins that lack FcRn binding capacity (Chaudhury et al. (2003) The major histocompatibility complex-related Fc receptor for IgG (FcRn) binds albumin and prolongs its lifespan. J Exp Med 197, 315-322). In contrast, target-mediated CL processes are nonlinear and can dominate overall mAb CL depending on dose (Ovacik, M., and Lin, K. (2018) Tutorial on Monoclonal Antibody Pharmacokinetics and Its Considerations in Early Development. Clin Transl Sci 11, 540-552; Ryman, J. T. and Meibohm, B. (2017) Pharmacokinetics of Monoclonal Antibodies. CPT Pharmacometrics Syst Pharmacol 6, 576-588; Huisinga et al., Target-Driven Pharmacokinetics of Biotherapeutics. In Pharmaceutical Sciences Encyclopedia. pp 1-15; and Meno-Tetang, G. M. L. Target-Driven Pharmacokinetics of Biotherapeutics. In Pharmaceutical Sciences Encyclopedia. pp 1-12; and Peletier, L. A. and Gabrielsson, J.(2012) Dynamics of target-mediated drug disposition: characteristic profiles and parameter identification. J Pharmacokinet Pharmacodyn 39, 429-451). Target-mediated drug disposition (TMDD) is driven by high-affinity mAb-target binding on the cell surface, with subsequent internalization of the mAb-target complex leading to intracellular catabolism (Ovacik, M. and Lin, K. (2018) Tutorial on Monoclonal Antibody Pharmacokinetics and Its Considerations in Early Development. Clin Transl Sci 11, 540-552; Ryman, J. T. and Meibohm, B. (2017) Pharmacokinetics of Monoclonal Antibodies. CPT Pharmacometrics Syst Pharmacol 6, 576-588; Huisinga et al., Target-Driven Pharmacokinetics of Biotherapeutics. In Pharmaceutical Sciences Encyclopedia. pp 1-15; and Meno-Tetang, G. M. L. Target-Driven Pharmacokinetics of Biotherapeutics. In Pharmaceutical Sciences Encyclopedia. pp 1-12; and Peletier, L. A. and Gabrielsson, J.(2012) Dynamics of target-mediated drug disposition: characteristic profiles and parameter identification. J Pharmacokinet Pharmacodyn 39, 429-451).

[0005] CL of mAbs (even those with overlapping target specificities) ind Variability can occur for several reasons. For example, changes in FcRn binding due to differences in the physicochemical properties of the variable region or other non-specific endocytic mechanisms can broadly affect the PK profile of a candidate therapeutic (Kelly et al. (2016) Target-independent variable region mediated effects on antibody clearance can be FcRn independent. MAbs 8, 1269-1275; Piche-Nicholas et al. (2018) Changes in complementarity-determining regions significantly alter IgG binding to the neonatal Fc receptor (FcRn) and pharmacokinetics. MAbs 10, 81-94; and Grevys et al. (2022) Antibody variable sequences have a pronounced effect on cellular transport and plasma half-life. iScience 25, 103746). Thus, a key goal within the field of macromolecular PK is the ability to predict in vivo PK parameters of early preclinical mAbs through in vitro experimental measurements to define in vitro to in vivo correlation. Recent work on mAb CL indMethods for performing the classification have encompassed a number of biophysical interaction assays for both specific and non-specific binding. For example, surrogate assays for non-specific pinocytosis including heparin chromatography and baculovirus particle binding have been commonly employed (Kraft et al. (2020) Heparin chromatography as an in vitro predictor for antibody clearance rate through pinocytosis. MAbs 12, 1683432; Hotzel et al. (2012) A strategy for risk mitigation of antibodies with fast clearance. MAbs 4, 753-760; Jain et al. (2017) Biophysical properties of the clinical-stage antibody landscape. Proc Natl Acad Sci U S A 114, 944-949; and Datta-Mannan et al. (2016) Aberrant bispecific antibody pharmacokinetics linked to liver sinusoidal endothelium clearance mechanism in cynomolgus monkeys. MAbs 8, 969-982).Evaluation of Fc-FcRn interactions is routinely accomplished using surface plasmon resonance and FcRn column chromatography (Schlothauer et al. (2013) Analytical FcRn affinity chromatography for functional characterization of monoclonal antibodies. MAbs 5, 576-586; Vaughn, D. E. and Bjorkman, P. J. (1997) High-affinity binding of the neonatal Fc receptor to its IgG ligand requires receptor immobilization. Biochemistry 36, 9374-9380; and Borrok et al. (2015) pH-dependent binding engineering reveals an FcRn affinity threshold that governs IgG recycling. J Biol Chem 290, 4282-4290). The inference drawn from the application of these techniques is that both non-specific uptake and FcRn affinity influence mAb CL. ind and should be considered for successful CL ind Prediction (Grevys et al. (2022) Antibody variable sequences have a pronounced effect on cellular transport and plasma half-life. iScience 25, 103746).

[0006] Cells endogenously or ectopically expressing human FcRn (hFcRn) and human β2m (hβ2m) can also be used to directly interrogate FcRn function, and multiple reports highlight the utility of cell-based platforms to study FcRn transport kinetics and biology (Praetor, A. and Hunziker, W. (2002) beta(2)-Microglobulin is important for cell surface expression and pH-dependent IgG binding of human FcRn. J Cell Sci 115, 2389-2397; Antohe et al. (2001) Expression of functionally active FcRn and the differentiated bidirectional transport of IgG in human placental endothelial cells. Hum Immunol 62, 93-105; Roberts et al. (1990) Isolation and characterization of the Fc receptor from the fetal yolk sac of the rat. J Cell Biol 111, 1867-1876; Dickinson et al. (1999) Bidirectional FcRn-dependent IgG transport in a polarized human intestinal epithelial cell line. J Clin Invest 104, 903-911; Ober et al. (2004) Visualizing the site and dynamics of IgG salvage by the MHC class I-related receptor, FcRn.J Immunol 172, 2021-2029; Ober et al. (2004) Exocytosis of IgG as mediated by the receptor, FcRn: an analysis at the single-molecule level. Proc Natl Acad Sci U S A 101, 11076-11081; Kim et al. (2004) Net absorption of IgG via FcRn-mediated transcytosis across rat alveolar epithelial cell monolayers. Am J Physiol Lung Cell Mol Physiol 287, L616-622; Schmidt et al. (2017) Direct demonstration of a neonatal Fc receptor (FcRn)-driven endosomal sorting pathway for cellular recycling of albumin. J Biol Chem 292, 13312-13322; Bern et al. (2020) An engineered human albumin enhances half-life and transmucosal delivery when fused to protein-based biologics. Sci Transl Med 12; Grevys et al. (2022) Antibody variable sequences have a pronounced effect on cellular transport and plasma half-life.iScience [InterScience] 25, 103746; Tzaban et al. (2009) The recycling and transcytotic pathways for IgG transport by FcRn are distinct and display an inherent polarity. J Cell Biol [Cell Biology Journal] 185, 673-684; Tesar et al. (2006) Ligand valency affects transcytosis, recycling and intracellular trafficking mediated by the neonatal Fc receptor. Traffic [Transport] 7, 1127-1142; Claypool et al. (2004) Bidirectional transepithelial IgG transport by a strongly polarized basolateral membrane Fcgamma-receptor. Mol Biol Cell [Molecular Biology Cell] 15, 1746-1759; Goebl et al. (2008) Neonatal Fc receptor mediates internalization of Fc in transfected human endothelial cells. Mol Biol Cell [Molecular Biology Cell] 19, 5490-5505; Sockolosky et al. (2012) Engineering neonatal Fc receptor-mediated recycling and transcytosis in recombinant proteins by short terminal peptide extensions.Proc Natl Acad Sci U S A 109, 16095-16100; Ying et al. (2015) Engineered antibody domains with significantly increased transcytosis and half-life in macaques mediated by FcRn. MAbs 7, 922-930; Chung et al. (2019) An in vitro FcRn-dependent transcytosis assay as a screening tool for predictive assessment of nonspecific clearance of antibody therapeutics in humans. MAbs 11, 942-955; Liu et al. (2021) A cell-based FcRn-dependent recycling assay for predictive pharmacokinetic assessment of therapeutic antibodies. Bioanalysis 13, 1135-1144; Jaramillo et al. (2017) Toward in vitro-to-in vivo translation of monoclonal antibody pharmacokinetics: Application of a neonatal Fc receptor-mediated transcytosis assay to understand the interplaying clearance mechanisms.MAbs [Monoclonal Antibodies] 9, 781-791; Grevys et al. (2018) A human endothelial cell-based recycling assay for screening of FcRn targeted molecules. Nat Commun [Nature Communications] 9, 621; and Gjolberg et al. (2022) Biophysical differences in IgG1 Fc-based therapeutics relate to their cellular handling, interaction with FcRn and plasma half-life. Commun Biol [Communications Biology] 5, 832. Detailed in vitro experimental strategies can be used to evaluate the CL of mAbs. indand now several published studies have demonstrated that cell-based approaches offer the advantage of additional biological context not collected from standard biophysical methods (Grevys et al. (2022) Antibody variable sequences have a pronounced effect on cellular transport and plasma half-life. iScience 25, 103746; Chung et al. (2019) An in vitro FcRn-dependent transcytosis assay as a screening tool for predictive assessment of nonspecific clearance of antibody therapeutics in humans. MAbs 11, 942-955; Liu et al. (2021) A cell-based FcRn-dependent recycling assay for predictive pharmacokinetic assessment of therapeutic antibodies. Bioanalysis 13, 1083-1155; Jaramillo et al. (2017) Toward in vitro-to-in vivo translation of monoclonal antibody pharmacokinetics: Application of a neonatal Fc receptor-mediated transcytosis assay to understand the interplaying clearance mechanisms.MAbs 9, 781-791; Grevys et al. (2018) A human endothelial cell-based recycling assay for screening of FcRn targeted molecules. Nat Commun 9, 621; Gjolberg et al. (2022) Biophysical differences in IgGl Fc-based therapeutics relate to their cellular handling, interaction with FcRn and plasma half-life. Commun Biol 5, 832; Chung et al. (2022) Methods for Functional Characterization of FcRn Interactions with Therapeutic Antibodies and Fc-Fusion Proteins. Methods Mol Biol 2313, 295-303; Chung et al. (2018) Development of a label-free FcRn-mediated transcytosis assay for in vitro characterization of FcRn interactions with therapeutic antibodies and Fc-fusion proteins. J Immunol Methods 462, 101-105). This includes direct measurements of cellular internalization, endosomal FcRn binding, and subsequent intracellular trafficking, which expands our understanding of the CL. indmechanistic understanding of the process (Chung et al. (2019) An in vitro FcRn- dependent transcytosis assay as a screening tool for predictive assessment of nonspecific clearance of antibody therapeutics in humans. MAbs 11, 942-955; Gjolberg et al. (2022) Biophysical differences in IgG1 Fc-based therapeutics relate to their cellular handling, interaction with FcRn and plasma half-life. Commun Biol 5, 832; Gurbaxani et al. (2013) Are endosomal trafficking parameters better targets for improving mAb pharmacokinetics than FcRn binding affinity? Mol Immunol 56, 660-674; and Brinkhaus et al. (2022) The Fab region of IgG impairs the internalization pathway of FcRn upon Fc engagement. Nat Commun 13, 6073).

[0007] However, the human CL of mAbs indPredictive power remains deficient. The range of biophysical assays that identify the physicochemical determinants of mAb PK alone typically fail to capture biological frameworks and are often based on subjective determination of critical points. Combining readouts from multiple technologies can provide more robust interpretation and the ability to extend findings to in vivo behavior (Kraft et al. (2020) Heparin chromatography as an in vitro predictor for antibody clearance rate through pinocytosis. MAbs 12, 1683432). Cell-based methods can simultaneously collate multiple biological processes and physicochemical aspects into endpoints, which can facilitate a deeper understanding of mAb PK behavior. But limitations remain. For example, one study demonstrated a strong correlation between FcRn-mediated transcytosis and mAb CL, whereby higher transcytosis indicated elevated in vivo CL. The underlying mechanistic principles of this trend have not been explored, and the authors highlighted that the assay could not successfully extrapolate the CL of mAbs with enhanced engineered FcRn affinity (Chung et al. (2019) An in vitro FcRn-dependent transcytosis assay as a screening tool for predictive assessment of nonspecific clearance of antibody therapeutics in humans. MAbs 11, 942-955). Separate studies demonstrated that the extent of FcRn recycling depends largely on the amount of mAb internalized by specific or non-specific processes, and that the charge profile of the Fv region is a key contributing factor to the latter (Grevys et al. (2022) Antibody variable sequences have a pronounced effect on cellular transport and plasma half-life. iScience 25, 103746). However, the scope of this work was limited due to the number of compounds examined.

[0008] The present disclosure is directed to overcoming these problems and other deficiencies in the art. SUMMARY

[0009] A first aspect of the disclosure relates to a method of predicting in vivo non-target- mediated clearance (CL ind ) of a biomolecule. The method involves providing a cell preparation, wherein the cells of the preparation do not express a target of the biomolecule. The method further involves incubating the cell preparation with a medium containing the biomolecule under conditions that mimic in vivo physiological conditions, and determining the amount of the biomolecule taken up by the cells of the preparation after incubation. The method further involves predicting in vivo non-target-mediated clearance of the biomolecule based on said determining.

[0010] A further aspect of the disclosure relates to a method of predicting in vivo non-target- mediated clearance of an FcRn interacting molecule. The method involves providing a first cell preparation, wherein the cells of the first preparation do not express human neonatal Fc receptor (hFcRn), and providing a second cell preparation, wherein the cells of the second preparation express a heterodimer of hFcRn and human beta-2-microglobulin (hβ2m). The cells of the first cell preparation and the second cell preparation do not express a target of the FcRn interacting molecule. The method further involves subjecting the first cell preparation and the second cell preparation to a first incubation period and a second incubation period, wherein the first incubation period comprises incubating the cell preparations with a medium containing the FcRn interacting molecule under acidic and / or non-acidic conditions. The second incubation period comprises incubating said cell preparations with a medium lacking the FcRn interacting molecule under non-acidic conditions after the first incubation. The method further involves determining the amount of the FcRn interacting molecule taken up by the cells of the first preparation and the second preparation after said subjecting to the first incubation period and / or the second incubation period, and measuring the amount of the FcRn interacting molecule in the medium after the second incubation period. The method further involves quantifying non-specific endocytosis and FcRn recycling of the FcRn interacting molecule based on the determining and measuring steps. In vivo non-target-mediated clearance of the FcRn interacting molecule is predicted based on the quantifying step.

[0011] A further aspect of the disclosure relates to a method of predicting subcutaneous bioavailability of a biomolecule based on the extent of non-specific endocytosis of the biomolecule predicted according to the methods disclosed herein.

[0012] In vivo clearance mechanisms of biomolecules encompass both target-mediated processes and target-independent processes. Independent of target-mediated influences, two distinct determinants of biomolecule clearance are pH-dependent recycling via FcRn and non-specific endocytosis, each of which varies in rate and extent between molecules. Methods to quantify these kinetics have shown strong utility for establishing in vitro-in vivo correlations, but are mechanistically limited or limited in the number of analytes.

[0013] Several biophysical techniques, including baculovirus particle binding and heparin chromatography, are typically used to investigate the potential of biomolecules, like mAbs, to undergo non-specific endocytosis. However, because these methods do not provide insight into the specific rate of cell turnover, a reproducible and robust mammalian cell-based method to quantify biomolecule non-specific endocytosis is described herein. Data generated with this cell-based method can be used to identify overall pharmacokinetic (PK) liabilities during preclinical drug development and inform mechanistic PK models for human translation.

[0014] A functional cell-based FcRn recycling assay using mammalian cells is also disclosed herein. A series of pH-dependent internalization studies using model antibodies have confirmed the functionality of the human FcRn complex in this assay. In addition, non-specific endocytosis was observed to be the primary endocytic pathway for test antibodies when the receptor was not present. Application of these cell assays to evaluate FcRn and non-specific interactions in a collection of clinical antibodies, multispecific antibodies, and Fc fusion proteins with a range of PK behaviors. The results demonstrate that non-specific endocytosis rates, pH-dependent non-specific interactions, and engagement with FcRn all contribute to the overall recycling efficiency of these therapeutic molecules.

[0015] By determining the successful identification of all antibodies in humans with CL ind greater than 5 mL / kg / day, the predictive power of the assays described herein is highlighted. These results demonstrate that the combination of cell assays can identify individual mechanisms underlying the overall in vivo recycling efficiency and CL ind of biomolecules. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figures 1A-1C Serum concentration-time profiles of ASA and anti-IL-4Ra mAb in wild-type mice are depicted. Data were obtained from separate studies with different intravenous bolus doses of ASA (3 mg / kg) ( Figure 1A ) and anti-IL-4Ra mAb (1 mg / kg) ( Figure 1B ). Figure 1C The predicted values in Figure 1A and Figure 1B were obtained via computational fitting using a two-compartment pharmacokinetic model with linear elimination (CL) from the central compartment and distribution (CL D ) between the vascular and non-vascular compartments. Parameter estimates are reported in Table 1. Cl and VI, serum concentration and central compartment volume; C2 and V2, serum concentration and non-vascular compartment volume; ASA, anti-streptavidin antibody.

[0017] Figures 2A-2DThe internalization kinetics of anti-IL-4Rα mAb and ASA in CHO-K1 cells, obtained by flow cytometry and high-content confocal microscopy, are shown. Following uptake studies, cells were washed, trypsin-digested, stained with live / dead dyes, fixed, permeabilized, stained with Alexa Fluor 647-conjugated anti-human Fc mouse IgG, washed, and analyzed by flow cytometry. Representative gating strategies are shown in… Figure 2A In the mean time interval, the median fluorescence intensity of a single live-cell event was obtained for each sample. CHO-K1 cells were incubated together with 100 µg / mL anti-IL-4Rα mAb or ASA at 37°C or 4°C for a gradually increasing time interval. For anti-IL-4Rα mAb, but only at 37°C, a two-stage time dependence was observed. Figure 2B ) and linear concentration dependence ( Figure 2C Internalization. In contrast, ASA endocytosis was negligible at 37°C. Minimal cell surface binding of anti-IL-4Rα mAb and ASA was detected at 4°C relative to the untreated control. Figure 2D Using anti-IL-4Rα mAb ( Figure 2D (See right image) or ASA ( Figure 2D (Middle image) A set of confocal micrographs of CHO-K1 cells after incubation and similar post-experimental treatments. Untreated cells are shown in... Figure 2D In the left inset, cells were stained with CellMask Blue and Hoechst to visualize whole cells (cytoplasm and nucleus) and stained with a fluorescently labeled anti-Fc antibody (red). These images demonstrate the practicality of confocal microscopy as an alternative to flow cytometry for high-throughput quantification of therapeutic protein internalization kinetics. ASA, anti-streptavidin antibody; CHO-K1, Chinese hamster ovary cells.

[0018] Figures 3A-3D An overview of the development of quantitative endocytosis assays using flow cytometry is provided. Figure 3A Histograms of five superimposed bead populations when stained with 5 or 10 µg / mL of anti-human Fc mouse IgG1 conjugated with Alexa Fluor 647 are shown. A plot of median fluorescence intensity versus antibody binding capacity (ABC) is also shown, indicating that both staining conditions produced r 2 A linear curve with a value of 0.999. Figure 3Bis a graph showing the determination of reproducibility evaluated by uptake studies in CHO-K1 cells incubated with 100 pg / mL of anti-IL-4Ra mAb or ASA for 60 min at 37°C over several different experimental days. Mean anti-IL-4Ra mAb ABC: 40,839 (SD 6614, CV 16.2%); mean ASA ABC: 921 (SD 315, CV 34.2%). N = 20 for each sample group. Figure 3C is a representative histogram of single ASA and untreated samples, where gating is performed with 99% of the untreated population incorporated (corresponding to a "negative" signal). ASA endocytosis under the experimental setup is so low that about 92% of the ASA signal resides within the "negative" gate. Figure 3D is an illustrative overview of the method of quantifying endocytosis. ASA, anti-streptavidin antibody; IgG1, immunoglobulin G1; CHO-K1, Chinese hamster ovary cell; SD, standard deviation; CV, coefficient of variation.

[0019] Figures 4A to 4C shows the surface charge distribution on the anti-IL-4Ra mAb ( Figure 4A ) and ASA ( Figure 4B ), and demonstrates that removing exposed positive charges can reduce non-specific endocytosis. Figure 4A and Figure 4B include Fv ribbon diagram models depicting the heavy chain CDRs in orange (CDR1, CDR2) and red (CDR3). The light chain CDRs are shown in purple. Hydrophobic patches are shown in green. Negatively charged patches and positively charged patches are red and blue, respectively. Sites targeted for mutation are indicated. The anti-IL-4Ra mAb ( Figure 4A ) has more positively charged patches than the ASA ( Figure 4B ), with 2 positively charged patches in the heavy chain CDRs. Various point mutations were made on the anti-IL-4Ra mAb CDRs and Fv to reduce surface positive charge within the identified charge patches. Figure 4C The graph of shows that non-specific endocytosis was significantly reduced for all anti-IL-4Ra mAb mutants. CDR, complementarity determining region; WT, wild-type anti-IL-4Ra mAb; LC, light chain; CDR H1, first complementarity determining region of the heavy chain; CDR H3, third complementarity determining region of the heavy chain.

[0020] Figure 5This is a set of serum concentration-time curves for a group of preclinical antibodies (mAb B1-B5) that bind to the same target after a single intravenous bolus dose in wild-type mice. The data were fitted to mean serum concentrations using a two-compartment computational model graphically depicted in Figure 1.

[0021] Figures 6A-6D This demonstrates that nonspecific endocytosis is conserved across different species and cell types. In CHO-K1 (grey hamster (Cricetulus griseus) ovarian epithelial-like cells); Figure 6A ) or Vero cells (renal epithelial cells of African green monkeys (Cercopithecus aethiops); Figure 6B In this study, mice with different CL values ​​in wild-type mice were used. Figure 5 Uptake studies were conducted on the mAb group. A range of uptake levels independent of pI were observed. These results also demonstrate the superior sensitivity of endocytosis assays performed at 37°C compared to cell surface binding alone (i.e., the 4°C group). Nonspecific endocytosis in CHO-K1 or Vero cells is strongly correlated with CL, such as... Figure 6D As shown, this attribute serves as the CL for these mAbs. ind Important factors. Furthermore, the results for Vero and CHO-K1 cells strongly correlated with each other ( Figure 6C This supports the mechanism by which nonspecific endocytosis is used for mAb internalization. All data are plotted as mean ± SD, n = 3–4 / group. CL, clearance; CHO-K1, Chinese hamster ovary cells; CL ind Target-independent clearance.

[0022] Figures 7A-7C The co-expression of hFcRn-GFP and hβ2m in stably transfected MDCK II cells is shown. Maximum projection of the confocal image after anti-FcRn immunofluorescence staining confirms hFcRn-GFP (green, yellow) expression in sorted hFcRn-GFP / hβ2m MDCK II cells. Figure 7A Cells were stained with rabbit anti-FcRn antibody using a goat anti-rabbit secondary antibody conjugated to Alexa Fluor 594 (yellow). Parental MDCK II cells showed only positive nuclear staining (Hoechst, blue). Figure 7A The top (i) and bottom (ii) images have and do not have the depicted GFP channels, respectively. Scale bar, 20 µm. Figure 7BThe co-staining of parental and hFcRn-GFP / hβ2m MDCKII cells with targeted anti-hFcRn and anti-hβ2m antibodies or corresponding allotype controls demonstrates robust transgene co-expression in transfected, sorted hFcRn-GFP / hβ2m MDCKII cells. Cells were stained with (total) or without (cell surface) fixation / infiltration. Under maintained selection pressure, visual inspection (e.g.) was performed. Figure 7A The GFP-hFcRn-GFP / hβ2mMDCK II cell population was observed consistently by flow cytometry. As determined by flow cytometry, it comprised less than 5% of the total population. Therefore, all subsequent fluorescence detection analyses, including those shown in the current figure, were performed on GFP+ hFcRn-GFP / hβ2mMDCK II cells. Figure 7B The median fluorescence intensity of hFcRn and hβ2m signals from hFcRn-GFP / hβ2m MDCK II cells indicates that most hFcRn-GFP and hβ2m are intracellular, such as... Figure 7C The figure is shown. N = 3, mean ± SD. GFP, green fluorescent protein; BF, bright field; hFcRn, human neonatal Fc receptor; hβ2m, human β2-microglobulin; MDCK II Madin-Darby canine kidney subclone II cells.

[0023] Figures 8A-8C ASA was showcased WT Internalization kinetics of -DL650 and HSA-DL650 in hFcRn-GFP / hβ2m and parental MDCK II cells. Using 10 or 100 µg / mL ASA at pH 5.8, 7.4, or 8.0. WT -DL650 was used to study time-dependent endocytosis in order to confirm the function of hFcRn-GFP / hβ2m ( Figure 8A The results show 100 µg / mL ASA at pH 5.8. WT - Representative histogram of DL650 time-dependent uptake results ( Figure 8A (Top small image). hFcRn-GFP / hβ2m MDCK II cells showed that ASA levels decreased with decreasing pH. WT -DL650 increases endocytosis in a time- and concentration-dependent manner. Figure 8A (See bottom left image). No ASA was observed in parental MDCK II cells between the tested pH conditions. WT -Difference in DL650 endocytosis rate ( Figure 8A(See bottom right figure). With or without 50 mg / mL of unlabeled analyte as a competitive inhibitor, ASA was used in FcRn-GFP / β2m MDCK II cells at pH 5.8. WT -DL650 ( Figure 8B ) or HSA-DL650 ( Figure 8C The concentration-dependent endocytosis mediated by FcRn was studied over a period of 20 min. Specific internalization data, expressed as rates per minute, were fitted to the Michaelis-Menten equation to provide the half-maximal uptake rate (K0) within the corresponding experimental system. m ) and maximum speed (V max ASA WT Estimates of -DL650 and HSA-DL650 concentrations (see...) Figure 8B (ii) and Figure 8C (ii)). Figure 8B and Figure 8C The illustration at the bottom (iii) is from the results of a study conducted in the presence of excess unlabeled protein. Figure 8B (i) and Figure 8C (i) shows linear concentration-dependent uptake. Data were fitted using linear regression to support the observation of nonspecific endocytosis in the absence of receptor-mediated internalization. N = 3–4 / point, mean ± SD. MeFI, median fluorescence intensity; HSA-DL650, human serum albumin conjugated with DyLight 650 fluorescence.

[0024] Figures 9A-9C It shows the relationship with ASA WT Following time-dependent incubation with DL650, hFcRn-GFP was intracellularly localized in hFcRn-GFP / hβ2m-MDCK II cells. One day prior to the uptake study, human FcRn-GFP / hβ2m-MDCK II cells were loaded with 10 kDa Texas red dextran (TR-dextran) to label lysosomes. On the day of imaging, cells were incubated with 10 or 100 µg / mL ASA at specified time points at pH 5.8 or 7.4. WT - Incubate together with DL650. Figure 9A The results showed that 100 µg / mL ASA was used at pH 5.8. WT A representative set of images of hFcRn-GFP / hβ2m-MDCK II cells treated with DL650. Hoechst was used as the nuclear staining agent. Arrows indicate GFP lacking hFcRn-GFP. -Cells. They provide a further confirmation of the correct transgene function, as GFP - ASA WT -DL650 was consistently negative. Intracellular ASA WT -DL650 mean fluorescence intensity (MFI) (Fig. 3B) Figure 9B , which is in agreement with the observations using flow cytometry. p < 0.0001 indicates comparison to untreated control MFI after two-way ANOVA with Tukey’s multiple comparison. N = 3 / group, mean ± SD. Figure 9C hFcRn-GFP, ASA WT -DL650 and lysosomal intracellular signals only resulted in positive colocalization between hFcRn-GFP and ASA WT -DL650 over the duration of the study. Relative to the 15 min time point, one-way ANOVA with Dunnett’s multiple comparison on the hFcRn vs. ASA WT groups, , , p < 0.05, <0.001, < 0.0001. N = 3 / group, mean ± SD.

[0025] Figures 10A to 10E Development of the cell-based hFcRn recycling assay as described and claimed herein is depicted. Figure 10A is a schematic depiction of the experimental workflow. Parental MDCK II cells were included to provide an assessment of non-specific mAb interactions. Because negligible intracellular trafficking can occur at 4 °C conditions, the 4 h recycling phase at this temperature provides both confirmation of active processes in the 37 °C group and a measure of the total amount of mAb internalized during the loading phase. MDCK II hFcRn recycling studies were performed with ASA WT or mutants with deleted hFcRn affinity (ASA AAA ). It was found that for ASA WT , human FcRn-mediated uptake and recycling was higher at pH 5.8 compared to pH 7.4, for ASA AAAmAb was essentially zero (no difference between parental MDCK II and hFcRn-GFP / hβ2m-MDCK II), and increased upon incubation at 37°C compared to 4°C, supporting active cell transport. Figure 10B (Left figure). Furthermore, based on minimal internalization in parental MDCK II at any pH, the nonspecific endocytosis of all tested ASA mAbs was determined to be negligible. Figure 10B (Right figure). These findings suggest that mAbs with low nonspecific uptake (i.e., low endocytosis in parental MDCK II) at pH 7.4 will exhibit very low hFcRn-mediated recycling, as the recycling fraction will be determined by the initial amount internalized. Therefore, evaluating mAbs with favorable PK behavior using only pH 7.4 loading conditions will be challenging. Incubation at pH 5.8 provides a way to load sufficient mAbs intracellularly to elucidate hFcRn recycling efficiency. The significance of the differences in hFcRn-GFP / hβ2m-MDCK II cells was measured by two-way ANOVA and Tuki's multiple comparison test. This indicates that the adjusted p < 0.0001. The bar chart represents the mean ± SD, n = 6-8 replicates / group. Figure 10C and Figure 10D This demonstrates the intravenous administration of Tg32 ( Figure 10C ) or Tg276 ( Figure 10D ASA after hFcRn transgenic mouse model WT and ASA AAA A serum concentration-time curve was plotted to evaluate the treatment. ASA AAA It exhibits fast CL, consistent with its lack of hFcRn binding. Mean ± SD, for each time point and group n = 3. When compared with ASA AAA In comparison, using data from ASA WT The hFcRn recycling score was significantly higher in the pH 5.8 loading phase, as shown in the results. Figure 10E The figure is shown below. The scoring results are... Figure 10C and Figure 10D The in vivo CL values ​​were consistent with those determined in the study. p < 0.001 Unpaired t-test, mean ± SD. Green, yellow, and red dashed lines represent 100%, 50%, and 25% of the ASAWT recirculation score, respectively.

[0026] Figures 11A-11D This shows the corresponding high CL in humans indhFcRn recycling score. Figure 11A Representative data sets for mAb 1 from hFcRn recycling studies in transfected and parental MDCK II cells are shown, encompassing all conditions used to derive the hFcRn recycling score. The amount internalized during the loading phase was obtained via the remaining 4°C fraction, as negligible intracellular transport would be expected at this temperature. The mAb 1 reference exhibited pH-dependent uptake in hFcRn-GFP / hβ2m, which is consistent with the extent of recycling at matching pH values. Furthermore, low non-specific uptake was measured in parental MDCK II cells. The hFcRn recycling score (FREMS) was obtained for 8 mAbs, and these scores were plotted against their human CL ind values as shown in Figure 11B . Low recycling scores indicate high non-specific uptake and / or inefficient hFcRn-mediated recycling, with values below 0.25 strongly indicating rapid CL ind in vivo. Scoring was performed using pH 5.8 loading conditions, as CL ind was below 5 mL / d / kg for mAbs with very low internalization amounts. MAb 1 served as a reference mAb for the full mAb group comparison. Following one-way ANOVA with Dunnett’s multiple comparison on FcRn recycling scores compared to mAb 1, , , p < 0.05, 0.01, 0.001. Green, yellow, and red dashed lines represent 100%, 50%, or 25% of the recycling score of mAb 1, respectively. Loading phase results (indicating internalization amounts) from parental MDCK II cells at 4°C demonstrated significantly higher uptake of anti-IL-4Ra mAbs at pH 7.4 compared to mAb 1 Figure 11C (p < 0.0001, one-way ANOVA with Dunnett’s multiple comparison). At pH 5.8, both anti-IL-4Ra mAbs mAb 9 exhibited significantly higher non-specific uptake compared to mAb 1 (one-way ANOVA with Dunnett’s multiple comparison). MAb 9 and mAb 1 also showed a significant increase in non-specific uptake at pH 5.8 compared to pH 7.4 (after multiple t-tests , p < 0.05, 0.0001). Non-specific pinocytosis of the mAb panel was measured by flow cytometry with anti-human Fc detection on fixed and permeabilized cells after incubation of CHO-K1 cells with 100 pg / mL mAb at pH 5.8 or 7.4 for 60 min at 37°C. Figure 11D ). When compared to ASA WT , only anti-IL-4Ra mAb (p < 0.0001) and mAb9 (p < 0.01) exhibited significantly higher non-specific uptake at pH 7.4 (ordinary one-way ANOVA with Dunnett’s multiple comparison test). In addition, the extent of anti-IL-4Ra mAb internalization was significantly higher than that of mAb9 at pH 7.4. At pH 5.8, all mAbs exhibited significantly higher non-specific pinocytosis of CHO-K1 cells when compared to the pH 7.4 condition, with mAb9 showing the largest change (multiple unpaired t-tests, p at least < 0.05). In summary, these results demonstrate that the mAbs tested at pH 5.8 have a significantly increased propensity for non-specific behavior relative to pH 7.4 due to a lack of high non-specific interactions caused by physiochemical properties of the mAbs such as local charge patches.

[0027] Figure 12A- Figure 12B An illustrative summary is provided. FIG. 12A is a graph depicting low CL ind mAbs cellular processes. Due to a lack of high non-specific interactions caused by physiochemical properties of the mAbs such as local charge patches, a small amount of mAbs enter non-targeted vascular endothelial cells via non-specific pinocytosis at pH 7.4. The endosomes containing internalized mAbs will undergo gradual acidification during transport. As shown herein, all of the tested mAbs exhibit increased non-specific interactions at acidic pH relative to pH 7.4. However, for low CL ind compounds, these non-specific behaviors can have minimal impact on mAb interaction with hFcRn, resulting in a high percentage of endosomal hFcRn binding. The bound mAbs will subsequently be transported to the plasma membrane and dissociate from hFcRn near neutral pH. The result is highly efficient hFcRn recycling, which was observed in the current work as high hFcRn recycling scores. Examples of low CL ind mAbs from this study include mAb1 and ASA WT . Figure 12B is a graph depicting how multiple cellular mechanisms can drive high mAb CL indImages. Due to unfavorable physicochemical characteristics (including local patches of strong charge at neutral pH), some mAbs (such as anti-IL-4Rα mAbs) exhibit high rates of nonspecific endocytosis. This results in a relatively large amount of mAbs being internalized into non-target cell populations via nonspecific endocytosis. Nonspecific increases may occur after endosome acidification. This pH-dependent change can be significant, as observed in the current study of mAb9. These nonspecific aspects may impair binding to hFcRn and potentially lead to other nonspecific interactions, such as interactions with endosome membrane components. The results are (1) greater total mAb in the endosome for each hFcRn receptor, (2) hFcRn binding dysfunction due to altered charge state, and / or (3) the effect of counteracting the increased nonspecific interactions of hFcRn-mAb binding. Additionally, if the affinity of mAbs for receptors is too high at pH 7.4, the dissociation of mAbs from hFcRn at the cell surface may be impaired. Since a smaller fraction of internalized mAbs undergoes hFcRn-mediated recycling, these factors can lead to a higher rate of intracellular catabolism and thus result in high CL. ind This was experimentally validated in the study described herein, where a low hFcRn recycling score indicated the occurrence of one or more of these processes. Test mAbs that did not show obvious negative behavior (e.g., mAb8) were affected by varying degrees of nonspecific behavior, where the amount of hFcRn interaction was negatively canceled out by the amount of nonspecificity relative to the reference mAb1. Images were created using BioRender.com.

[0028] Figure 13A The phylogenetic schemes for all endocytosis studies are shown, with sequential subpopulations indicated by underlined numbers (top left of each figure). In the parent (GFP) - ) or hFcRn-GFP / hβ2m (GFP + Median fluorescence intensity was measured in a single live-cell event in MDCK II cells. Histograms from two separate samples were overlaid in inset 4 to demonstrate the difference in GFP expression. Figure 13A The bottom of the chart shows two representative histograms of individual MDCK II cell types from time-dependent uptake results of 100 µg / mL ASAWT-DL650 at pH 5.8. Figure 13B In the meantime, the uptake results using pH 5.8 conditions were removed to allow visualization of the results at pH 7.4.

[0029] Figures 14A-14B The figure shows the amount of mAb recycled in the hFcRn recycling study tested in transfected and parental MDCK II cells. Figure 14A ) and remaining amount (Figure 14B ), encompassing all conditions used to derive hFcRn recycling scores. The 37°C and 4°C groups are appropriate for the respective temperatures at which the plates are measured during the recycling phase of the study. The amount internalized during the loading phase is obtained via the remaining 4°C group, as negligible intracellular transport would be expected at this temperature. The non-specific uptake coefficient (NUC) and FcRn recycling efficiency metric (FREM) scores are calculated using the amount internalized (uptake) and recycling after the pH 5.8 loading phase, respectively, and the remaining concentration.

[0030] Figures 15A-15B is the measurement of non-specific transcytosis in CHO-K1 cells at pH 7.4 ( Figure 15A ) and 5.8 ( Figure 15B ) for the expanded panel of anti-IL-4Ra mAbs with single point mutations and double point mutations. Non-specific transcytosis was significantly different for all WT and YTE mutants when compared to the control anti-IL-4Ra wild-type mAb at either pH value (ordinary one-way ANOVA with Dunnett’s multiple comparison test; #: P < 0.001, and : P < 0.0001). Each bar graph represents the mean ± SD, N = 3-4 / group.

[0031] Figures 16A-16D hFcRn-mediated concentration-dependent transcytosis studies with ASA WT -hIgG2-DL650 ( Figures 16A-16B ) or ASA WT -hIgG2-YTE-DL650 ( Figures 16C-16D ) were performed in hFcRn-GFP / hb2m MDCK II cells for 30 min at pH 5.8. Non-specific transcytosis measurements in parental MDCK II cells were subtracted from the hFcRn-specific internalization data, and the resulting values were expressed as rates per minute. These data were fit to the Michaelis-Menten equation to provide estimates of ASA WT -hIgG2-DL650 ( Figure 16B ) and ASA WT -hIgG2-YTE-DL650 ( Figure 16D ) concentrations that achieve half-maximal uptake velocity (Km) and maximum velocity (Vmax) within the respective experimental system. N = 3-4 / point, mean ± SD. MeFI, median fluorescence intensity; ASA WT -hIgG2-DL650, anti-Streptavidin human immunoglobulin G2 antibody conjugated to DyLight 650; ASA WT- hlgG2-YTE-DL650, anti-Streptavidin human immunoglobulin G2 YTE antibody conjugated with DyLight 650.

[0032] Figures 17A-17C The ability of the cell-based hFcRn recycling assay to successfully and simultaneously rank mAbs with wild-type or engineered Fc regions was demonstrated. Figure 17A hFcRn efficiency metric (FREM) scores of anti-IL-4Ra WT and YTE charge mutant mAbs are shown. Scores were normalized against anti-IL-4Ra mAb-EEES-YTE. The assay was optimized to simultaneously compare both Fc engineered YTE and wild-type Fc (i.e., hlgG2) charge mutant mAbs. The half-life extended YTE mAbs exhibited better FREM scores relative to their WT counterparts. Each bar represents the mean ± SD, and N = 6-8 replicates / group, anti-IL-4Ra mAb-EEES-YTE varied, N = 16. The YTE mutant mAbs were tested in two batches on different days, and the FREM scores from each day were normalized against the EEES-YTE mutant mAb from that day as a relative control. Ordinary one-way ANOVA with Dunnett’s multiple comparison test was performed between groups, and significance was compared against anti-IL-4Ra mAb as a control : P < 0.05, and : P < 0.0001). Figure 17B Serum concentration time profiles of tested anti-IL-4Ra mAb WT and YTE charge mutants in male homozygous immunodeficient SCID hFcRn Tg32 transgenic mice (N = 3 / group) are shown. Figure 17C CL ind vs. FREM scores are shown. It was observed that mAbs with relatively lower FREM scores exhibited higher CL ind .

[0033] Figures 18A-18F Recycling amounts, uptake amounts, and remaining amounts of anti-IL-4Ra WT mAb mutants in hFcRn-GFP / β2M-transfected ( Figures 18A-18C ) and parental MDCK II cells ( Figures 18D-18F ) in hFcRn recycling studies to derive FREM scores are shown. Loading and recycling phases of mAbs were tested at 37°C. The amounts internalized (uptake) and recycled after the loading phase at pH 5.8 and the remaining concentrations were used to calculate the non-specific uptake coefficient (NUC) and the FcRn efficiency metric (FREM) scores, respectively.

[0034] Figures 19A-19FRecycling, uptake, and residual amounts of anti-IL-4Ra YTE mAb mutants in hFcRn recycling studies in hFcRn-GFP / 2M transfected (A-C) and parental MDCK II cells (D-F) of four YTE mutants and EEES-YTE as control mAb are shown, which were used to derive FREM scores. Experimental data of Figure 20 were collected on a separate day from Figure 19. This experiment was performed together with the WT mutants from the same cell flasks mentioned in Figure 18. Loading and recycling phases of mAbs were tested at 37 °C. The amounts internalized (uptake) and recycled after the loading phase at pH 5.8 and the residual concentrations were used to calculate the non-specific uptake coefficient (NUC) and the FcRn recycling efficiency measure (FREM) scores, respectively. Figures 19A-19C Figures 19D-19F

[0035] Figures 20A-20F

[0036] Figure 21A is a plot showing non-specific endocytosis results from CHO-K1 uptake assays of a set of fully human and humanized clinical mAbs or derivatives thereof with known clinical PK data. The mAbs were grouped by CL ind below 4.5 mL / kg / d, CL ind above 4.5 mL / kg / d, or CL ind was not obtained (e.g. dose level was not sufficient to reach target saturation). This was done because the presently disclosed assay cannot provide information on mAb target-mediated kinetics. The value of 4.5 mL / kg / d was chosen because this corresponds to an end- serum half-life of approximately 10 days in an 80 kg human. The generated assay threshold is also shown on the plot. The “low risk” group contains mAbs with high CL ind and / or low F SQ due to non-specific endocytosis. It is defined as F​​​SQ Above 50% and / or CL ind The upper limit of the 95% confidence interval for the mean ABC value of mAb <4.5 mL / kg / d. The "intermediate risk" group contains individuals who may or may not exhibit high CL. ind and / or low F SQ The mAbs were set at two standard deviations above the mean ABC directly mentioned above. The "high-risk" group highlighted mAbs with significant nonspecific endocytosis, which could be detrimental to their disposal in humans. Figure 21A Having CL ind All mAbs of the estimated values ​​are binned to their respective risk categories, as described in this paper and plotted on [the map]. Figure 21B Medium. Average CL ind The gradual increase in values ​​was associated with an increase in risk categorization in the CHO-K1 assay. Additionally, it was found that individuals with low CL... ind However, mAbs that still exhibit high nonspecific CHO-K1 endocytosis (i.e., high ABC value) have low F in humans. SQ (e.g., mAb 28). Therefore, mAb in the high-risk category does not have a favorable PK in individuals.

[0037] Figures 22A-22B This highlights how nonspecific endocytosis can identify high-risk multispecific antibodies with increased target-independent clearance. Forty-eight multispecific antibodies were obtained, which exhibited broad-spectrum target-independent clearance (CL) in Tg32 human FcRn transgenic mice. ind (0.2 to 2000 mL / kg / h) Figure 22A Proteins are derived from various molecular forms. Nonspecific endocytosis of the multispecific proteome was measured by obtaining antibody binding capacity (ABC) in CHO-K1 cells. Then, using the same parameters as the human mAb group in Figure 15, the compounds were binned into low-risk, intermediate-risk, or high-risk categories ( ). Figure 22B (That is, low risk in the current instance is defined as having the same risk as...) Figure 21A (The same ABC values ​​as the human mAb group depicted in the text). This allows for multispecific identification of intermediate-risk and high-risk CL as elevated in Tg32 mice. ind The propensity to [specific protein type] increased, with 94% of proteins in the high-risk chamber and 92% of proteins in the medium-risk chamber having a concentration (CL) higher than 0.5 mL / kg / h in Tg32 mice. ind However, 63% of low-risk multispecific antibodies exhibited CL values ​​higher than 0.5 mL / kg / h in Tg32 mice. indThis highlights alternative clearance mechanisms besides specific endocytosis. This suggests a need for supporting assays of alternative mechanisms, such as the cellular human FcRn recycling assay defined in the current work.

[0038] Figures 23A-23F This study demonstrates how nonspecific endocytosis directs elevated target-independent clearance across a wide range of protein structures. A group of Fc-fusion proteins containing five distinct protein structures unrelated to human immunoglobulins was generated. Each construct contains two separate proteins fused to a human Fc domain. A single-dose study (2 mg / kg) in wild-type mice demonstrated a broad range of CL... ind (0.81 to 1260 mL / h / kg), such as Figure 23A As shown. All Fc-fusion proteins were analyzed using the CHO-K1 cell endocytosis method. At pH 7.4 ( Figure 23B ) or pH 5.8 ( Figure 23C The antibody binding capacity (ABC) was obtained under the condition that it targeted its corresponding CL in wild-type mice. ind Plotting was performed. Then, using the ABC cutoff points derived from the human mAb group depicted in Figure 21, the mean ABC values ​​from the CHO-K1 nonspecific endocytosis studies conducted at pH 7.4 were placed in low-risk, medium-risk, or high-risk bins. Figure 23D Ninety-four percent of the Fc-fusion proteins in the high-risk chamber had a CL greater than 3 mL / kg / h in wild-type mice. ind Fc-fusion proteins deemed low-risk or intermediate-risk after endocytosis assays at pH 7.4 will be placed in a new risk threshold based on their pH 5.8 ABC values. Figure 23E The following three proteins were found to exhibit a wide range of pH-dependent, non-specific hopping behaviors: Fc-fusions 7, 8, and 10. Two-thirds of these proteins exhibited very high CL values ​​in mice. ind ( Figure 23F This supports the undesirable property of high pH-dependent nonspecificity as a therapeutic protein. Detailed Implementation

[0039] This disclosure relates to in vitro cell-based methods that can be used to predict the in vivo pharmacokinetic properties of candidate therapeutic biomolecules. In particular, the cell-based assays described herein help predict or estimate the in vivo non-target-mediated clearance of candidate therapeutic molecules. The cell-based assays described herein can also be used to rank the in vivo non-target-mediated clearance of candidate molecules to support lead candidate selection and optimization, while reducing the number of molecules tested in animal models.

[0040] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the application as claimed.

[0041] Unless otherwise defined, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Generally, nomenclature used in connection with, and techniques of, cell culture, molecular biology, genetics, and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. Unless specifically indicated otherwise, the methods and techniques of the present disclosure are generally performed according to conventional methods in the art and as described in various general and more specific references that are cited throughout this specification. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992), and Harlow and Lane Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990), all of which are incorporated herein by reference.

[0042] In this disclosure, the use of singular terms includes the plural and the use of plural terms includes the singular, unless explicitly stated otherwise. As used herein, the singular forms "a," "an," and "the" include singular and plural referents unless the context clearly dictates otherwise.

[0043] In this disclosure, the use of "or" means "and / or" unless specifically stated otherwise. Moreover, the use of the term "including" as well as other forms such as "includes" and "included" is not limiting. Also, terms such as "component" encompass both components including one unit as well as components including more than one subunit, unless specifically stated otherwise.

[0044] As used herein, the terms "comprising," "comprises" and "comprised of" shall be synonymous with "including," "includes," "containing" or "contains," is inclusive or open-ended and does not exclude additional, unrecited members, compounds, products, elements, or method steps. The expression "consisting essentially of" when used in the context of compositions or methods (e.g., a "method consisting essentially of") means that additional elements, ingredients or steps can be present, but only if such additional elements, ingredients or steps do not alter / modify the essential characteristics / activities / functions of the product, composition or method. The expression "consisting of" when used in the context of compositions or methods means that the composition or method includes only the elements, steps or ingredients specifically recited in the particular embodiment or claim.

[0045] In embodiments or claims where the term "comprising" is used as an open-ended transitional phrase, such embodiments and claims also contemplate the replacement of the term "comprising" with the transitional phrases "consisting of" or "consisting essentially of."

[0046] Numerical ranges expressed in ranges from about and / or from approximately are understood to include all values and amounts encompassed there between, including the approximated or stated values and amounts.

[0047] The term "about" as used herein when referring to a measurable value such as a parameter, a quantity, a duration, and the like, is meant to encompass variations that can occur in such a value due to experimental error, measurement error, and variations in the manufacture and / or use of the compositions and / or methods disclosed herein. It is further to be understood that the value of the modifier "about" that is used in connection with a given variable will also encompass variations that fall within the experimental error, measurement error, and variations in the manufacture and / or use of the compositions and / or methods disclosed herein.

[0048] A first aspect of the present disclosure relates to a method of predicting in vivo non-target mediated clearance of a biomolecule. The method involves providing a cell preparation, wherein the cells of the preparation do not express a target of the biomolecule. The method further involves incubating the cell preparation with a medium containing the biomolecule under conditions that mimic in vivo physiological conditions, and determining the amount of the biomolecule taken up by the cells of the preparation after incubation. The method further involves predicting in vivo non-target mediated clearance of the biomolecule based on said determination.

[0049] As referred to herein, "clearance" or "CL" is defined as the volume of plasma corresponding to the drug clearance over a specified time period. Thus, the unit of measurement for drug clearance is volume / time. Clearance is equal to the rate of removal of a drug (e.g., a biomolecule such as a therapeutic protein) from plasma (mg / mL) divided by the concentration of that drug in plasma (mg / mL). For therapeutic biomolecules (such as antibodies, antigen-binding antibody fragments, antibody derivatives, multi-specific engineered antibodies and proteins, and fusion proteins), clearance is determined by both target-dependent and non-target-dependent pathways.

[0050] Target-dependent clearance due to biomolecule interaction with its target is dependent on the rate of internalization of the therapeutic biomolecule, the density of binding to the target (e.g., antigen), the binding affinity, and the turnover kinetics of binding to the target. Non-target-dependent or target-independent clearance (CL ind ) of a biomolecule is mediated by non-specific cellular uptake of the biomolecule via adsorptive endocytosis and / or fluid phase endocytosis. Once in the endosomal compartment of the cell, two competing processes (i.e., intracellular lysosomal catabolism and neonatal Fc receptor (FcRn)-mediated recycling or transcytosis) determine the clearance of the biomolecule. From a drug development perspective, candidate therapeutics that exhibit high levels of non-specific endocytosis and / or poor FcRn capture and recycling will have increased non-target-dependent clearance. Additionally, as described herein, high non-specific endocytosis has been identified as a key parameter that predicts low subcutaneous bioavailability. Thus, the cell-based assays described herein are quantitative tools that can be used to assess non-specific endocytosis and FcRn recycling efficiency to identify pharmacokinetic liabilities, including increased non-target-dependent clearance and low subcutaneous bioavailability, early in the drug development process.

[0051] In any embodiment, the methods described herein can be used to predict non-target-mediated clearance of a biomolecule in vivo. As referred to herein, a "biomolecule" includes, without limitation, any therapeutic protein, polypeptide, or nucleic acid molecule. In any embodiment, the biomolecule interacts with or binds to a particular or defined target (e.g., a cellular target). Such biomolecules of the disclosure are referred to as "binding molecules," and these encompass therapeutic proteins, polypeptides, or nucleic acid molecules that comprise a domain having binding specificity for a target molecule (i.e., a "binding target") or are coupled to the same. The binding target or target molecule can be a toxin, a drug, a protein, a nucleic acid molecule, or other biomolecule. In any embodiment, the binding target is a protein expressed by a cell, e.g., a cell surface protein or cell surface receptor. In any embodiment, the biomolecule has binding specificity for a human target molecule (e.g., a human cell surface protein or human cell surface receptor).

[0052] Exemplary biomolecules include antigen-binding proteins, such as antibodies (e.g., human polyclonal or monoclonal antibodies), multispecific antibodies (e.g., human bispecific antibody proteins), antigen-binding fragments of antibodies (e.g., Fv, Fab', and (Fab')2), and antibody derivatives (e.g., single-chain antibodies, minibodies, diabodies). Biomolecules also include therapeutic proteins (e.g., multispecific engineered proteins, recombinant proteins, and fusion proteins) and nucleic acid molecules (e.g., siRNA molecules, antisense oligonucleotides, aptamer molecules, and mRNA molecules). Biomolecules of the present disclosure can also interact with or bind to the neonatal Fc receptor (FcRn). In one embodiment, a biomolecule naturally interacts with or binds to FcRn. In one embodiment, a biomolecule is engineered to bind to FcRn. In one embodiment, a biomolecule comprises a fragment crystallizable (Fc) region that interacts with FcRn. In one embodiment, a biomolecule comprises an Fc region engineered to increase FcRn binding affinity. In one embodiment, a biomolecule comprises an Fc portion engineered to decrease FcRn binding affinity or engineered to remove or delete FcRn binding. In one embodiment, a biomolecule comprises an albumin domain (e.g., the C-terminal DIII region of human serum albumin) and variants thereof that are capable of binding to FcRn (see, e.g., Andersen et al., J. Biol. Chem. 289(19): 13492-13502 (2014), which is hereby incorporated by reference in its entirety). In one embodiment, a biomolecule comprises an albumin-binding protein domain, such as the albumin-binding domains B2A3 and B1A2B2A3 from Streptococcal protein G, which indirectly bind to FcRn via binding to albumin (see, e.g., Andersen et al., J. Biol. Chem. 286(7):5234-5241 (2011), which is hereby incorporated by reference in its entirety). In one embodiment, a biomolecule comprises an affibody molecule, which is a small affinity protein that directly binds to FcRn in a pH-dependent manner (see Seijsing et al., Applied Biol. Sci. 111(48): 17110-17115 (2014), which is hereby incorporated by reference in its entirety).

[0053] According to this and all aspects of the disclosure, the method of predicting in vivo non-target mediated clearance of a biomolecule described herein involves providing a cell preparation that does not express a target of the biomolecule (e.g., a binding target). Suitable cell preparations include any mammalian cell preparation, such as a human cell preparation, a primate cell preparation, a canine cell preparation, a feline cell preparation, a porcine cell preparation, a rodent cell preparation, or any other suitable mammalian cell preparation. Suitable cells can be derived from any mammalian tissue, including but not limited to skin (dermal and epidermal tissue), epithelium, vascular tissue (endothelial cells), heart, lung, kidney, liver, intestine, stomach, pancreas, colon, ovary, lymphoid tissue, bone, cartilage, tumor, and can be a primary cell preparation or an immortalized cell preparation, such as a cell line. Exemplary cell line preparations suitable for use in the methods described herein include, without limitation, Chinese hamster ovary (CHO) cells, Madin-Darby canine kidney (MDCK) cells, Vero cells, HEK-293 cells, HeLa cells, 3T6 cells, A549 cells, BHK-21 cells, MCF-7 cells, Saos-2 cells, PC3 cells, HepG2 cells, and human umbilical vein endothelial cells (HUVECs).

[0054] Cell preparations useful in this aspect of the disclosure do not express a target molecule of the biomolecule, i.e., the cells of the preparation do not express a cell surface protein or receptor that binds or interacts with the biomolecule. The cells of the preparation can not naturally express any protein that is bound by or interacts with the target of the biomolecule. Alternatively, the cells of the preparation can express a variant of the target that is not bound by the biomolecule, such as a canine cell preparation that expresses a target homolog that is not bound by a human biomolecule having binding specificity for the human target. In another embodiment, the cells of the preparation are modified so as not to express the target of the biomolecule. For example, siRNA, shRNA, or other inhibitory nucleic acid molecules can be used to silence expression of the target. Alternatively, gene editing techniques can be used to genetically silence expression of the target.

[0055] According to this aspect of the disclosure, if the biomolecule is one that interacts or binds with an Fc receptor, then the cells of the preparation also do not express an Fc receptor or protein. For example, when the biomolecule is a human immunoglobulin comprising an Fc region, the cells of the preparation do not express a cell surface receptor or protein that binds to the Fc region of the human immunoglobulin. In any embodiment, the cells of the preparation do not naturally express a human Fc receptor (such as FcaR, FcaRI, FcyRI, FcyRII, FcyRIII, FcRn, FcsRI, or FcsRII), or any species equivalent that binds to the Fc portion of a human immunoglobulin. In any embodiment, the cells of the preparation do not express the human FcRn protein, and thus do not express the active human FcRn / hb2m complex. The cells of the preparation can either naturally lack expression of Fc receptors or be engineered to not express such receptors (e.g., genetically modified to knockdown or silence Fc receptor expression). In any embodiment, suitable cell preparations include those engineered to not express hFcRn or hb2m, with the absence of either protein rendering the cells of the preparation incapable of expressing the active FcRn / hb2m complex.

[0056] Cell preparations suitable for use in the methods and assays described herein are cultured in suitable cell culture media under standard tissue culture conditions suitable for cell survival, growth, and proliferation. Appropriate growth and culture conditions for various mammalian cell types are well known in the art. The cells of the preparation can be seeded onto and / or within a substrate such that they are uniformly distributed at a relatively high surface and / or volumetric density. The cell suspension can comprise about about 1 x 10 4 to about 5 x 10 7 cells / ml of media, or about about 2 x 10 6 cells / ml to about 2 x 10 7 cells / ml, or about about 5 x 10 6 cells / ml. Optimal concentrations and absolute numbers of cells will vary with cell type, growth rate of the cells, substrate material, and various other parameters. The suspension can be formed in any physiologically acceptable media, preferably one that does not impair the cells or impair the ability of the cells to adhere to the substrate. Suitable media include standard cell growth media, such as DMEM with 10% FBS.

[0057] In methods of predicting in vivo non-target mediated clearance, the biomolecule of interest is added to a cell culture medium, and the cells are cultured in the presence of the biomolecule under otherwise normal culture conditions (e.g., 37 °C, 5% C02, and 20% 02) for a suitable duration of time, e.g., 30 minutes, 60 minutes, 90 minutes, 120 minutes, 180 minutes, 3 hours, 4 hours, 5 hours, or more, to allow non-target mediated cellular uptake to occur. In any embodiment, the cells are cultured in the presence of the culture medium containing the biomolecule for about 60 to about 90 minutes.

[0058] The cells are cultured in the presence of the culture medium containing the biomolecule under conditions that mimic in vivo physiological conditions, including physiological pH. In any embodiment, the physiological pH is a neutral pH, e.g., the pH of blood. A suitable neutral pH for the culture medium containing the biomolecule is a pH of about 7.0 to about 8.0. In any embodiment, the culture medium containing the biomolecule has a pH of about 7.0, a pH of about 7.1, a pH of about 7.2, a pH of about 7.3, a pH of about 7.4, a pH of about 7.5, a pH of about 7.6, a pH of about 7.7, a pH of about 7.8, a pH of about 7.9, or a pH of about 8.0.

[0059] In another embodiment, the physiological pH is an acidic pH that mimics the typical pH of endosomal acidification. In any embodiment, the culture medium containing the biomolecule has a pH of about 5.6 to about 6.9. For example, the biomolecule has a pH of about 5.6, a pH of about 5.7, a pH of about 5.8, a pH of about 5.9, a pH of about 6.0, a pH of about 6.1, a pH of about 6.2, a pH of about 6.3, a pH of about 6.4, a pH of about 6.5, a pH of about 6.6, a pH of about 6.7, a pH of about 6.8, or a pH of about 6.9.

[0060] After culturing the cell preparation in the presence of the culture medium containing the biomolecule under conditions that mimic in vivo physiological conditions, the amount of biomolecule taken up by the cells of the preparation is determined. In any embodiment, a first cell preparation is cultured in the presence of a culture medium containing a biomolecule, wherein the culture medium has a neutral pH, and a second cell preparation that is otherwise identical to the first cell preparation is cultured in the presence of a culture medium containing the biomolecule, wherein the culture medium has an acidic pH. Comparison of the amount of biomolecule taken up by the cells exposed to the biomolecule under neutral pH conditions to the amount of biomolecule taken up by the cells exposed to the biomolecule under acidic pH conditions provides information on the pH dependence of non-target mediated uptake of the biomolecule.

[0061] In one embodiment, determining the amount of biomolecules taken up by cells of the formulation involves permeabilizing the cells of the formulation and detecting the amount of biomolecules taken up by the cells. To detect the biomolecules taken up by the cells, the biomolecules can be labeled directly or indirectly with a detectable portion. For example, in one embodiment, the biomolecules are directly conjugated to a detectable portion (such as a fluorescent portion) before being cultured with cells, and the amount of the detectable portion in the permeabilized cells is detected to determine the amount of biomolecules taken up by the cells. Alternatively, the biomolecules can be directly conjugated or coupled to an enzyme label or a small molecule label (e.g., biotin), and the amount of biomolecules taken up by the cells is determined by incubating the permeabilized cells with a reactant suitable for detecting the enzyme label or small molecule label. In another embodiment, biomolecules are indirectly labeled with a detectable moiety by incubating permeated cells with an antibody (e.g., IgG or IgM molecule), antibody fragment (e.g., Fab F(ab'), Fc fragment, single-domain antibody, γ chain of IgG, Fc5µ of IgM, or Mu chain of IgM), or antibody derivative (e.g., scFv) that has binding specificity to the biomolecule, wherein the antibody is conjugated to the detectable moiety (i.e., the labeled secondary antibody). Suitable detectable moieties include, but are not limited to, fluorescent molecules, small molecules, enzyme labels, and radioisotopes, as readily known in the art. After this incubation, the amount of the detectable moieties is detected to determine the amount of biomolecule taken up by the cells.

[0062] Suitable methods for detecting detectable portions include those known in the art, readily available, and described in the examples herein. For example, in any embodiment, the detectable portion is a fluorescent portion, such as Alexa Fluor dye, fluorescein, Oregon Green dye, rhodamine dye, Texas Red dye, and / or derivatives thereof, and is detected via a fluorometer in an immunoassay (e.g., ELISA), fluorescence microscopy, or flow cytometry. Other suitable detectable portions include radioisotopes (e.g., [missing information]) that can be detected using immunoassays, flow cytometry, spectroscopy, microscopy, confocal microscopy, liquid chromatography-tandem mass spectrometry, or scintillation imaging methods well known in the art. 14 C 125 I, 32 P, 35 S), luminescent proteins, enzyme proteins (such as horseradish peroxidase, alkaline phosphatase, glucose oxidase, etc.), and small molecules (such as biotin).

[0063] Once the amount of detectable moiety is detected, it is compared to one or more reference values to quantify the amount of biomolecule taken up by the cell, thereby providing a value for non-specific endocytosis. For example, as described herein, a standard curve or calibration curve representing the binding capacity of the biomolecule can be generated. The amount of biomolecule detected in the cell is compared to or plotted against the standard curve to quantify the amount of biomolecule taken up by the cell. As described herein, a standard curve can be generated using commercially available microspheres coated with an anti-Fc antibody (e.g., anti-IgG antibody) that binds to the biomolecule of interest. The level of non-specific endocytosis of the biomolecule as determined from the calibration curve is compared to the level of non-specific endocytosis of one or more internal reference antibodies to predict non-target mediated clearance of the biomolecule. In preferred embodiments, the amount of biomolecule taken up by non-specific endocytosis as determined from the calibration curve is compared to the corresponding value of at least two reference antibodies, one reference antibody having low non-target clearance (e.g., < 4.5 mL / kg / d) and one having high non-target clearance (e.g., > 4.5 mL / kg / d).

[0064] Alternatively, the amount of biomolecule detected in the cell is compared to one or more internal reference antibodies included in the assay and detected with the biomolecule, where the non-specific endocytosis and in vivo non-target dependent clearance of the reference antibodies have been previously quantified and are known. The level of non-specific endocytosis of the biomolecule being tested is compared to the level of non-specific endocytosis of one or more internal reference antibodies to predict non-target mediated clearance of the biomolecule of interest. In preferred embodiments, the amount of biomolecule taken up by non-specific endocytosis is compared to at least two reference antibodies, one reference antibody having low non-target clearance (e.g., < 4.5 mL / kg / d in humans) and one having high non-target clearance (e.g., > 4.5 mL / kg / d in humans).

[0065] The level of non-specific endosomal uptake can also be used to provide information and predict human subcutaneous bioavailability (F SQ ) where a high level of non-specific endocytosis indicates low F SQ (e.g., < 50%) and a low level of non-specific endocytosis indicates high F SQ (e.g., > 50%). In any embodiment, thresholds for "high," "medium," and "low" levels of non-specific endocytosis can be generated using a plurality of reference antibodies as described herein (see, e.g., Example 9 and FIG. 21). For example, a low non-specific endocytosis threshold is identified by defining the average quantified value of non-specific endocytosis of one or more reference mAbs that have a CL in humans of less than 4.5 mL / kg / d.ind and / or F > 50% SQ The upper limit of the 95% confidence interval of this mean value can be used as a reference range for low non-specific uptake. The low CL ind Moderate non-specific uptake can be established via the non-specific endocytosis value of the mean value of endocytosis. Test biomolecules with non-specific uptake in the moderate range are expected to have a higher chance of having increased CL ind and / or F < 50% SQ Any case above the moderate uptake threshold is considered to have high non-specific endocytosis, and a corresponding greatly enhanced chance of high CL ind and / or F < 50% SQ .

[0066] Another aspect of the present disclosure relates to a method of predicting in vivo non-target mediated clearance of a neonatal Fc receptor (FcRn) interacting molecule. This method assesses both non-specific endocytosis and FcRn recycling to enhance in vivo prediction of non-target mediated clearance of a biomolecule. This method involves providing a first cell preparation, wherein the cells of the first preparation do not express human FcRn, and providing a second cell preparation, wherein the cells of the second preparation express a heterodimer of human neonatal Fc receptor (hFcRn) and human β2m (hβ2m). The cells of the first cell preparation and the second cell preparation do not express the target of the FcRn interacting molecule. The method further involves subjecting the first cell preparation and the second cell preparation to a first incubation period and a second incubation period, wherein the first incubation period comprises incubating the cell preparations with media containing the FcRn interacting molecule under acidic conditions, under non-acidic conditions, or under both acidic and non-acidic conditions. The second incubation period comprises incubating the cell preparations with media lacking the FcRn interacting molecule under non-acidic conditions after the first incubation. The method further involves determining the amount of FcRn interacting molecule taken up by the cells of the first preparation and the second preparation after the first incubation period and / or the second incubation period, and measuring the amount of FcRn interacting molecule in the media after the second incubation period. The method further involves quantifying non-specific endocytosis and FcRn recycling of the FcRn interacting molecule based on the determining and measuring steps, i.e., quantifying non-specific endocytosis based on the amount of FcRn interacting molecule determined to be taken up by the cells, and quantifying FcRn recycling of the FcRn interacting molecule based on the amount of FcRn interacting molecule measured in the media after the second incubation. The in vivo non-target mediated clearance of the FcRn interacting molecule is predicted based on the quantifying step.

[0067] This method of the disclosure provides a tool for evaluating in vitro the potential propensity of a candidate therapeutic molecule in terms of non-target mediated clearance. This method helps to predict or estimate the in vivo non-target mediated clearance of a particular candidate therapeutic molecule or can be used to rank the in vivo non-target mediated clearance of candidate molecules to support lead candidate selection and optimization while reducing the number of molecules tested in animal models.

[0068] According to this aspect of the disclosure, as referred to herein, an "FcRn interacting molecule" is any therapeutic protein, polypeptide, or nucleic acid molecule comprising a domain or sequence portion that binds to the neonatal Fc receptor (FcRn). FcRn is an MHC class I-like molecule that is a heterodimer of an alpha chain non-covalently associated with a beta2-microglobulin. FcRn plays a critical role in maintaining the in vivo levels of IgG and albumin by preventing their lysosomal degradation within cells. Thus, for the purposes of the disclosure, exemplary FcRn interacting molecules include, without limitation, molecules comprising: an Fc domain (e.g., an Fc domain of an IgG), an albumin domain (e.g., full-length albumin or its C-terminal DIII fragment), an albumin domain variant with enhanced FcRn binding affinity (see, e.g., Andersen et al., J. Biol. Chem. 289(19): 13492-13502 (2014), which is hereby incorporated by reference in its entirety), an albumin binding protein domain from Streptococcal protein G or other gamma-positive bacteria (see, e.g., Andersen et al., J. Biol. Chem. 286(7):5234-5241 (2011), which is hereby incorporated by reference in its entirety), or a small affinity protein (affibody) that directly binds FcRn in a pH-dependent manner (see Seijsing et al., Applied Biol. Sci. 111(48): 17110-17115 (2014), which is hereby incorporated by reference in its entirety).

[0069] In one embodiment, the FcRn-interacting molecule comprises a domain or sequence portion that binds to human FcRn. Suitable FcRn-interacting molecules for purposes of the present disclosure also encompass molecules comprising an FcRn-binding domain that is engineered (i.e., comprises one or more amino acid substitutions, insertions, or deletions) to have an enhanced FcRn binding affinity relative to the binding affinity of a non-engineered form of the domain. FcRn-interacting molecules also encompass molecules comprising an FcRn-binding domain that is engineered to have a reduced FcRn binding affinity relative to the FcRn binding affinity of a non-engineered form of the domain. FcRn-interacting molecules further encompass molecules comprising an FcRn-binding domain that is engineered to lack or remove FcRn binding. In any embodiment, the FcRn-interacting molecule can also interact with or bind to a non-FcRn cellular target (e.g., a cell surface receptor or a cell surface ligand).

[0070] Exemplary FcRn-interacting molecules include antigen-binding proteins, such as antibodies (e.g., human immunoglobulins, including IgGl, IgG2, IgG3, and IgG4 immunoglobulins), multispecific antibodies (e.g., human bispecific or trispecific antibody proteins comprising an IgG Fc domain or other FcRn-binding domain), antigen-binding fragments of antibodies comprising an IgG Fc domain or other FcRn-binding domain, antigen-binding fragments of multispecific antibodies comprising an IgG Fc domain or other FcRn-binding domain, and antibody derivatives comprising an IgG Fc region or other FcRn-binding domain. FcRn-interacting molecules also include therapeutic and candidate therapeutic proteins (e.g., multispecific engineered proteins, recombinant proteins, Fc-fusion proteins, albumin fusion proteins), and nucleic acid molecules comprising an FcRn-binding domain (e.g., siRNA molecules, antisense oligonucleotides, aptamer molecules, and mRNA molecules).

[0071] According to this aspect of the disclosure, suitable first and second cell preparations include any of the mammalian cell preparations described above, e.g., a human cell preparation, a primate cell preparation, a canine cell preparation, a feline cell preparation, a porcine cell preparation, or a rodent cell preparation. Suitable cells can be derived from any tissue, including but not limited to skin (dermal and epidermal tissue), epithelium, vascular tissue (endothelial cells), heart, lung, kidney, liver, intestine, pancreas, colon, ovary, lymphoid tissue, bone, cartilage, tumor, and can be a primary cell preparation or an immortalized cell preparation, e.g., a cell line. Exemplary cell line preparations include, without limitation, Chinese hamster ovary (CHO) cells, Madin-Darby canine kidney (MDCK) cells, Vero cells, HEK-293 cells, HeLa cells, 3T6 cells, A549 cells, BHK-21 cells, MCF-7 cells, Saos-2 cells, PC3 cells, HepG2 cells, human umbilical vein endothelial cells (HUVEC).

[0072] This aspect of the disclosure entails the use of a first cell preparation and a second cell preparation, wherein the first and second preparations differ in their expression of the human neonatal Fc receptor (FcRn). For example, the cells of the first cell preparation do not express hFcRn, while the cells of the second cell preparation express functional FcRn, i.e., a heterodimer of human FcRn and human b2m (h b2m).

[0073] The cells of the first cell preparation can be cells that naturally do not express hFcRn, e.g., CHO cells are hamster kidney cells that do not express human FcRn. Alternatively, the cells of the preparation can be modified to silence expression of hFcRn for the purposes of performing this method of the disclosure. In any embodiment, cells that express hFcRn can be adapted for use as the first cell preparation by silencing expression of hFcRn and / or h b2m using siRNA or other inhibitory nucleic acid molecules. Alternatively, cells that express hFcRn and h b2m can be genetically modified using gene editing techniques to “knock down” or “knock out” expression of hFcRn and / or h b2m.

[0074] The cells of the second cell preparation can naturally express functional hFcRn protein. For example, a suitable second cell preparation includes a human cell preparation that expresses hFcRn and h b2m. Alternatively, the cells of the second preparation can not naturally express functional hFcRn protein and need to be modified to achieve such expression. In this embodiment, the cells of the second preparation can be modified to express one or more heterologous genes, e.g., the FCGRT gene encoding human neonatal Fc receptor and / or the B2M gene encoding human b-2-microglobulin.

[0075] According to this aspect of the disclosure, the first cell preparation and the second cell preparation do not express a target of the FcRn interacting molecule, e.g., a binding target of the FcRn interacting molecule. As noted above, suitable cells can naturally not express a target of the FcRn interacting protein. Alternatively, suitable cells include those cells that express a variant of the target (e.g., a species variant of the target) that does not bind or interact with the human FcRn interacting molecule. In another embodiment, however, suitable cells are those that have been modified to silence expression of the target.

[0076] As noted above, this method of predicting non-target mediated clearance of the FcRn interacting molecule in vivo involves a first incubation period and a second incubation period. In the first incubation period, the first cell preparation and the second cell preparation are incubated with media comprising the FcRn interacting molecule. This first incubation period is referred to herein as the "loading phase," in which non-target mediated cellular uptake of the FcRn interacting molecule is allowed to occur via non-specific endocytosis, FcRn-mediated uptake, or a combination of both. In one embodiment, this first incubation is performed under non-acidic, neutral pH conditions. Thus, in one embodiment, a subset of cells from each of the first cell preparation and the second cell preparation are incubated with media comprising the FcRn interacting molecule under neutral pH conditions. Suitable neutral pH conditions include a media pH of about 7.0 to about 8.0, preferably a pH of about 7.2, a pH of about 7.3, a pH of about 7.4, a pH of about 7.5, or a pH of about 7.6.

[0077] In some cases, the FcRn interacting molecule does not exhibit high levels of non-specific endocytosis at neutral pH. In other cases, it is desirable to use lower amounts of the FcRn interacting molecule, such as during early development stages where material quantities are limited. In these cases, the loading phase is performed under acidic pH conditions to enhance total endocytosis (i.e., non-specific and FcRn-mediated) to ensure that sufficient amounts of the FcRn interacting molecule are loaded into the cells to assess the FcRn recycling efficiency of the molecule. Thus, in one embodiment, a subset of cells from each of the first cell preparation and the second cell preparation are incubated with media comprising the FcRn interacting molecule under acidic pH conditions. As noted above, acidic pH conditions include a media pH of about 5.6 to about 6.9, preferably a pH of about 5.6, a pH of about 5.7, a pH of about 5.8, a pH of about 5.9, or a pH of about 6.0.

[0078] Where the first incubation period is performed under acidic pH conditions, it is preferred to employ a concentration of the FcRn-interacting molecule that does not saturate the FcRn receptor binding capacity within the cells expressing FcRn. As shown herein, FcRn-interacting molecules with high FcRn affinity (e.g., antibodies comprising the YTE substitution (M252Y / S254T / T256E)) can saturate FcRn binding under acidic conditions, which leads to inaccurate results. To accurately assess the non-specific transcytosis and FcRn recycling efficiency of these molecules, the concentration of the FcRn-interacting molecule added to the media used for the first incubation is optimized to achieve <100% FcRn binding (e.g., optimized to achieve about 90% FcRn binding occupancy).

[0079] In another embodiment, a subset of cells from each of the first and second cell preparations is incubated with media comprising an FcRn-interacting molecule under acidic pH conditions, and a second subset of cells from each of the first and second cell preparations is incubated with media comprising the FcRn-interacting molecule under non-acidic, neutral pH conditions. Assessing cellular uptake of novel FcRn-interacting molecules under neutral (non-acidic) and acidic conditions provides a way to determine the propensity of a molecule to exhibit high levels of non-specific transcytosis at physiologically relevant pHs while simultaneously measuring FcRn recycling. As described above, the FcRn-interacting molecule can be a molecule comprising an FcRn binding domain that has been engineered to reduce or remove / eliminate FcRn binding affinity. These FcRn-interacting molecules can be incubated with the first and second cell preparations under acidic and non-acidic conditions to confirm the reduction or elimination of FcRn-mediated uptake and / or recycling of the engineered molecule.

[0080] According to the disclosed method of predicting in vivo non-target mediated clearance of an FcRn interacting molecule, the first cell preparation and the second cell preparation are subjected to a first incubation period. During this first incubation period, the cell preparations are incubated with media containing a concentration of FcRn interacting molecules that does not saturate the FcRn mediated uptake capacity of the cells in the preparation. As demonstrated herein, FcRn interacting molecules that have been engineered to have enhanced FcRn binding can more easily saturate FcRn binding under acidic conditions. When the FcRn binding capacity per cell is saturated, non-specific endocytosis and FcRn recycling parameters cannot be assessed adequately or accurately. Thus, in one embodiment, the concentration of FcRn interacting molecules in the media during the first incubation is a concentration at which the cells of the second preparation achieve < 100% FcRn occupancy per cell. In one embodiment, the concentration of FcRn interacting molecules in the media is a concentration at which the cells of the second preparation achieve < 99% FcRn occupancy per cell. In one embodiment, the concentration of FcRn interacting molecules in the media is a concentration at which the cells of the second preparation achieve > 25% FcRn occupancy per cell. In one embodiment, the concentration of FcRn interacting molecules in the media is a concentration at which the cells of the second preparation achieve between 25% and 99% FcRn occupancy per cell. In one embodiment, the concentration of FcRn interacting molecules in the media is a concentration at which the cells of the second preparation achieve between 25% and 90% FcRn occupancy per cell. In one embodiment, the concentration of FcRn interacting molecules in the media is a concentration at which the cells of the second preparation achieve between 50% and 99% FcRn occupancy per cell. In one embodiment, the concentration of FcRn interacting molecules in the media is a concentration at which the cells of the second preparation achieve between 50% and 90% FcRn occupancy per cell.

[0081] A suitable concentration of the FcRn-interacting molecule can be determined by performing a concentration-dependent FcRn interaction study that provides information for the concentration of the FcRn-interacting molecule that will achieve a defined level of FcRn occupancy (e.g., 50% FcRn occupancy per cell of cells within a cell preparation). This is preferably determined for each type or form of FcRn molecule being tested, where the type or form refers to the FcRn-interacting portion of the molecule (e.g., the Fc portion). Exemplary types or forms of FcRn-interacting molecules include, without limitation, wild-type IgG Fc portions, IgG Fc portions engineered to have enhanced FcRn binding, IgG Fc portions engineered to have reduced or eliminated FcRn binding. When comparing non-target mediated cellular uptake of FcRn-interacting molecules having different forms using the assays described herein, it is important to optimize the concentration of each molecule form being tested to balance the amount of each FcRn-interacting molecule loaded into cells via FcRn-mediated means.

[0082] In one embodiment, a suitable concentration-dependent FcRn interaction study includes a concentration-dependent FcRn binding study. The binding affinity curve generated in a concentration-dependent FcRn binding study can be used to determine the equilibrium dissociation constant (K D ) between the FcRn-interacting molecule and FcRn. K D is the concentration of the FcRn-interacting molecule at which 50% of the total FcRn receptors are bound by the FcRn-interacting molecule. Thus, in one embodiment, a suitable concentration of the FcRn-interacting molecule in the medium used for the first incubation is K D In one embodiment, a suitable concentration of the FcRn-interacting molecule in the medium used for the first incubation is 1 / 2 K D to achieve at least 25% FcRn occupancy. In one embodiment, a suitable concentration of the FcRn-interacting molecule in the medium used for the first incubation is 2X K D to achieve about 90% FcRn occupancy. In one embodiment, a suitable concentration of the FcRn-interacting molecule in the medium used for the first incubation is between 2X and 10X K D to achieve about 90-99% FcRn occupancy.

[0083] In one embodiment, a suitable concentration-dependent FcRn interaction study includes a concentration-dependent transcytosis assay as described herein. The Km of the FcRn interaction molecule can be determined using the cellular uptake rate curve generated in the concentration-dependent transcytosis study. As described herein, the Km is the concentration at which half-maximal velocity is achieved, or the concentration at which approximately 50% of FcRn binding capacity is achieved. Thus, in one embodiment, the suitable concentration of the FcRn interaction molecule in the medium for the first incubation is the Km determined from the transcytosis study. In one embodiment, the suitable concentration of the FcRn interaction molecule in the medium for the first incubation is ½ Km (to achieve at least 25% FcRn binding capacity). In one embodiment, the suitable concentration of the FcRn interaction molecule in the medium for the first incubation is 2X the Km (approximately 90% FcRn binding capacity). In one embodiment, the suitable concentration of the FcRn interaction molecule in the medium for the first incubation is between 2X and 10X the Km (approximately 90-99% FcRn binding capacity).

[0084] As described above, the first incubation period in the disclosed methods of predicting in vivo non-target mediated clearance of an FcRn interaction molecule is referred to as the "loading phase," in which non-target mediated cellular uptake occurs via non-specific transcytosis, FcRn-mediated uptake, or a combination of both. The first and second incubation periods are performed at 37°C for a duration sufficient for non-specific transcytosis and / or FcRn-mediated uptake to occur, for example, about 30 minutes, about 60 minutes, about 90 minutes, about 120 minutes, about 180 minutes, about 3 hours, about 4 hours, or longer. The amount of FcRn interaction molecule taken up by the cells after the first incubation period is referred to herein as the "total uptake" amount or "uptake concentration." This amount can be determined in a subset of the first and second cell preparations by permeabilizing the cells to detect and quantify the amount of FcRn interaction molecule present in the cells, as described above.

[0085] In the remaining first cell preparation and second cell preparation, the media containing the FcRn-interacting molecule is removed, and the cells are washed one or more times with media or a saline solution to remove FcRn-interacting molecules that have not been taken up by the cells. The cells are then subjected to a second incubation period to assess FcRn recycling of the FcRn-interacting molecule. This second incubation period is the "recycling phase," and is conducted for a sufficient amount of time to allow the FcRn-interacting molecule to be captured by FcRn in the endosomal compartment, transported to the cell surface by the endosome, and released / recycled back into the media by the FcRn. This incubation period can be about 30 minutes, about 60 minutes, about 90 minutes, about 120 minutes, about 180 minutes, about 3 hours, about 4 hours, about 5 hours, about 6 hours, or more. Following the second incubation period, the recycling phase media is collected, and the amount of FcRn-interacting molecule that is recycled and released into the media is detected and quantified. The amount of FcRn-interacting molecule detected is quantified via comparison to an internal reference value or standard curve generated as described above. The amount of FcRn-interacting molecule in the media of the second cell preparation (expressing FcRn) following the second incubation is subtracted from any amount of FcRn-interacting molecule detected and measured in the media of the first cell preparation (not expressing FcRn) to be the "recycled amount" or "recycled concentration" of FcRn-interacting molecule.

[0086] Finally, the amount of FcRn-interacting molecule remaining in the cells of the second cell preparation following the first and second incubations is determined following the second incubation period. This amount of FcRn-interacting molecule remaining in the cells is referred to herein as the "remaining amount" or "remaining concentration" of FcRn-interacting molecule. The FcRn-interacting molecule remaining in the cell preparation is detected and quantified as described above, i.e., by directly or indirectly labeling the FcRn-interacting molecule with a detectable moiety and detecting the detectable moiety by flow cytometry, microscopy, spectroscopy, scintigraphic imaging, or other immunoassay.

[0087] In any embodiment, the recycled concentration, remaining concentration, and uptake concentration of FcRn-interacting molecule obtained when performing the methods of the disclosure can be used to calculate a hFcRn recycling efficiency metric (FREM) score by applying the following formula:

[0088]

[0089] In the above formula, R X is the recycled concentration of FcRn-interacting molecule "X" from the 37°C sample following the loading phase and subsequent recycling phase, and R Xis the residual concentration of the FcRn-interacting molecule “X” from the 37°C sample after the loading phase and the subsequent recycling phase (Grevys et al. iScience 25, 103746 (2022); and Grevys et al. Nat Commun 9, 621 (2018), which are hereby incorporated by reference in their entirety.

[0090] The uptake concentration of each FcRn-interacting molecule is calculated using the first and second cell preparations (i.e., cells expressing FcRn - and FcRn + respectively) by the following formula:

[0091]

[0092] In the above formula, is the uptake concentration of molecule “X” in FcRn + cells (i.e., the second cell preparation), both FcRn-dependent and independent, and is the FcRn-independent uptake of molecule “X” in FcRn - cells (i.e., the first cell preparation) (Grevys et al. iScience 25, 103746 (2022), which is hereby incorporated by reference in its entirety).

[0093] In one embodiment, when the NUC value is between 0 and 1 (0 < NUC < 1), this indicates that the cellular uptake process is dominated by FcRn-independent, non-specific endocytosis (Grevys et al. iScience 25, 103746 (2022), which is hereby incorporated by reference in its entirety). In these cases, the FREM score is multiplied by NUC. When NUC > 1, it indicates that FcRn-mediated uptake of the molecule (“X”) has negligible non-specific endocytosis. For these molecules, the FREM score is not multiplied by the NUC value.

[0094] As demonstrated herein, the FREM score is negatively correlated with the target-independent clearance value, where a higher FREM score (i.e., higher recycling efficiency) predicts low target-independent clearance, and a lower FREM score (i.e., lower recycling efficiency) predicts high levels of target-independent clearance (see Figure 17C ).

[0095] Having described the present application, the following examples are provided by way of illustration and not limitation. Example

[0096] Materials and methods for examples 1-3

[0097] Cell culture: Parental Chinese hamster ovary-K1 (CHO-K1) cells were maintained in Ham’s F-12K media (Thermo Fisher, 21127022) containing 10% heat-inactivated fetal bovine serum (Thermo, 16140071). Vero cells were purchased from the American Type Culture Collection (ATCC; CCL-81, lot 70016956) and grown in ATCC-formulated EMEM with 10% heat-inactivated fetal bovine serum (ATCC; 30-2003). Cells were cultured at 37 °C in a humidified 5% CO2 incubator without antibiotics and passaged using 0.05% trypsin-EDTA (Thermo, 25300054). Mycoplasma testing was performed using the MycoAlert assay kit (Lonza). Cells were cryopreserved using complete growth medium containing 5% (v / v) DMSO.

[0098] Monoclonal antibodies (mAbs): Two fully human wild-type mAbs were used for initial characterization: an IgG2 anti-IL-4Ra mAb (AMG 317) and an IgGl control mAb raised against streptavidin (anti-streptavidin antibody, ASA). Both mAbs were constructed by recombinant DNA technology along with a series of anti-IL-4Ra mAb mutants and produced in stably transfected CHO cells using standardized protocols. The preclinical mAb panel included five antibodies raised against G protein-coupled receptors (mAbs B1-B5). Only the anti-IL-4Ra mAb and ASA were wild-type IgG. Other mAbs were produced in stably transfected CHO cells on a stable, effectorless Fc backbone as previously described (Liu et al., J. Biol. Chem 292(5): 1876-1883 (2016), which is hereby incorporated by reference in its entirety).

[0099] PK studies and analysis: PK data were obtained from three separate studies. Female wild-type C57BL / 6J (for ASA and anti-IL-4Ra mAbs) and Balb / C (for mAbs B1-B5 group) mice were purchased from Jackson Laboratory (Bar Harbor, ME). The proteins of interest were administered via intravenous bolus doses at 1 mg / kg (anti-IL-4Ra mAbs), 2 mg / kg (mAbs B1-B5 preclinical group), or 3 mg / kg (ASA) via lateral tail vein. Blood samples were collected at various times post-injection, incubated at ambient temperature for approximately 20 minutes or until fully clotted, and then centrifuged to isolate serum. All serum samples were stored at -70 °C (± 10 °C) until used in the analytical assays. Mice were cared for in accordance with the Guide for the Care and Use of Laboratory Animals, 8thEdition, at AAALAC internationally accredited facilities. All mouse protocols were approved by the Amgen, Inc. Institutional Animal Care and Use Committee (Thousand Oaks, CA).

[0100] Proteins in mouse serum were quantified via electrochemiluminescence immunoassay on a MSD Sector 600 instrument (Meso Scale Diagnostics, Rockville, MD) using anti-human Fc antibody as the capture reagent and detection reagent. In all assays, serum concentrations of the analytes were interpolated from the standard curve using the corresponding analyte prepared in pooled mouse serum using Watson LIMS software (Thermo Fisher). The following assays were used:

[0101] Murine PK for all tested mAbs was described using a two-compartment model with a central and a distribution compartment (Tang et al., J. Pharm Sci. 93(9): 2184-204 (2004), which is hereby incorporated by reference in its entirety). Parameters included concentrations and volumes within the respective compartments, elimination from the central compartment, and distribution between the compartments. Calculational modeling was performed in Phoenix (Certara, Princeton, NJ) using the mean of the serum concentration time curves, and plotted using GraphPad Prism (Dotmatics, Boston, MA). This approach was chosen because all mAbs were assumed to have linear clearance at the doses used due to lack of measurable target binding in mice. Specifically, to report human clearance for anti-IL-4Ra mAbs, previously reported CL indValues are normalized to 80 kg as this weight was used to model its PK (Kakkar et al., Pharm Res. 28(10:2530-42 (2011), which is hereby incorporated by reference in its entirety).

[0102] Cell Uptake Studies: CHO-K1 and Vero cells were seeded onto 96-well plates at a density of 150,000 cells / well 24 h prior to the experiment or 75,000 cells / well 48 h prior to the experiment. On the day of the study, cells were washed twice in pre-warmed (37 °C) or ice-cold (4 °C) Lactated Ringer’s Solution (pH 7.4, 122.5 mM NaCl, 5.4 mM KCl, 1.2 mM CaCl2, 0.8 mM MgCl2, 0.8 mM Na2HPO4, 0.2 mM NaH2PO4, 5.5 mM d-glucose, and 10 mM HEPES). Fresh Lactated Ringer’s Solution was added for the third time and cells were equilibrated at either temperature for 30 min. After this time, the intrawell liquid was aspirated and treatment was applied immediately thereafter. Studies utilized 37 °C (to enable cell surface binding, internalization, intracellular trafficking) and 4 °C (to enable cell surface binding) conditions. For time-dependent studies, CHO-K1 cells were incubated with 100 µg / mL mAb for 15, 30, 60, or 120 min. For concentration-dependent studies, CHO-K1 cells received concentrations diluted 1 : 2 (spanning 100-25 µg / mL) for 60 min. These conditions were based on preliminary data that found lower than saturation detection limits. Following incubation, cells were washed four times with ice-cold Lactated Ringer’s Solution on ice and then trypsinized for 4 min at 37 °C. Once cells were dissociated, complete ice-cold growth media was added at a 1 : 1 ratio (v / v) to inhibit trypsin. Cells were removed from cell culture plates and placed into V-bottom 96-well plates (Fisher #249944) and then centrifuged at 300 g for 5 min at 4 °C. Cells were stained in 100 µL 1X PBS containing 0.5% (v / v) Zombie UV Fixable Viability dye (BioLegend #423108) for 20 min on ice. Cells were washed once with 100 µL FACS Buffer (1X PBS, 2% w / v BSA, 1 mM EDTA, 0.1% w / v sodium azide), centrifuged at 300 g for 5 min at 4 °C, and then fixed / permeabilized using Cyto-Fast Fixation / Permeabilization Buffer (BioLegend #426803) for 20 min at room temperature in the dark. Cells were then washed twice using 1X Cyto-Fast Permeabilization Wash Solution (BioLegend #426803).Next, samples were incubated with 5 pg / mL (100 pL / well, Cyto-Fast Permeabilization Wash Solution) of mouse anti-human Fc monoclonal IgGl antibody conjugated with Alexa Fluor 647 (Ab35-AF647) fluorescence for 30 min on ice in the dark (Hall et al., J. Immunol. Methods 393(1-2):70-73 (2013), which is hereby incorporated by reference in its entirety). Samples were washed three times with Cyto-Fast Permeabilization Wash Solution, resuspended in 100 pL Cyto-Fast Permeabilization Wash Solution, and analyzed on a BD FACSymphony flow cytometer with 18-color, 5-laser configuration (UV-355 nm, Violet-405 nm, Blue-488 nm, Yellow / Green-561 nm, Red-637 nm) and a BD Biosciences high throughput sampler (catalog number 338301). Gating was performed as depicted in the figures, and 10,000 single cells Zombie were acquired using BD Diva software. - Target of (live) events. For Vero cell samples, approximately 5000 single live cell events were captured due to the difference in trypsinization rate between the Vero cell line and the CHO-K1 cell line, which resulted in fewer Vero cells collected. Data were analyzed using FlowJo software (Becton Dickson, Franklin Lakes, NJ) to obtain the median fluorescence intensity of the specified cell population.

[0103] Antibody non-specific pinocytosis (NSE) by fluorescence microscopy: Pinocytosis studies were performed as described directly above using 100 pg / mL of ASA or anti-IL-4Ra mAb for 60 min at 37°C, then cells were washed 4 times on ice with ice-cold Linger’s solution. Cells were then fixed using 4% paraformaldehyde in IX PBS for 15 min and then washed twice with IX PBS. Samples were then blocked and permeabilized in IX Cyto-Fast Permeabilization Wash Buffer for one hour at room temperature. After aspiration, wells were stained with 0.5 pg / mL Ab35-AF647 in IX Cyto-Fast Permeabilization Wash Buffer for 1 h at room temperature. Next, samples were washed three times with IX Cyto-Fast Permeabilization Wash Buffer at room temperature, then stained with 1 pg / mL Hoechst (Thermo #H1399, diluted in IX PBS) and 2 pg / mL HCS Cell Mask Blue (Thermo #H32720, diluted in DMSO) solution for 30 min at room temperature. Wells were then washed three times with IX PBS and imaged on an Opera Phenix High Content Screening System (PerkinElmer) using a 40x water objective with Z-stacking acquisition. Maximum projections were obtained using Columbus software (PerkinElmer).

[0104] Antibody binding capacity: Quantum™ Simply Cellular® Mouse IgG beads were obtained from Bio-Rad (#FCSC815; lot 15515; Hercules, CA). Initial evaluations used a final Ab35-AF647 concentration of 5 or 10 pg / mL, following the supplier’s product information exactly, with all procedures performed on ice or at 4°C. Subsequent work used a 5 pg / mL concentration to match the cell staining experiments, as this concentration was shown to saturate each bead population with antibody. For each uptake study, freshly stained beads were analyzed on the same flow cytometer using the same instrument parameters from that day’s cell samples. Median fluorescence intensity in beads was obtained using FlowJo software, then plotted against the known antibody binding capacity of each bead population provided by the supplier via GraphPad Prism. Data were fit as a straight line using a simple linear regression. Antibody binding capacity for each cell sample was then interpolated from the cell median fluorescence intensity.

[0105] Homology modeling and anti-IL-4Ra mAb mutant design: Antibody homology models were constructed using the molecular operating environment (MOE v2022.02, Chemical Computing Group; Montreal, Canada) and default settings in antibody modeler (Amber10 forcefield, template search by identity, highest scoring template used in model, number of models = 1). For each antibody, the Fv region was modeled using the template with the highest identity to the framework and complementarity determining regions (CDRs). After a structural template search of the PDB database by framework and CDR region similarity and identity (Kabat definition), the top scoring structural template was used for homology modeling. One homology model was then built for each antibody. Structure preparation was performed using default settings for charges, protonation, rotamers, and spatial clash minimization, and energy minimization. On the prepared homology model, protein properties were calculated at pH 7.4 and pH 5.2. Protein properties of interest included Fv charge separation (between heavy chain and light chain domains), isoelectric point (pi, based on structure), zeta potential, dipole moment, hydrophobic moment, and mobility. Protein patches greater than 50 angstroms in size (hydrophobic, positively charged, and negatively charged) were also calculated and visualized. Homology models were represented as ribbon structures with surface patches colored hydrophobic green, positively charged blue, and negatively charged red.

[0106] Comparison of anti-IL-4Ra mAb and ASA homology models with calculated properties highlights the high positive charge on the anti-IL-4Ra mAb Fv (+7) and the resulting higher pi (8.7). Targeted mutant designs were generated to reduce the charge patches and charge separation between the variable domains. Specifically, residues in the positively charged patches were mutated to negatively charged and neutral residues, and protein properties were calculated to evaluate the impact on the charge patches (reduced size) and protein properties (reduced Fv charge and pi). Triple and double mutants showed the greatest impact on the reduction of Fv charge and pi. Single mutants were also explored, but were expected to show small impact on overall protein characteristics.

[0107] Example 1: ASA and anti-IL-4Ra mAb have varying degrees of non-specific uptake and preclinical clearance

[0108] The anti-IL-4Ra mAb is a therapeutic protein that lacks wild-type mouse PK data due to previous preclinical rodent studies conducted with murine surrogate mAbs. To better understand the mechanisms driving the high rate of anti-IL-4Ra mAb CL, PK studies were conducted in wild-type animals to compare non-target mediated elimination of the anti-IL-4Ra mAb and a reference mAb (ASA). Single-dose PK studies in wild-type mice demonstrated that the anti-IL-4Ra mAb has approximately 3-fold higher CL than ASA (Table 1, Figure 1). This result can not be due to differences in IgG subclass between ASA and the anti-IL-4Ra mAb, as past studies have reported that IgG subclass has no significant impact on mAb linear PK (Grinshpun et al., (2021) “Identifying biophysical assays and in silico properties that enrich for slow clearance in clinical-stage therapeutic antibodies,” MAbs 13, 1932230 and Haraya et al., (2021) “Estimation of Clearance and Bioavailability of Therapeutic Monoclonal Antibodies from Only Subcutaneous Injection Data in Humans Based on Comprehensive Analysis of Clinical Data,” Clin Pharmacokinet 60 1325-1334, which are hereby incorporated by reference in their entirety). However, observations made in wild-type mice suggest a target-based non-specific elimination effect due to negligible cross-reactivity of the anti-IL-4Ra mAb with murine IL-4Ra.

[0109] Table 1: Human and rodent CL values for anti-IL-4Ra mAb and ASA

[0110]

[0111] CL of anti-IL-4Ra mAb 人is an estimated linear clearance obtained via pharmacokinetic modeling from Kakkar et al. (2011). The murine parameter estimates with coefficient of variation are reported. CL 人 , human clearance; N / A, not applicable; CL, central compartment clearance in wild-type mice; CL D , distribution clearance in wild-type mice; V1, volume of central compartment; V2, volume of non-vascular compartment; ASA, anti-streptavidin antibody.

[0112] Previous analyses of the anti-IL-4Ra mAb Phase 1 and 2 studies demonstrated that the anti-IL-4Ra mAb had non-dose proportionality in plasma exposure, indicating target-mediated kinetics at the evaluated doses. The clinical results were mathematically described via a two-compartment population PK model with parallel linear (target-independent) and nonlinear (target-mediated) CL pathways. From this report, it was shown that the anti-IL-4Ra mAb exhibited an estimated CL ind (Table 1) of 10.5 mL / d / kg, which is the average CL indmore than two-fold the value (4.84 ± 4.72 SD) (Kakkar et al., “Population PK and IgE pharmacodynamic analysis of a fully human monoclonal antibody against IL4 receptor,” Pharm Res 28 (2011) 2530-2542; Grinshpun et al., “Identifying biophysical assays and in silico properties that enrich for slow clearance in clinical-stage therapeutic antibodies,” MAbs 13 (2021) 1932230, which are hereby incorporated by reference in their entireties). This linear CL was also significantly higher when compared to dupilumab, a human IgG4 monoclonal antibody that binds the same target antigen (human IL-4R a) as the anti-IL-4Ra mAb (Kovalenko et al., “Exploratory Population PK Analysis of Dupilumab, a Fully Human Monoclonal Antibody Against IL-4Ralpha, in Atopic Dermatitis Patients and Normal Volunteers,” CPT Pharmacometrics Syst Pharmacol 5 (2016) 617-624, which is hereby incorporated by reference in its entirety). Taken together, these findings suggest that target-mediated disposition is not the only reason for the high CL of anti-IL-4Ra mAbs in humans.

[0113] To determine whether non-specific transcytosis contributes to the rapid CL of the anti-IL-4Ra mAb ind, using parental CHO-K1 cells for both ASA and anti-IL-4Ra mAb internalization kinetics. This line was chosen because it is highly amenable to flow cytometry, easy to grow, widely used, and non-human. Of the two mAbs tested, the anti-IL-4Ra mAb exhibited clear endocytosis under the conditions tested, with biphasic time-dependent uptake ( Figure 2B ) and linear concentration-dependent uptake ( Figure 2C ). The ASA was virtually indistinguishable from the untreated control, such that the two populations exhibited a strong overlap ( Figure 2A , bottom left panel). Additionally, very low signal was observed for both mAbs at 4°C, indicating that cell surface binding was negligible relative to the extent of internalization at 37°C. Confocal microscopy performed after a separate 60 min uptake study at 37°C using 100 µg / mL of either ASA or anti-IL-4Ra mAb confirmed extensive internalization of the anti-IL-4Ra mAb when compared to ASA and the untreated control ( Figure 2D ). These combined observations support extensive non-specific endocytosis of the anti-IL-4Ra mAb relative to ASA in CHO-K1 cells. Furthermore, non-specific uptake of ASA was essentially undetectable when compared to the untreated control. This result highlights a detectable influence factor that is believed to contribute to the rapid elimination of the anti-IL-4Ra mAb in vivo.

[0114] Example 2: Development of a quantitative flow cytometry method enabling day-to-day comparisons

[0115] One of the limitations of the initial endocytosis assay iteration was the inability to compare the extent of uptake across experimental days. This was due to the readout being median fluorescence intensity, a variable that is influenced by various factors including different voltage settings on the cytometer and use of separate instruments across days. To standardize the assay to allow for day-to-day comparisons and reliable database establishment of molecular attributes, the quantitative flow cytometry method was optimized using commercially available anti-mouse IgG microspheres. Each population of these beads binds a predetermined amount of antibody that can be used to calculate the antibody binding capacity (ABC) of a cell population (i.e., the amount of antibody associated with a single live cell event). Each bead standard was saturated under the experimental conditions. When stained with 5 or 10 µg / mL of an anti-human Fc mouse mAb directly conjugated to Alexa Fluor 647, the histograms of each bead population overlapped ( Figure 3A ). Both concentrations produced standard curves with similar degrees of linearity (r 2 = 0.999).

[0116] A concentration of 5 pg / mL was chosen because this is the same concentration used to stain CHO-K1 cells after uptake. Uptake studies were performed using anti-IL-4Ra mAb or ASA, over three separate days, two different cytometers, and two different scientists (n = 20 total samples) to evaluate assay reproducibility. Based on initial kinetic characterization, the antibody was incubated with cells at 100 pg / mL for 60 min at 37 °C (Figure 2). A 60 min incubation time was chosen to enable positive detection of any future mAbs that exhibit moderate non-specific uptake that can not be as fast or to the same extent as the anti-IL-4Ra mAb. Under these conditions, the average ABC was measured to be 40,839 (SD 6614, CV 16.2%) for the anti-IL-4Ra mAb and 921 (SD 315, CV 34.2%) for the ASA (Figure 2). Figure 3B A population histogram including representative ASA and untreated samples was included to demonstrate a significant amount of signal overlap, which indicates weak internalization of the ASA in CHO-K1 cells (Figure 3). Figure 3C A schematic overview of the assay is included in Figure 3D

[0117] Example 3: High non-specific adsorptive endocytosis is driven by charge-dependent interactions and is conserved across species and cell types

[0118] One possible mechanism that contributes to the high rate of anti-IL-4Ra mAb uptake is charge. Differences in protein charge have been shown to impact mAb PK, with positive and negative net charge extremes often correlating with the strongest impact (see, e.g., Liu et al., MAbs 13(1): 1993769 (2021); Datta-Mannan et al., MAbs 7(3):483-93 (2015); Igawa et al., Protein Eng. Des. Sel. 23(5):385-92 (2010); Boswell et al., Bioconjug. Chem. 21(12):2153-63 (2010)). It was previously shown that charge effects in more specific structural regions, like the CDRs, can impact non-specific mAb endocytosis, even if the isoelectric point (pi) is not measurably shifted (Datta-Mannan et al., MAbs 7(3):483-93 (2015)). To better understand the relationship between these variables and NSE, structural modeling was performed to examine the charge distribution of the anti-IL-4Ra mAb and the ASA (Figure 4). Figure 4A ​​Figure 4B The pI values ​​for anti-IL-4Rα mAb and ASA were calculated to be 8.75 and 7.24, respectively, based on their amino acid sequences. Antibody modeling identified several charged patches on anti-IL-4Rα mAb, including two positively charged patches in the heavy chain CDR. The calculated charge of anti-IL-4Rα mAb was higher (Fv(+7)) compared to that of ASA (Fv(+2)).

[0119] Therefore, the additional positive charge patch on the anti-IL-4Rα mAb, along with its higher Fv charge, may contribute to its high nonspecific uptake and CL. ind To confirm this, point mutations were performed within the CDR and light chain framework of anti-IL-4Rα mAb to mitigate its positive charge properties. Mutations were placed at amino acid sites exhibiting positive charge clusters and / or crucial for driving the protein's net charge (Table 2). ABC values ​​obtained via endocytosis assays in CHO-K1 cells demonstrated that each mutation resulted in a significant reduction in nonspecific adsorbent endocytosis. Figure 4C These findings support the view that the positive charge exposed on the anti-IL-4Rα mAb surface is a key factor leading to the increased rate of nonspecific adsorbent endocytosis.

[0120] Table 2. A series of point mutations in modified charge that resist IL-4Rα mAb

[0121]

[0122] The bolded letters correspond to the mutant markers in the diagram.

[0123] VH, variable heavy chain; VL, variable light chain.

[0124] Nonspecific endocytosis was evaluated in individual preclinical mAb groups (mAb B1-B5) targeting G protein-coupled cell surface receptors. The mAbs had similar pI values ​​and were expected to be unable to bind to mouse targets, but exhibited significantly different CLs in wild-type mice. ind ( Figure 5 (Tables 3 and 4). Basic Local Alignment Search (BLAST) analysis identified differences in amino acids 27 (light chain), 54, and 56 (both on the heavy chain) within the mAbs' CDRs. These amino acid variations resulted in the expected charge differences, with mAb B1 exhibiting the lowest positive charge and mAb B5 the highest. CHO-K1 endocytosis results were consistent with this trend, with mAb B1 showing the lowest ABC and mAb B5 showing the highest ABC. Figure 6A). Experiments repeated in Vero cells (African green monkey) a cell unrelated to CHO-K1 (Hamster) both in species and tissue origin (kidney vs. ovary) directly agreed with the results using CHO-K1 confirming that internalization was non-specific Figure 6B , Figure 6C ). In addition, uptake studies performed at 4°C support the lack of target expression in either cell line and also highlight the magnitude of non-specific adsorptive endocytosis versus cell surface binding. The in vivo-in vitro correlation was then derived using the ABC readout from either CHO-K1 or Vero cells at 37°C demonstrating a strong relationship with CL ind,小鼠 Figure 6D ). In summary, these results confirm that surface exposed positive charges of therapeutic proteins lead to a higher rate of non-specific adsorptive endocytosis, reveal that the non-specific adsorptive internalization mode is shared across apparently different cell types, and infer the ability to provide information for CL ind using the presently disclosed methods.

[0125] Table 3. Characteristics of preclinical mAb series

[0126]

[0127] Table 4. Mouse PK parameter estimates for preclinical mAb B1-B5 series

[0128]

[0129] Discussion of Examples 1-3

[0130] The studies herein have demonstrated the sensitivity of the cell-based assays of the present disclosure to identify different rates of non-specific endocytosis of different mAbs in mammalian cells.

[0131] ​Kinetic characterization of ASA and anti-IL-4Ra mAb supports non-specific uptake of the mAb in the cell-based experimental platform in the absence of the target for a number of reasons. First is the linear relationship between the concentration of anti-IL-4Ra mAb and the extent of its endocytosis. Second, a large difference between the cell-associated amounts of anti-IL-4Ra mAb was observed at separate temperatures. However, 37°C allows for cell binding, endocytosis, and transport, while 4°C allows only for surface binding. Thus, the low signal at 4°C indicates that anti-IL-4Ra mAb is internalized at 37°C and supports the conclusion that the target is not present within CHO-K1 cells. Of note is the biphasic time-dependent uptake of anti-IL-4Ra mAb.This same observation was previously made by others and attributed to rapid and constitutive analyte exocytosis following initial non-specific internalization (Besterman et al., Endocytosis: a review of mechanisms and plasma membrane dynamics, Biochem. J. 210 (1983) 1-13; Besterman et al., Exocytosis of pinocytosed fluid in cultured cells: kinetic evidence for rapid turnover and compartmentation, J. Cell Biol. 91 (1981) 716-727; van Deurs et al., Kinetics of pinocytosis studied by flow cytometry, Eur. J. Cell Biol. 34 (1984) 96-102; and Scharschmidt et al., Fluid phase endocytosis by cultured rat hepatocytes and perfused rat liver: implications for plasma membrane turnover and vesicular trafficking of fluid phase markers, Proc. Nat'l Acad. Sci. U S A 83 (1986) 9488-9492, which are hereby incorporated by reference in their entireties). Thus, the combined results with anti-IL-4Ra mAbs are consistent with previous reports describing the non-specific kinetics of various solutes in several different cell types.

[0132] In addition to non-specific uptake, FcRn-facilitated recycling is another major component of mAb linear PK (Ovacik and Lin, Tutorial on Monoclonal Antibody Pharmacokinetics and Its Considerations in Early Development, Clin. Transl. Sci. 11 (2018) 540-552 and L. Liu, Pharmacokinetics of monoclonal antibodies and Fc-fusion proteins, Protein Cell 9 (2018) 15-32, which are hereby incorporated by reference in their entirety). Due to the pH-dependent association of FcRn with the Fc region, FcRn is critical for the long half-life of Fc-bearing therapeutics. Following endocytosis, FcRn binds to its ligand during endosomal acidification, then mediates transport of the ligand to the plasma membrane where dissociation from FcRn occurs due to the low affinity of the FcRn-Fc interaction at near neutral pH (Challa et al., FcRn: from molecular interactions to regulation of IgG pharmacokinetics and functions, Curr. Top. Microbiol. Immunol. 382 (2014) 249-272, which is hereby incorporated by reference in its entirety). Although FcRn is an important aspect of mAb PK, and changes in preclinical mAb CL can be caused by differences in murine FcRn binding, efforts here focused on determining the relationship between non-specific uptake and CL. However, as described herein (Examples 4-8), combined evaluation of non-specific and FcRn interactions of mAbs can provide additional insights into potential PK liabilities.

[0133] The cell-based assays described herein are useful in vitro tools for identifying high-risk compounds in preclinical macromolecule development. The current CHO-K1 format has moderate throughput, with a single user able to run approximately 50 samples per day. This can be increased by removing the 4°C panel, which is used here in part to help identify the presence of target binding. This panel can be replaced with publicly available transcriptomic / genomic level information to assess target expression.

[0134] Because many variables can influence mAb PK, cell-based measurements of non-specific uptake can be used in conjunction with accompanying methods to aid in preclinical candidate selection and de-risking strategies. Current technology provides per-cell quantification outputs that can be incorporated into computational models to aid in a more in-depth, biologically relevant assessment of preclinical compounds. For example, readouts from current assays can directly yield analyte-specific single-cell internalization rates for use in PK modeling efforts, rather than inferring this parameter with biophysical techniques or from a general measure of non-specific endocytosis. Additionally, other cells can be used in place of CHO cells to tailor internalization kinetics measurements to the tissue being modeled, which can provide a more representative level of site-specific information.

[0135] Materials and methods for examples 4-8

[0136] Antibodies and corresponding CL values. ASA WT and ASA AAA are fully human IgGl mAbs raised against streptavidin, which contain either wild-type Fc regions or H310A, I253A, H435A (ASA AAAmutations (Kim et al. (1999) Mapping the site on human IgG for binding of the MHC class I- related receptor, FcRn. Eur J Immunol 29, 2819-2825; Qiao et al. (2008) Dependence of antibody-mediated presentation of antigen on FcRn. Proc Natl Acad Sci U S A 105, 9337- 9342, which are hereby incorporated by reference in their entireties). The clinical mAbs include commercialized antibody products, including anti-CD20 antibody (mAb 5), anti-PD-1 antibody (mAb 3), and anti-CD38 antibody (mAb 2), as well as several investigational analogs of clinical antibodies, including anti-PCSK9 antibody (mAb 4), anti-IL-15 antibody (mAb 6), IL-4R antibody (AMG-317), IL-12 / IL-23 antibody (mAb 1 and mAb 9), EGFR / HER3 antibody (mAb 7), IL-2R alpha antibody (mAb 8), and phosphatidylserine antibody (mAb 10). As previously described, the mAb analogs were produced in stably transfected CHO cells on stable effector- functionless Fc scaffolds (Chaudhury et al. (2003) The major histocompatibility complex- related Fc receptor for IgG (FcRn) binds albumin and prolongs its lifespan. J Exp Med 197, 315-322, which is hereby incorporated by reference in its entirety).

[0137] In addition to the mAbs of Table 2, a series of fully human wild-type hlgG2 anti-IL-4R alpha WT and M252Y / S254T / T256E (YTE) charge mutant mAbs were produced to investigate the impact of Fv mutations as well as the half-life extending (HLE) Fc engineering YTE mutation. In addition, two hlgG2 control mAbs, ASA WT - hlgG2-DL650 and ASA WT-hIgG2-YTE was used for characterization. All mAbs were constructed using recombinant DNA technology and produced in stably transfected CHO cells using a standardized protocol. A list of these mAbs, along with their pI and net charge, is provided in Table 8.

[0138] The CL value of many clinical mAbs is estimated from the CL values ​​of multiple mAbs. ind Internal data, FDA monographs, or previous reports were obtained (Grinshpun et al., Identifying biophysical assays and in silicoproperties that enrich for slow clearance in clinical-stage therapeutic antibodies), MAbs [Monoclonal Antibodies] 13 (2021) 1932230, which is hereby incorporated by reference in its entirety). Normalization was performed using a body weight of 80 kg. Publicly available clinical data for some antibodies are limited. For these cases, the following equation was used, via the reported distribution volume (V) obtained under linear PK conditions. D ) and terminal half-life (t 1 / 2β ) value to estimate CL ind (Schoenwald, RD (2002) Basic Principles. Pharmacokinetics in Drug Discovery and Development (Schoenwald, RD, ed.), 1st edition. pp. 3-33, hereby incorporated in its entirety by reference):

[0139]

[0140] Cell culture. Parental MDCK II cells (Sigma #MTOX1300) were maintained in 5% heat-inactivated FBS EMEM (ATCC #30-2003) and passaged using 0.25% trypsin-EDTA. Maintaining subconfluence was found to be crucial for proper monolayer formation. Optimal passage conditions were determined to be 2.5-3e6 cells / 75 cm² every 2-3 days. 2The density of the cells was adjusted to maintain them below 90% confluency. Parental MDCK II cells were seeded at 100,000 cells / well in flat bottom 96 well plates (Corning #353072). Transfected MDCK II cells (see below) were seeded at 120,000 cells / well in the same plates. Monolayers of both cells were achieved within 2 days. CHO-K1 cells were seeded and grown as described in reference to Examples 1-3 herein. All cells were cultured at 37°C in a 5% CO2-95% O2 humidified incubator. Mycoplasma negativity was confirmed using the Lonza MycoAlert Assay Kit (Lonza, Walkersville, MD, #LT07-318). Freezing medium was complete supplemented growth medium with 5% DMSO (v / v).

[0141] Plasmids and transfection. The hFcRn construct was developed on a pcDNA3.1 vector with an N-terminal HA tag followed by hFcRn, a G4S linker, and mEGFP, conferring resistance to G418. The hβ2m construct was developed on a pEF6 / V5-His vector containing a puromycin resistance gene.

[0142] In a sterile 1.5 mL Eppendorf tube A, the two plasmids at a 1 : 1 molar ratio were diluted to a total of 4 μg DNA in 250 μl of serum-free Opti-MEM I reduced serum medium and mixed gently. Lipofectamine 2000 (10 μΐ) was diluted in 250 μΐ of Opti-MEM I medium and incubated at room temperature for 5 minutes. After the 5 min incubation, the diluted DNA was combined with the diluted Lipofectamine 2000 (total volume = 500 μΐ), mixed gently, and incubated at room temperature for 20 minutes. Five hundred microliters of the complex was added dropwise to each well containing cells and mixed gently by rocking the plate back and forth. Cells were incubated at 37°C in a humidified CO2incubator for 48 hours prior to testing for transgene expression. The hFcRn-GFP / hβ2m-MDCK II were maintained constantly in selection medium (1000 μg / mL G418, 7.5 μg / mL puromycin) as transgene loss was observed if the cells were cultured in normal growth medium.

[0143] Immunostaining was performed using flow cytometry. Immunostaining was performed using the fluorescently conjugated antibodies indicated in Table 5. Parental and FcRn-GFP / β2m-MDCK II cells grown to confluency were resuspended in IX PBS containing Zombie UV fixable viability dye (Biolegend # 423108) under the manufacturer’s recommended conditions, then incubated at room temperature for 20 min. Cells were washed once in FACS buffer (IX PBS, 2% w / v BSA, 1 mM EDTA, 0.1% w / v sodium azide), then (1) maintained on ice for cell surface staining, or (2) fixed and permeabilized using Cyto-Fast Fix / Perm Buffer (Biolegend #426803) for 20 min at room temperature in the dark. Fixed cells were washed twice in IX Cyto-Fast Perm Wash Solution (Biolegend # 426803). All cells were then transferred to V-bottom 96-well plates (Fisher #249944) and stained with the antibody amounts (100 µL volume) indicated in Table 5 for 30 min at ice in the dark. Staining was performed using FACS buffer (for unfixed cells) or IX Cyto-Fast Perm Wash Solution (for fixed cells). Cells were then washed three times with the respective buffer, then analyzed on a BD FACSymphony flow cytometer with an 18-color, 5-laser configuration (UV-355 nm, Violet-405 nm, Blue-488 nm, Yellow / Green-561 nm, Red-637 nm) and a BD Biosciences high throughput sampler (catalog number 338301). Where needed, spectral compensation was completed using single stain UltraComp eBeads (Thermo #01-2222-42), ArC™ amine-reactive compensation beads (Thermo #A10346), and / or AcGFP flow cytometer calibration beads (Takara, #632594) based on the supplier’s instructions.

[0144] Table 5. List of antibodies and dilutions for flow cytometry

[0145]

[0146] Fluorescence-activated cell sorting (FACS). Based on preliminary assessments, clone #8 FcRn-GFP / β2m-MDCK II cells exhibited high transgene expression, so this clone was selected for expansion. Different GFP + and GFP - populations (corresponding to hFcRn expression) were initially observed. FACS was therefore performed to enrich for GFP +Population. Clone #8 cells were subcultured, resuspended at 7e6 cells / mL in azide-free FACS buffer, stained through a 70 pm filter 12 mm x 75 mm polystyrene tube, and sorted for GFP + expression within the single cell clone #8 population via a BD FACSMelody cell sorter (equipped with violet-405 nm, blue-488 nm, and red-640 nm lasers) at the University of Michigan Flow Cytometry Core Facility. However, subsequent evaluation demonstrated insufficient hβ2m expression (multiple populations). GFP + Clone #8 FcRn-GFP / β2m-MDCK II cells were expanded and sorted for the highest 10% GFP + , hFcRn + , hβ2m + expression via cell surface staining with anti-hFcRn-AF647 and anti-hβ2m-PerCP / Cy5.5 (Table 6) on a BD FACSAria Fusion cell sorter (18-color, 5-laser configuration (UV-355 nm, violet-405 nm, blue-488 nm, yellow / green-561 nm, red-640 nm)) at the University of Michigan Flow Cytometry Core Facility. Single cells were again sorted. Even after FACS and constant selection, approximately 2-3% of the total FcRn-GFP / β2m-MDCK II cell population was consistently GFP - . Therefore, internalization analysis using fluorescently conjugated proteins was performed on GFP + cells.

[0147] Table 6. ASA mAb PK estimates following single intravenous bolus in hFcRn transgenic mice

[0148]

[0149] Normalized using an average mouse weight of 0.028 kg. Data was fit using a two-compartment model.

[0150] Fluorescent conjugation. Human serum albumin (Sigma #A3782) and ASA WT (2 mg / mL final concentration each reaction; 1 mL volume) were conjugated with DyLight 650-NHS (Thermo #62265) at a protein:dye ratio of 10: 1 in 50 mM borate buffer, pH 8.5 (Thermo #28384). Proteins were dialyzed against 1X PBS in 7 kDa cutoff molecular weight cassettes (Thermo #66372) at 4 °C overnight for 3 L (1 L increments, total of three changes). Final protein concentration was obtained by spectrophotometry as described by the manufacturer.

[0151] Flow cytometry was used for ASA internalization studies. Parental and FcRn-GFP / β2m- MDCK II cells were seeded into 96-well plates and grown to confluent monolayers as described above. On the day of the study, cells were washed twice with pre-warmed pH 7.4 Ringer’s solution (122.5 mM NaCl, 5.4 mM KCl, 1.2 mM CaCl2, 0.8 mM MgCl2, 0.8 mM Na2HPO4, 0.2 mM NaH2PO4, 5.5 mM d-glucose, and 10 mM HEPES) and then incubated in pH 7.4 Ringer’s solution at 37 °C for 30 min. For time-dependent studies, cells were aspirated and immediately incubated with 10 or 100 µg / mL DyLight 650-conjugated ASA WT (ASA WT -DL650) in pH 5.8, 7.4, or 8.0 Ringer’s solution for 2 h. For ASA WT -DL650 and DyLight 650-conjugated human serum albumin (HSA-DL650) concentration-dependent studies, cells were incubated at pH 5.8 in the presence or absence of an excess of unlabelled protein (50 mg / mL; IgG from human serum for ASA WT -DL650, Sigma #I4506) to confirm receptor-mediated endocytosis. Upon completion of the treatment, cells were placed on ice and washed four times with ice-cold pH 7.4 Ringer’s solution (200 µL / well / wash) and then incubated with 100 µL / well 0.25% EDTA-trypsin at 37 °C for 8-10 min. Once the cells rounded, the wells were first agitated by gentle but rapid pipetting and then 100 µL of MDCK II growth media (without selection reagent) was added per well to inhibit trypsin. Cells were transferred to V-bottom 96-well plates, centrifuged at 300 g for 5 min at 4 °C, and then stained with 0.5% v / v UV Zombie dye in IX PBS for 20 min on ice. Cells were washed once with 100 µL FACS buffer / well, centrifuged at 300 g for 5 min at 4 °C, and then fixed with Cyto-Fast Fixation / Permeabilization Buffer (50 µL / well) in the dark at room temperature. Fixed cells were washed twice in IX Cyto-Fast Permeabilization Wash Solution (150 µL / well), resuspended in 100 µL FACS buffer, and then analyzed on a BD FACSymphony flow cytometer. Targeted 10,000 single-cell live GFP +Median fluorescence intensity of (FcRn-GFP / β2m-MDCK II only) events. Data files were analyzed using FlowJo (BD). Concentration-dependent receptor-specific uptake data (i.e., total signal minus mean (+) unlabelled condition and untreated control median fluorescence intensity for each concentration) was divided by total incubation time and then fit using the Michaelis-Menten equation in GraphPad Prism (GraphPad Software, Boston, MA) with:

[0152]

[0153] V max is the maximum rate achieved within the experimental system, [S] is the ASA WT - concentration of DL650 or HSA-DL650, and K m is the concentration at which half-maximal rate is achieved.

[0154] ASA WT - hlgG2-DL650 and ASA WT - hlgG2-YTE-DL650 concentration-dependent studies, except that cells were incubated at pH 5.8 for 30 min in both parental and hFcRn-GFP / β2m-MDCK II cells. After analyzing data files using FlowJo (BD), concentration-dependent receptor-specific uptake data (i.e., total signal in hFcRn-GFP / β2m-MDCK II cells minus mean signal of parental MDCK II cells and median fluorescence intensity of untreated parental control cells for each concentration) was divided by total incubation time and then fit using the Michaelis-Menten equation as mentioned in the previous paragraph, where V max is the maximum rate achieved within the experimental system, [S] is the ASA WT - hlgG2-DL650 or ASA WT - concentration of hlgG2-YTE-DL650, and K m is the concentration at which half-maximal rate is achieved.

[0155] FcRn-ASA WT - Lysosome imaging and colocalization analysis. Human FcRn-GFP / β2m-MDCK II cells were seeded into PerkinElmer CellCarrier Ultra 96-well plates (cat# 6055302) as described above. The confluent cell monolayers were incubated with 0.25 mg / mL of 10 kDa Texas Red-conjugated dextran (Sigma #D1828) in selection media for 6 h the day before imaging studies. The dextran concentration was based on a pilot titration study the previous week. Following incubation, cells were washed with HBSS++ The cells were washed three times in PBS and then incubated in the selection media overnight. This method will allow internalized dextran to accumulate within the lysosome and thus label the lysosome. The next day, time-dependent ASA internalization studies were performed in either pH 5.8 or 7.4 Ringer’s solution as described in “ASA Internalization Studies with Flow Cytometry”. The cells were washed three times in PBS and then incubated in the selection media overnight. This method will allow internalized dextran to accumulate within the lysosome and thus label the lysosome. The next day, time-dependent ASA internalization studies were performed in either pH 5.8 or 7.4 Ringer’s solution as described in “ASA Internalization Studies with Flow Cytometry”. WT - DL650 Uptake Studies (10 and 100 µg / mL). After incubation was complete, the FcRn-GFP / β2m-MDCK II cells were washed four times with ice-cold pH 7.4 Ringer’s solution and then fixed in 4% v / v paraformaldehyde in IX PBS for 15 min at room temperature. After fixation, the cells were washed twice with IX PBS and then stained with 1 µg / mL Hoechst (Thermo #H1399) diluted in IX PBS for 30 min at room temperature. The cells were washed twice with IX PBS and then stored in 200 µL IX PBS / well at 4°C. The next day, images were captured on an Opera Phenix High Content Screening System (PerkinElmer) using a 63x water objective with Z-stacking acquisition. The Columbus software (PerkinElmer) was used to analyze ASA WT - Mean Fluorescence Intensity (MFI) measurements on the DL650 channel. Analysis sequences were prepared to segment the cytoplasmic region of individual cells and collect MFI across different time points and treatments. Colocalization image analysis was performed via Acapella software (PerkinElmer). Briefly, the Hoechst channel was used for nucleus and cytoplasm segmentation. Within the cytoplasmic region, Pearson’s correlation coefficient was used to measure colocalization between different channel pairs (ASA WT - Colocalization between DL650, hFcRn-GFP, and lysosomes loaded with Texas Red-dextran. The Costes correction method was used to help distinguish labeled regions from background. GraphPad software was used to plot MFI and colocalization metrics.

[0156] Immunofluorescence and confocal microscopy. Parental and hFcRn-GFP / β2m- MDCK II cells were seeded as described above and grown to confluent monolayers in PerkinElmer CellCarrier Ultra. Cells were washed twice with IX PBS, fixed in 4% PFA (IX PBS), and blocked / permeabilized in IX Cyto-Fast Permeabilization Wash Solution for 1 h at room temperature. All subsequent volumes were 100 µL / well. Anti-FcRn antibody (Sigma #HPA012122, 0.2 mg / mL stock concentration) was added at a 1 : 5000 dilution in IX Cyto-Fast Permeabilization Wash Solution and incubated for 1 h at room temperature. Cells were washed twice with IX Cyto-Fast Permeabilization Wash Solution and then incubated with Alexa Fluor 594-conjugated goat anti-rabbit secondary antibody (Thermo #A11037, 2 mg / mL stock concentration, 1 : 1000 dilution in IX Cyto-Fast Permeabilization Wash Solution) for 1 h at room temperature. Cells were washed twice with IX Cyto-Fast Permeabilization Wash Solution and then stained with 1 µg / mL Hoechst plus 2 µg / mL CellMask Blue (Thermo #H32720) both diluted in IX Cyto-Fast Permeabilization Wash Solution for 30 min at room temperature. Cells were washed twice with IX PBS and imaged on an Opera Phenix High Content Screening System using a 40x water objective with Z-stack acquisition.

[0157] Human FcRn mAb recycling studies for Examples 5 and 6 and Figures 10 and 11. Parental and FcRn-GFP / β2m MDCK II cells were seeded at 1e5 cells and 1.2e5 cells / well, respectively, in 200 µL of their respective growth media in 96-well plates and cultured for 48 h. Cell media was aspirated and then cells were washed twice with pre-warmed (37°C) pH 7.4 Ringer’s solution followed by equilibration in Ringer’s solution pH 7.4 supplemented with MEM Non-Essential Amino Acids 1X (Corning #25-025-CI) and sodium pyruvate 1 mM solution (Corning #25-000-CI) for 30 min at 37°C. Then, intrawell liquid was aspirated and cells were incubated with mAb solutions prepared at 666.67 nM (100 µg / mL) in pH 5.8 and pH 7.4 Ringer’s solution TM ) at 37°C for 30 min. Cells were then washed twice with pre-warmed (37°C) pH 7.4 Ringer’s solution and then incubated with mAb solutions prepared at 666.67 nM (100 µg / mL) in pH 5.8 and pH 7.4 Ringer’s solution ++ ) at 37°C for 30 min. Cells were then washed twice with pre-warmed (37°C) pH 7.4 Ringer’s solution and then incubated with mAb solutions prepared at 666.67 nM (100 µg / mL) in pH 5.8 and pH 7.4 Ringer’s solution ++The cells were incubated for 2 hours. This is referred to as the loading phase. After incubation, the cells were washed 4 times with room temperature Ringer's solution pH 7.4 at 200 pL / well at 20 °C. Then, serum-free EMEM growth medium (150 pL / well) was added to the cells and the plates were incubated at 37 °C or 4 °C for 4 hours. This is denoted as the recirculation phase. Since there is no active recirculation at 4 °C, the remaining data at 4 °C indicates the uptake process after a loading phase of 2 h at 37 °C using 100 pg / mL mAb. Next, the supernatant (recirculation sample) was collected in a 96-deep well plate (Axygen, #P-DW-500-C) and stored at -80 °C until analysis. Subsequently, the cells were re-washed 4 times on ice with 4 °C Ringer's solution. The cells were then lysed with 150 pL / well of ice-cold MOPS lysis buffer pH 7.4 containing microcOmplete EDTA-free protease inhibitor cocktail (Roche #04693159001) in 0.1% (v / v) Triton X-100. To prepare the lysis buffer, 1 microcOmplete EDTA-free tablet containing protease inhibitor cocktail was added to every 10 mL of MOPS lysis buffer and vortexed. The plates were incubated at 4 °C on ice with 600 rpm shaking for 30 min, followed by 30 inversions to uniformly remove the cells from the bottom of the plate using a multichannel pipette. The lysates were collected in a 96-deep well plate and stored at -80 °C until further analysis.

[0158] Example 8 and Figure 17 Anti-IL-4Ra WT and YTE mutant mAbs human FcRn recirculation study: This human FcRn recirculation study was performed at 37 °C for loading of the anti-IL-4Ra WT and YTE mutant mAbs, since no active recirculation was observed at 4 °C in previous recirculation studies. Parental and FcRn-GFP / 32m MDCK II cells were seeded at 75,000 and 85,000 cells / well, respectively, in 200 pL of their individual growth medium in 96-well plates and cultured for 72 h. The cell medium was aspirated and the cells were then washed twice with pre-warmed (37 °C) Ringer's solution pH 7.4, followed by equilibration in Ringer's solution pH 7.4 supplemented with IX GlutaMAX™ supplement (Cat. No. 35050-061, Gibco TM ) and 1 mM sodium pyruvate solution (Corning #25-000-CI) (referred to as Ringer's solution + / + ) for 30 min. This was followed by incubation with 100 pg / mL of the respective mAbs for 2 h at 37 °C. After the incubation, the cells were washed 4 times with room temperature Ringer's solution pH 7.4 at 200 pL / well at 20 °C. Then, serum-free EMEM growth medium (150 pL / well) was added to the cells and the plates were incubated at 37 °C or 4 °C for 4 h. This is denoted as the recirculation phase. Since there is no active recirculation at 4 °C, the remaining data at 4 °C indicates the uptake process after a loading phase of 2 h at 37 °C using 100 pg / mL mAb. Next, the supernatant (recirculation sample) was collected in a 96-deep well plate (Axygen, #P-DW-500-C) and stored at -80 °C until analysis. Subsequently, the cells were re-washed 4 times on ice with 4 °C Ringer's solution. The cells were then lysed with 150 pL / well of ice-cold MOPS lysis buffer pH 7.4 containing microcOmplete EDTA-free protease inhibitor cocktail (Roche #04693159001) in 0.1% (v / v) Triton X-100. To prepare the lysis buffer, 1 microcOmplete EDTA-free tablet containing protease inhibitor cocktail was added to every 10 mL of MOPS lysis buffer and vortexed. The plates were incubated at 4 °C on ice with 600 rpm shaking for 30 min, followed by 30 inversions to uniformly remove the cells from the bottom of the plate using a multichannel pipette. The lysates were collected in a 96-deep well plate and stored at -80 °C until further analysis. ++The difference is the lack of non-essential amino acids, which were not found to be essential for cell viability. After that, the liquid in the wells was aspirated and the cells were incubated with a solution of anti-IL-4Ra WT mutant mAb prepared at 666.67 nM (100 pg / mL) or anti-IL-4Ra YTE mutant mAb prepared at 166.67 nM (25 pg / mL) in pH 5.8 Ringer's solution + / + The incubation period is referred to as the uptake phase, which is the net result of both uptake and recycling that occurs during this loading phase. After incubation, the cells were washed four times on ice with 200 pL / well ice-cold Ringer's solution pH 7.4. The cells were then lysed with 200 pL / well of ice-cold lysis buffer pH 8.0 containing protease inhibitors without EDTA (10 mM Tris HC1 pH 8.0, 1% IGEPAL CA-630, 0.5% sodium deoxycholate, 0.1% SDS, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA) (herein referred to as ULS lysis buffer). To prepare the final ULS lysis buffer, 1 mini tablet of protease inhibitor cocktail without EDTA (Roche #04693159001) was added to each 10 mL of ULS lysis buffer and vortexed. The plates were incubated for 30 minutes at 4°C with 600 rpm shaking on ice, followed by 50 repeated pipetting strokes in a Tecan Freedom EVO 200 liquid handler controlled by Freedom EVOware® version 2.8 software to dislodge the cells uniformly from the bottom of the plate. The lysates were collected in a 96-deep well plate and stored at -80°C until further analysis. These samples constitute the uptake portion of the FcRn score. To obtain the recycling samples, the exact uptake procedure mentioned above was followed, with the difference that after the loading phase, the cells were washed 4 times at 20°C with 200 pL / well room temperature Ringer's solution pH 7.4. Then, serum-free EMEM growth medium (200 pL / well) was added to the cells and the plates were incubated at 37°C for 4 hours. This represents the recycling phase. Next, the supernatant (recycling sample) was collected in a 96-deep well plate (Axygen, #P-DW-500-C) and stored at -80°C until analysis. Subsequently, the cells were re-washed 4 times on ice with 4°C Ringer's solution. The cells were then lysed after the addition of 200 pL / well ice-cold ULS pH 8.0 lysis buffer in the same manner as mentioned above. The lysates were collected in a 96-deep well plate and stored at -80°C until further analysis. These lysates are represented as the remaining sample.

[0159] Recycle assay sample analysis was performed by MesoScale Discovery immunoassay. Similar to previously developed protocols (91), electrochemiluminescence (ECL) immunoassays were performed using a MesoScale Discovery (MSD) Sector S 600 instrument (Meso Scale Diagnostics, Rockville, MD, USA). First, plates stored at -80 °C were thawed on ice. Thawed lysate samples were centrifuged at 1,000 g for 10 min at 4 °C to remove cell debris, and the supernatant was transferred to a new 96-well plate. For MSD experiments, 96-well MSD Gold ® Streptavidin SECTOR ®Assay plates (Meso Scale Diagnostics, #L15SA-1) were coated with 50 µL of biotinylated proprietary anti-human Fc antibody (clone 35; Ab35) at 2 µg / mL in blocking buffer (Blocker™ BLOTTO in Tris-buffered saline [TBS], Thermo #37530) for 1 hour at room temperature with shaking at approximately 600 rpm. Plates were then washed 4 times in a plate washer (Biotek 405 Select Microplate Washer) with IX KPL wash buffer (LGC seracare). Standards, quality control samples (100, 10, and 1 ng / mL), and diluted recirculation samples were prepared in the following matching media: serum-free EMEM media for recirculation samples, EDTA-free complete protease inhibitor-containing MOPS lysis buffer pH 7.4 (as described above), or ULS lysis buffer pH 8.0 (as described above) for remaining samples. The lysis buffer mix used to prepare standards and quality control samples for remaining sample analysis was generated based on the average protein content of the remaining samples for that plate using matching amounts of lysate. Protein content was measured by Bradford (Examples 5 and 6, Bryniarski et al., Am. J. Physiol Renal Physiol. 315(5): F1191-F1207 (2018), which is hereby incorporated by reference in its entirety) or BCA according to the manufacturer’s protocol (Example 8). To generate the matrix for the remaining sample standards, equal amounts (approximately le6 cells) of hFcRn-GFP / hP2M MDCK II and parental MDCK II cells were grown into confluent monolayers on 100 mm round culture dishes (Corning #353003), and lysed with 1 mL of EDTA-free complete protease inhibitor-containing MOPS lysis buffer pH 7.4 or ULS lysis buffer pH 8.0. The mixtures were then centrifuged at 1,000 g for 10 min to remove cell debris. The supernatants were collected, and appropriate amounts of lysate were added to the standard and quality control sample lysis buffer mix (v / v) from this stock to match the average protein amount of the remaining samples for the plate. Standards, quality controls, and recirculation assay samples (50 µL / well) were added to the plates and incubated for 2 hours at room temperature with shaking, followed by an additional 4 washes. Plates were then incubated with 50 µL of 2 µg / mL sulfotagged Ab35 (ruthenium) in BLOTTO blocking buffer as the detection reagent for 1 h at room temperature.Next, the plates were washed 4 times and read in the MSD instrument after adding 150 µL 2X MSD read buffer (diluted from 4X MSD read buffer T with surfactant (Cat# R92TC-1, Meso Scale Diagnostics) with DI water) in each well. In GraphPad Prism, the analyte concentration was interpolated from the sigmoidal four-parameter least-squares fit of the antibody-specific standard curve within the same plate, and verified using the quality control samples.

[0160] Human FcRn recycling efficiency metric (FREM) score. The hFcRn recycling efficiency metric (FREM) score was calculated using the recycling, remaining, and uptake (4°C recycling phase) concentrations from the initial pH 5.8 loading phase of the mAb (Example 5 and Example 6) by the following equation:

[0161]

[0162]

[0163] Here, R X is the recycling concentration of mAb “X” from the 37°C sample after the pH 5.8 loading phase (the mean of the 4°C recycling concentrations was subtracted from each sample readout to account for active recycling processes), and RA X is the remaining concentration of mAb “X” from the 37°C sample after the pH 5.8 loading phase (Grevys et al. (2022) Antibody variable sequences have a pronounced effect on cellular transport and plasma half-life. iScience 25, 103746; Grevys et al. (2018) A human endothelial cell-based recycling assay for screening of FcRn targeted molecules. Nat Commun 9, 621, which are hereby incorporated by reference in their entireties). The same equation was used to calculate the FREM score in Example 8, where R X is the recycling concentration of mAb “X” from the 37°C sample after the pH 5.8 loading phase, and RA X is the remaining concentration of mAb “X” from the 37°C sample after the pH 5.8 loading phase.

[0164] The non-specific uptake coefficient (NUC) was calculated using the uptake concentrations of each mAb’s FcRn-GFP / hP2m MDCK II and parental MDCK II cells by the following formula:

[0165]

[0166] is the total uptake (both FcRn-dependent and independent) of mAb “X” in FcRn-GFP / hP2m MDCK II cells and is the FcRn-independent uptake of mAb “X” in parental MDCK II cells (Grevys et al. (2022) Antibody variable sequences have a pronounced effect on cellular transport and plasma half-life. iScience 25, 103746, which is hereby incorporated by reference in its entirety).

[0167] When the NUC value is between 0 and 1 (0 < NUC < 1), it indicates that the cellular uptake process is dominated by FcRn-independent non-specific endocytosis. In these cases, the FREM score is multiplied by NUC. When NUC > 1, it indicates FcRn-mediated uptake of mAb “X” with negligible non-specific endocytosis. For these mAbs, the FREM score is not multiplied by the NUC value. Outliers of the assay were identified by using the Grubbs method (when n = 4 (mAb group) and / or the Rout method (when n = 8; ASA hlgG1 as well as anti-IL-4Ra WT and YTE mutant mAbs) using GraphPad Prism software with an alpha value of 0.05.

[0168] CHO-K1 non-specific uptake studies. Non-specific endocytosis studies were performed at pH 7.4 or 5.8 as described in Examples 1-3 above. Antibody binding capacity (ABC) values are a quantitative technique facilitated inter-day comparisons between mAbs via the generation of a standard curve with IgG-binding microspheres.

[0169] ASA and anti-IL-4Ra mutant mAb PK study and analysis. Mice were housed in an AALAC-International accredited facility. Animals were cared for in accordance with the Guide for the Care and Use of Laboratory Animals, 8thedition. All study protocols were reviewed and approved by the American Home Products Institutional Animal Care and Use Committee. Six to eight-week-old male homozygous Tg32 (Strain #014565), homozygous Tg276 (Strain #004919), and immunodeficient homozygous hFcRn SCID Tg32 (Strain #018441) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). ASA WT (1 mg / kg), ASA YTE (1 mg / kg), and ASA AAA (3 mg / kg), anti-IL-4Ra WT and YTE mutant mAbs (1 mg / kg each) were administered at the indicated doses via intravenous bolus via lateral tail vein. Blood samples were collected at various times post-injection, incubated at ambient temperature for approximately 20 minutes or until fully clotted, and then centrifuged to isolate serum. All serum samples were stored at -70 °C (± 10 °C) until used in the analytical assay.

[0170] Proteins in mouse serum were quantified via electrochemiluminescence immunoassay on a MSD Sector 600 instrument using Ab35 anti-human Fc antibody as capture and detection reagent, similar to past reports (Poon-Andersen et al., (2022) Development of an immunoassay for aglycosylated murine IgG1 in mouse serum via generation of a specific tool antibody. Bioanalysis 14, 581-588, which is hereby incorporated by reference in its entirety). In all assays, analyte serum concentrations were interpolated from standard curves using the corresponding analyte prepared in pooled mouse serum in the Watson LIMS software.

[0171] Due to lack of target expression in mice, CL of ASA variants was expected to be linear. Serum concentration data were fitted individually using a two-compartment disposition model with a central and a peripheral compartment (Tang et al., (2004) Pharmacokinetic aspects of biotechnology products. J Pharm Sci 93, 2184-2204, hereby incorporated by reference in its entirety). Parameterization included first-order elimination from the central compartment (clearance, CL), distribution clearance between the compartments (CL D ), and volumes of the central (V1) and peripheral (V2) compartments. Modeling was performed using Ubiquity package workflow within R (v4.2.2).

[0172] Example 4: Generation and functional characterization of MDCK II cells with stable co-expression of hFcRn-GFP and hβ2m

[0173] MDCK II cells were stably transfected with constructs for hβ2m and hFcRn-GFP. Clones co-expressing substantial cell surface hβ2m and hFcRn-GFP were isolated by fluorescence-activated cell sorting (FACS). Protein expression was then confirmed in the resulting sorted cell line (designated hFcRn-GFP / hβ2m-MDCK II cells) via immunofluorescence derived from confocal microscopy ( Figure 7A ) and flow cytometry ( Figure 7B , Figure 7C ).

[0174] Because FcRn exhibits pH-dependent affinity for its ligand, internalization studies were performed at different pH values to assess the functionality of the co-transfected hFcRn-GFP / hβ2m complex. Parental and hFcRn-GFP / hβ2m-MDCK II cells were incubated with 10 or 100 µg / mL of wild-type hlgG1 anti-streptavidin antibody (ASA WT -DL650) conjugated with DyLight 650 fluorescence for increasing time periods at pH 5.8, 7.4, or 8.0. Analysis was performed via flow cytometry ( Figure 13A ). Only hFcRn-GFP / hβ2m-MDCK II cells exhibited concentration- and time-dependent uptake in a pH-dependent manner, whereby acidic conditions resulted in extensive ASA WT -DL650 endocytosis ( Figure 8A and Figure 13B ). This behavior is consistent with the enhanced affinity of FcRn for its ligand within acidified endosomes. Additionally, no ASA WT- differences in DL650 uptake, thus suggesting no detectable influence on endocytosis at different pH levels Figure 8A ). Thus, under the tested conditions, these results demonstrate that hFcRn-dependent ASA WT - DL650 endocytosis decreased with increasing media pH. In addition, non-specific uptake was observed as the endocytic mechanism for IgG in parental MDCK II cells.

[0175] To provide further evidence for FcRn-mediated endocytosis, ASA WT - DL650 or fluorescently labeled human serum albumin (HSA-DL650) were subjected to concentration-dependent uptake studies at pH 5.8. Both compounds were competitively inhibited by an excess of unlabeled protein ( Figure 8B and Figure 8C ). The Michaelis-Menten equation was used as a function to fit the specific uptake data, and the concentration at which half-maximal uptake was achieved within the experimental system was estimated to be 50 µg / mL [95% confidence interval] for ASA WT -DL650 and 44 µg / mL for HSA-DL650. These are equivalent to approximately 333 µM ASA WT -DL650 and 660 µM HSA-DL650. In addition, non-specific internalization was observed to be linear with concentration, consistent with non-specific internalization kinetics ( Figure 8B and Figure 8C , bottom panel). When considered together, this data indicates that (1) hFcRn-GFP / hβ2m is functional in co-transfected MDCK II cells and (2) non-specific endocytosis of albumin and ASA WT occurs in the absence of a target receptor.

[0176] The next goal was to assess the cellular localization and trafficking behavior of hFcRn-GFP. To this end, concentration, pH, and time-dependent uptake studies were performed with high-content confocal microscopy in hFcRn-GFP / hβ2m-MDCK II cells. ASA WT -DL650 (10 or 100 µg / mL) internalization was measured at pH 5.8 and 7.4. Imaging was performed on fixed cells after 15, 30, 60, or 120 min of incubation. Lysosomes were labeled by preloading cells the day before with 10 kDa Texas Red-conjugated dextran ( Figure 9A ). Intracellular ASA WT -DL650 imaging results were consistent with those reported in Figure 8, where time, concentration, and pH dependence were quantified ( Figure 9B ). Via the use of hFcRn-GFP, ASAWT Co-localization analysis using Costes method of image signal thresholding of DL650 and Texas Red dextran (i.e. lysosomes) indicated only hFcRn-GFP and ASA WT Pearson's correlation coefficient score of apparent co-localization of DL650 Figure 9C ). Very low degree of lysosomal co-localization of hFcRn-GFP and ASA WT was observed. These findings are consistent with past evaluations of GFP-tagged FcRn behavior and provide further support for normal hFcRn-GFP and hβ2m function in stably transfected MDCK II cells.

[0177] Example 5: Development of Quantitative FcRn Recycling Assay Using hFcRn-GFP / hβ2m-MDCK II Cells

[0178] The hFcRn-GFP / hβ2m-MDCK II recycling assay described herein was developed in standard 96-well plates optimized for ASA WT -DL650 kinetic analysis Figure 10A ). Following a 2 h loading phase at pH 5.8 or 7.4, cells were washed and then incubated in serum-free cell culture media at 37°C or 4°C for 4 h. The 4°C group served as a control to provide a means to estimate the amount of mAb internalized during the loading period, as intracellular processing would be halted on ice. Additionally, this control group also served to demonstrate active recycling when compared to the parallel 37°C group (i.e. 4 h recycling phase at 37°C). In summary, the workflow allowed for the quantification of specific (hFcRn-GFP / hβ2m-MDCK II) and non-specific (parental MDCK II) amounts of test mAb internalized and recycled under various assay conditions.

[0179] Initial studies to assess assay performance were completed using ASA WT and mutants with abrogated hFcRn affinity (H310A, I253A, H435A; ASA AAA ). FcRn recycling studies in parental and hFcRn-GFP / hβ2m-MDCK II cells demonstrated that (1) ASA WT uptake / recycling was higher at pH 5.8 compared to pH 7.4, (2) there was no difference in ASA AAA uptake / recycling at either pH condition when compared to parental MDCK II data, (3) recycling was higher during the 4 h 37°C condition compared to 4°C (when detectable uptake was observed), and (4) non-specific uptake of any ASA hIgGl in parental MDCK II cells was negligibleFigure 10B It is worth noting that after loading at pH 7.4, ASA... WT The very low recirculation signal is not a result of inefficient hFcRn-mediated recirculation, but rather due to the minimal amount of mAb entering the cell during the loading phase.

[0180] Rodent PK studies were conducted to characterize the differences in total CL of ASA mAb ( Figure 10C For in vivo studies, hFcRn transgenic mice (Tg32) were chosen to preserve the species-dependent interaction between ASA hIgG1 and FcRn, and because this preclinical model has been previously reported to be reliable for the conversion of human mAb PK. When compared with ASA... WT In comparison, ASA in Tg32 mice AAA They exhibited higher systemic CL (Table 6). Matched studies were conducted in homozygous Tg276 hFcRn mice to confirm these results. Tg276 has a promoter different from that in Tg32 and exhibits changes in hFcRn tissue expression, but can provide alternative humanized mouse models to elucidate changes in hIgG treatment. Similarly, ASA AAA Showing better than ASA WT Much faster CL (Table 6).

[0181] One goal of this project is to develop an in vitro assay that can sequence a range of compounds for mAb CL. indTo provide information. Previous reports outlined a metric to promote the efficiency of hFcRn-mediated recycling in cultured endothelial cells. Their scoring system was recently extended to also consider non-specific behavior, as the initial approach could not be used alone to describe cellular disposition of mAbs with extensive non-specific characteristics (Grevys et al. (2022) Antibody variable sequences have a pronounced effect on cellular transport and plasma half-life. iScience 25, 103746; Grevys et al. (2018) A human endothelial cell-based recycling assay for screening of FcRn targeted molecules. Nat Commun 9, 621, which are hereby incorporated by reference in their entirety). These published studies utilized pH 7.4 loading conditions to recapitulate physiological conditions within the vasculature. However, based on internalization results, here an analogous scoring approach was applied using pH 5.8 loading conditions in MDCK II cell studies to improve the ASA WT sensitivity of the signal Figure 8A , Figure 8B and Figure 10B ). As described above, the human FcRn recycling efficiency metric (FREM) score in the current study was calculated by multiplying the recycling fraction by the non-specific uptake coefficient (NUC) (Grevys et al. (2022) Antibody variable sequences have a pronounced effect on cellular transport and plasma half-life. iScience 25, 103746, which is hereby incorporated by reference in its entirety). The NUC was only incorporated when the analyzed mAb exhibited elevated non-specific endocytosis in hFcRn-GFP / hβ2m-MDCK II cells relative to uptake in parental MDCK II. The FREM score for ASA WT , ASA AAA was negligible, highlighting the lack of hFcRn interaction in the ASA AAA mutant Figure 10E). These results are in line with the trend of CL values obtained from two hFcRn transgenic mouse models ( Figure 10C and Figure 10D ).

[0182] In summary, to validate the cellular system, both parental and transfected MDCK II cells were used to define hFcRn specific and non-specific transport of two hlgG1 mAbs with different hFcRn affinities. The scoring system of ASA WT and ASA AAA was applied. Moreover, in hFcRn transgenic mice, the calculated hFcRn recycling score was in line with the measured CL values in single dose PK studies.

[0183] Example 6: Human FcRn recycling score from MDCK II cells can identify mAbs with fast human CL

[0184] Next, the ability of the MDCK II cell system to detect mAbs with high non-target mediated CL (i.e. CL ind ) was examined. A total of 10 mAbs with human CL values between 2.45 and 32.2 mL / d / kg were selected (Table 7). All mAbs were assayed in parental and hFcRn-GFP / hβ2m-MDCK II cells under the same conditions as for the ASA hlgG1 series (Figure 14). The clinical study analogue mAb (mAb1) was selected as a reference for all subsequent comparisons because it performed well in the MDCK II cell study (normal hFcRn interaction, low non-specific uptake) and because it is a FDA-approved drug with low CL (CL = 2.45 mL / d / kg, Table 7). As mentioned above for the ASA mAbs, the NUC metric was only incorporated when the NUC was below a value of 1, which indicates that non-specific uptake exceeds the uptake by hFcRn internalization. The average value of FREM scores below that of 25% mAb1 was associated with a human CL value above 4.8 mL / kg / d, thus highlighting the potential of this approach to identify mAbs with PK liabilities (CL > 4.8 mL / kg / d) (Table 7). Figure 11A Figure 11B ).

[0185] Table 7. Human CL, MDCK II FcRn recycling and biophysical assay metrics of mAb panel

[0186]

[0187] 1 CL and pi values were determined from relevant FDA labels, journal publications or internal data.

[0188] ​FREM and NUC scores of antibodies were normalized relative to mAb1

[0189] Example 7: High mAb CL ind of different mechanisms

[0190] The observations from the hFcRn recycling studies were a correlation between non-specific interactions (via NUC values) and the final hFcRn recycling score (Table 7), indicating a strong role of non-specific behavior in hFcRn recycling efficiency. Examination of the parental MDCK II uptake results revealed a unique outlier of anti-IL-4Ra mAbs and mAb9 as a non-specific feature. Anti-IL-4Ra mAbs had significantly higher endocytosis in parental MDCK II cells at pH 7.4 when compared to mAb1 analogs ( Figure 11C , p < 0.0001). This is consistent with Examples 1-3, demonstrating extensive non-specific cellular uptake of anti-IL-4Ra mAbs at this pH, which can contribute to its rapid CL in wild-type mice and humans. Large charge patches were identified within the Fv region of anti-IL-4Ra mAbs, which can be determinants of this observation. Therefore, for anti-IL-4Ra mAbs, extensive non-specific endocytosis from extracellular fluids (e.g., blood) can drive a large amount of mAbs into non-target cell populations, which would increase the probability of intracellular catabolism.

[0191] mAb9 is an anti-IL-12 / 23 antibody that has aberrant non-specific and hFcRn interactions, resulting in its high CL rate, as reported in the literature. Compared to anti-IL-4Ra mAbs, mAb9 exhibited elevated, but not statistically significant, parental MDCK II uptake at pH 7.4 relative to mAb1, which is also an anti-IL-12 / 23 antibody ( Figure 11C ). However, at pH 5.8, a significant increase in non-specificity was measured for mAb9 ( , p < 0.001). It was recently shown that the high positive charge surface area within the Fv region of mAb9 distinguishes its anti-IL12 / 23 variable domain from that of mAb1 and is also pH sensitive, with increased dissociation under acidic conditions. This supports the current findings: where the previously reported charge difference between the Fv domains of mAb1 and mAb9 can contribute to the elevated non-specific uptake at pH 7.4, the non-specific uptake becomes higher and significantly different at pH 5.8. Thus, mAb9 has pH-dependent charge patches that can not only affect its association / dissociation with hFcRn and the initial rate of non-specific endocytosis at pH 7.4, but also can lead to increased non-specific interactions within the endosome upon acidification, impairing the kinetics of mAb9 with hFcRn and reducing its recycling efficiency.

[0192] In addition, a small increase in the pH-dependent non-specific uptake of mAb1 in parental MDCK II cells was observed ( Figure 11C , p < 0.05). To better characterize these results across the mAb series, a follow-up study was performed that directly measured mAb non-specific endocytosis in CHO-K1 cells (as described above) ( Figure 11D ). The procedure for this method was to incubate parental CHO-K1 with one or more mAbs of interest at 37°C for 60 min. Analysis of cell-associated mAbs (i.e., intracellular and surface) was accomplished via anti-human Fc immunostaining and flow cytometry. A standard curve was generated each day using a fixed amount of detection antibody-bound quantitation microspheres to enable inter-day comparisons (antibody binding capacity, ABC). The use of CHO-K1 assays provided a way to recapitulate the potential for altered non-specific interactions within the endosomal compartment by performing incubations at acidic pH. Higher cell uptake rates using this method would indicate increased non-specific interactions at the pH values tested.

[0193] In CHO-K1 cells, elevated non-specific endocytosis was measured for mAb9 (p < 0.01) and an anti-IL-4Rα mAb (p < 0.0001) at pH 7.4 when compared to ASA WT Figure 11D At pH 5.8, each mAb exhibited increased endocytosis when compared to the pH 7.4 condition, indicating that non-specific interactions increased as pH decreased. Similar to the observations in parental MDCK II cells ( Figure 11C ), mAb9 non-specific increased to comparable amounts to the anti-IL-4Rα mAb only at pH 5.8. These results provide an elevated CL ind ​of two different mechanisms. The first is a non-specific uptake from the extracellular fluid and high general non-specificity within the endosome at pH 7.4 that can mitigate mAb-FcRn interactions (e.g., anti-IL-4Ra mAbs). The second (e.g., mAb9) is a relative low CL ind mAbs exhibit moderate non-specific endocytosis at pH 7.4 due to elevated non-specific interactions extracellularly. Following endocytosis, these non-specific behaviors increase during endosomal trafficking, which can lead to inadvertent association with endosomal membranes and / or impaired endosomal interactions with FcRn, both of which can result in increased intracellular catabolism. For higher CL ind mAbs with these aspects not to an extreme degree, the rate of extracellular non-specific endocytosis and / or the degree of acidic non-specificity can exceed the ability of the mAb to effectively interact with FcRn within the endosome, resulting in reduced FcRn recycling efficiency (Figure 12).

[0194] Example 8: Cell hFcRn Recycling Scores can successfully and simultaneously rank WT and Fc-engineered mAbs in order of decreasing murine CL ind of Fc-engineered mAbs

[0195] Decreasing the CL of Fc-interacting molecules (e.g., Fc-biologies) ind One approach to reduce the CL and extend the serum half-life of Fc-interacting molecules (e.g., Fc-biologies) is to enhance the affinity of the Fc region for FcRn. Doing so must selectively increase Fc-FcRn binding at acidic pH values while still maintaining the minimal possible interaction at near neutral pH to preserve the pH-dependent dissociation of the Fc region from FcRn (Dall’Acqua et al., J. Biol. Chem. 281(33):23514-24 (2006); Borrok et al., J. Biol. Chem. 290(7):4282-90 (2015); Mackness et al., MAbs 11(7): 1276-1288 (2019)). The cell-based FcRn recycling assays described herein provide a key in vitro tool to gauge the potential success of Fc-engineering strategies. However, to properly assess therapeutic proteins with altered FcRn interactions, the assay conditions should be specifically optimized. Failure to do so can result in assay failures that are not relevant to in vivo behavior. For example, previous studies employing cell-based FcRn endocytic transport assays were not successful in ranking the CL ind provided information but did not provide clear interpretation (Chung et al., MAbs 11(5):942-955 (2019)).

[0196] To demonstrate that the cell hFcRn recycling assay described in this paper accurately provides CL of Fc-interacting molecules with enhanced FcRn affinity. ind The feasibility of this information expands the physicochemical diversity of the anti-IL-4Rα mAb group detailed in Example 3. Specifically, additional single-point and double-point mutants with higher positive charge properties within the variable (Fv) domain were generated relative to the quadruple mutant in Example 3 (Table 8). NSE measurements in parental CHO-K1 cells at pH 7.4 and 5.8 demonstrated a significantly higher diffusion of nonspecific behavior compared to that observed against the original group. Figures 15A to 15B Then, the M252Y / S254T / T256E (YTE) mutation (EU: European Union number) was incorporated into each anti-IL-4Rα mAb variant as a clinically proven extension of half-life and CL. ind Decreased FcRn affinity increased model (Dall'Acqua et al., J. Biol. Chem. 281(33):23514-24 (2006); Borrok et al., J. Biol. Chem. 290(7): 4282-90 (2015); Mackness et al., MAbs 11(7): 1276-1288 (2019)). When compared with parental WT Fc mAb, the addition of YTE generally maintained the overall nonspecific trend between mAb and YTE, but led to increased NSE at pH 7.4 and decreased nonspecificity at pH 5.8. Figures 15A to 15B ).

[0197] Enhanced Fc-FcRn interaction strategies (like the YTE mutant) will lead to higher internalization across acidic to near-neutral pH in FcRn-expressing cells, where net extent is driven by relative affinity changes under these conditions. This will result in higher amounts of mAb-YTE entering hFcRn-GFP / hβ2m MDCK II cells at any given concentration relative to non-YTE mAb. This could saturate FcRn capacity per cell and lead to inaccurate assessment, thus causing the YTE mutant to incorrectly exhibit worse behavior than its matched WT parent after cellular hFcRn recycling studies. To counteract this affinity effect and equalize intracellular loading conditions, a control mAb ASA conjugated with DyLight 650 was used at pH 5.8. WT -hIgG2 and ASA WT -hIgG2-YTE concentration-dependent endocytosis study ( Figures 16A to 16DIntake studies and K were conducted as described above. m and V max Value estimation. Observations of ASA. WT -hIgG2-YTE-DL650's K m (15 [10, 21] µg / mL) is ASA WT -hIgG2-DL650 K m (41 [26, 64] µg / mL) nearly two-sevenths. Based on this data, subsequent FcRn recycling studies using hFcRn-GFP / hβ2m MDCK II cells utilized approximately 2 x K m Acidic uptake incubation was performed at concentrations (WT (100 μg / mL) and YTE (25 µg / mL)) to ensure that similar amounts of WT and YTE mAb were loaded into cells in an FcRn-dependent manner.

[0198] Cell-based hFcRn recycling studies were conducted using 6 WT / YTE pairs of anti-IL-4Rα mAb. FREM scores were calculated by analyzing uptake, recycling, and residual samples, and these scores were based on either one or both of the FcRn recycling efficiency (RE) and nonspecific uptake coefficient (NUC) scores as outlined above. Figure 17A The assay was optimized to compare both Fc-engineered YTE and WT mAb by normalizing all results for anti-IL-4Rα EEES-YTE mAb.

[0199] Table 8. A series of IL-4Rα-resistant WT and YTE mAb point mutants with modified charges

[0200]

[0201] The higher non-specificity and / or lower FcRn recycling efficiency of the mAbs resulted in lower FREM scores, as exemplified by the WT anti-IL-4Ra-mAb. The single point mutation at position R20 did not significantly improve the FREM score of the 20S-WT mutant (0.006 ± 0.001) compared to WT (0.004 ± 0.001). However, the FREM score (0.155 ± 0.048) was improved with the R33S mutation at the heavy chain (HC) CDR1 compared to the R20S mutation. The double point mutations at both the HC CDR1 and CDR3 significantly improved the FREM score (0.504 ± 0.085) of the anti-IL-4Ra mAb-33S,110L compared to the single point mutations at the mAb Fv region. The anti-IL-4Ra mAb-SSLS mAb exhibited a slightly lower FREM score (0.400 ± 0.095) compared to the double point mutations. The WT mAb mutant anti-IL-4Ra mAb-EEES, which exhibited the highest degree of exposed charge modification, demonstrated a higher FREM score (0.584 ± 0.127) compared to all other WT mAbs. The relative FREM score improvement trend between the WT and YTE mutant mAb series was conserved. The half-life extended YTE mAbs exhibited better FREM scores relative to their WT counterparts when tested alongside the WT mAbs, with the exception of the SSLS-YTE mutant, which improved minimally compared to its WT counterpart. When compared head-to-head, the anti-IL-4Ra mAb-EEES-YTE exhibited the highest FcRn recycling efficiency metric score (1.000 ± 0.196).

[0202] Table 9. CL from SCID hFcRn Tg32 mice ind FREM scores from the MDCK II hFcRn recycling assay and pi values of the anti-IL-4Ra WT and -YTE mutant mAbs.

[0203]

[0204] 1 CL ind was calculated from the serum concentration time curves of the mAb mutants using non-compartmental analysis (NCA).

[0205] FREM scores were normalized relative to the anti-IL-4Ra mAb-EEES-YTE

[0206] 2 pi values were collected from internal data.

[0207] PK studies were performed in male homozygous immunodeficient SCID hFcRn Tg32 transgenic mice. This mouse model facilitates the ability to obtain PK over an extended sampling time without interference from anti-drug antibody due to the lack of mature T or B cells. Serum concentration-time profiles of mAbs are shown in Figure 17B From the PK results it is apparent that mAbs with higher NSE and lower FcRn recycling efficiency have overall worse PK (e.g. anti-IL-4Ra mAbs). The FREM score was then utilized to simultaneously rank both WT and YTE mAbs. We observed a very strong relationship between CL ind and the FREM score, with lower FREM scores correlating with higher CL ind . Figure 17C .

[0208] Discussion of Examples 4-8

[0209] The current work provides a quantitative mechanistic examination of transcytosis and intracellular processing of a panel of mAbs to describe differences in hFcRn-mediated recycling efficiency. The experimental overview provides a way to simultaneously assess hFcRn-dependent transcytosis and recycling and non-specific transcytosis characteristics of the mAbs under evaluation in a cellular assay. The extent of non-specific interactions strongly dictates the recycling outcome, which is distinguished via the mass balance approach performed in this study. These findings highlight the need to consider non-specific transcytosis rates, total mAb internalization, and hFcRn recycling when studying mAb CL ind .

[0210] ASA WT was used as a model compound to measure mAb internalization kinetics in parental and hFcRn-GFP / hβ2m MDCK II cells as it has a low CL ind in wild-type mice and negligible non-specific transcytosis in mammalian cells. The current study demonstrates that time-dependent uptake of ASA WT -DL650 at pH 7.4 in parental MDCK II is very low when compared to receptor-mediated conditions at pH 5.8 in transfected MDCK II cells. Concentration-dependent transcytosis in hFcRn-GFP / hβ2m MDCK II cells at pH 5.8 in a competitive environment is directly proportional to extracellular ASA WT -DL650 concentration, which is consistent with non-specific transcytosis of ASA WT in the absence of receptor-mediated kinetics.

[0211] These measurements are important for studying hFcRn-recycling efficiency because they confirm that mAbs with low CL ind of 7.4 should exhibit minimal non-specific uptake. If 100 µg / mL is used, this would result in negligible recycling amounts. The data herein indicate that pH 5.8 promotes FcRn-mediated endocytosis in hFcRn-GFP / hβ2m MDCK II cells and results in significantly more ASA WT -DL650 into cells when compared to internalization via non-specific endocytosis alone. In terms of material usage for ASA WT or any other mAb that exhibits low non-specific uptake, the concentration required to achieve detectable recycling after a pH 7.4 loading phase would be impractical. Therefore, these results provide information for subsequent recycling studies by indicating that an acidic loading would be required to reach detectable concentrations for mAbs with limited non-specific endocytosis.

[0212] ASA WT -DL650 kinetics studies also demonstrate the contribution of hFcRn to initial mAb internalization at pH 7.4. At every time point examined at pH 7.4, the ASA WT -DL650 uptake of parental MDCK II cells was lower than the transfected cells, and pH 8.0 resulted in decreased ASA WT -DL650 endocytosis in hFcRn-GFP / hβ2m MDCK II cells when compared to pH 7.4.

[0213] While cell surface FcRn can contribute to endocytosis of IgG outside of acidic environments, this is not the focus of the current project. The data herein indicate that non-specific endocytosis is the primary endocytosis mechanism for IgG at near neutral pH in the absence of targets including FcRn or IgG receptors. The results for ASA WT -DL650 indicate that FcRn can promote internalization of IgG at pH 7.4 to some extent, but the results from the mAb set recycling studies indicate minimal FcRn involvement in internalization at this pH. The extent of FcRn contribution in vivo at pH 7.4 is still unknown. Any future work to assess this question should consider utilizing other physiologically relevant cell systems.

[0214] The success with acid incubation was confirmed with a clinically relevant panel of mAbs, resulting in very low levels of multiple mAbs internalizing into hFcRn-GFP / hβ2m MDCK II cells at pH 7.4, leading to essentially undetectable concentrations of recycled material after 100 pg / mL incubation. Importantly, it is important to note that cell-based FcRn recycling studies at near neutral pH are more physiologically relevant and can be performed as we have shown above. However, ASA WT - Internalization kinetics of DL650 (Figures 8 and 13), which for mAbs with low non-specific endocytosis and low CL ind at pH 7.4 would require at least 30-fold higher concentrations to achieve similar levels of internalization and thus similar assay resolution for the same mAb at pH 5.8. This observation and interpretation are a key difference from previously described work (Grevys et al. (2022) Antibody variable sequences have a pronounced effect on cellular transport and plasma half-life. iScience 25, 103746; Grevys et al. (2018) A human endothelial cell-based recycling assay for screening of FcRn targeted molecules. Nat Commun 9, 621, which are hereby incorporated by reference in their entirety).

[0215] Examination of ASA hIgG1 established the specificity of the MDCK II hFcRn recycling assay. ASA AAA did not exhibit discernible differences between parental cells and hFcRn-GFP / hβ2m MDCK II cells under any condition, supporting the loss of hFcRn binding affinity at the mutated amino acid. Furthermore, inclusion of several experimental groups for MDCK II cell studies fully elucidated the driving mAb CL indmechanisms. This framework facilitates mAb internalization, intracellular trafficking, hFcRn-mediated recycling, residual accumulation, and capture of non-specific interactions. The in vitro hFcRn recycling score from a previous report was modified to provide a way to pool cellular datasets into a cumulative metric to describe hFcRn recycling efficiency. An average FREM score above 0.5 relative to the mAb1 analog indicates that the mAb has attributes associated with a low elimination rate in humans, including low non-specific uptake and the ability to efficiently undergo hFcRn-mediated recycling. An hFcRn recycling score below 0.25 strongly indicates that the mAb has a CL in excess of 5 mL / d / kg. FREM scores between 0.25 and 0.5 suggest a potential for elevated CL ind

[0216] When tested in the in vitro cell-based recycling assays described herein, it was apparent that the FREM scores for the YTE molecules were relatively higher compared to the WT mutants. In general, the target-independent PK profiles for these engineered mAbs were well-explained with the in vitro cell-based assays discussed in this example. The interpretation of both NSE and FcRn engagement via the FREM score strengthened the conclusions and increased the PK predictive power of these engineered proteins. Furthermore, the use of acidic conditions necessitated optimization of the experimental workflow to accommodate the evaluation of Fc-engineered mAbs with increased FcRn affinity. Even with the use of physiologically relevant endocytosis conditions (i.e., approximately pH 7.4), this maneuver was required because of the inherent increase in FcRn affinity across a range of pH values (Dall’Acqua et al., J. Biol. Chem. 281(33):23514-24 (2006); Borrok et al., J. Biol. Chem. 290(7):4282-90 (2015); Mackness et al., MAbs 11(7):1276-1288 (2019)).

[0217] The results support the importance of non-specific and hFcRn interactions in determining mAb CL ind in humans, while also providing further insight into the mechanistic diversity that determines these cellular processes (summarized in Figure 12A- Figure 12B ​Here, a significant influence of pH on non-specific interactions of all mAbs was demonstrated, as shown by a higher rate of non-specific pinocytosis in CHO-K1 cells at pH 5.8 compared to 7.4. Observations suggest that internalized mAbs can exhibit non-specific interactions in a different degree and form than those exhibited on the cell surface at a pH close to 7.4. The isoelectric points (pi) of all tested mAbs exceed 7.0, which would lead to an increased positive charge at acidic pH (Table 7). This can result in a generally elevated non-specific pinocytosis due to increased interactions with the negatively charged plasma membrane. However, the pi alone cannot fully explain the huge difference in non-specific pinocytosis of anti-IL-4Ra mAbs or mAb9 relative to other mAbs.

[0218] In terms of cellular consequences, the observed pH-dependent features can lead to detrimental non-specific associations with endosomal membranes as well as with hFcRn itself, which reduces hFcRn recycling efficiency and leads to higher CL ind rates (Figure 12). It can be concluded at present that the incorporation of appropriate experimental controls is essential when assessing mAb cellular disposition and hFcRn interactions. hFcRn recycling scores are obtained at a pH of 5.8, and the NUC metric is utilized to assess the degree of non-specific uptake associated with mAb interactions with hFcRn. While measurements of non-specific interactions alone provide partial insights (e.g., CHO-K1 pinocytosis), NUC provides a way to define the manner in which mAbs interact with hFcRn and non-specific interactions in the same experimental setting. This is crucial for the ability to outline the relationship between in vitro performance and CL ind .

[0219] Single-parameter in vitro assessments that predict mAb in vivo behavior often do not directly correlate with mAb CL ind . This can be due to their inability to fully recapitulate the biokinetics important for mAb disposition, or because they focus on a single aspect only. A key advantage of the cellular hFcRn recycling assay described herein is that it integrates multiple processes important for mAb disposition. The results reported herein correlate with mAb CL indThe ability demonstrated by the other cell-based assessments of IgG-FcRn is consistent with the capacity to further emphasize the multifaceted relationship between cellular uptake and FcRn-mediated recycling. Cell-based systems provide their readouts within a biological framework, which has been shown directly to be advantageous for studying FcRn-IgG and FcRn-Fc interactions compared to standard biophysical methods. Subsequently, a physiologically-based PK model will play a key role in extending in vitro results to patients by helping to describe the net impact of mAb disposition on in vitro behavior. As the biopharmaceutical industry transitions to more complex structures to advance their biologic pipelines, there will be an urgent need for a combined approach that leverages known drug-like attributes of mAbs to develop low-CL Fc-HLEs.

[0220] Example 9: High non-specific endocytosis predicts low subcutaneous bioavailability in humans

[0221] Subcutaneous administration remains the preferred injection route for therapeutic proteins as it facilitates drug administration in a more convenient physical location for the patient. However, drugs administered subcutaneously must first be absorbed from the injection site into the blood. Relative to intravenous administration, the fraction of dose that reaches the systemic circulation (subcutaneous bioavailability, F SQ ) depends on several factors, including temperature, solubility, lymph flow at the injection site, and tissue composition (Richter et al., (2012) Mechanistic determinants of biotherapeutics adsorption following SC administration. AAPS J. 14(3): 559-70, which is hereby incorporated by reference in its entirety). The complexity of these factors and poor mechanistic understanding of their underpinnings has led to a lack of ability to use in vitro techniques to provide information for human F SQ .

[0222] As demonstrated herein, non-specific endocytosis and FcRn recycling are key determinants of CL ind for intravenous dosed monoclonal antibodies (mAbs). Defining the cellular mechanisms that dictate these processes and establishing a quantitative experimental workflow leads to enhanced ability to identify in vitro therapeutic mAbs with fast CL ind . It is hypothesized that these same factors, particularly non-specific endocytosis, will also be important determinants for F SQ . This is because high rates of non-specific endocytosis at the injection site will lead to elevated catabolism of the therapeutic protein, resulting in less drug available for absorption into the circulation. Additionally, the mechanisms that drive high non-specific endocytosis can also lead to other adverse effects on F SQunwanted in vivo characteristics, such as non-specific interaction with subcutaneous extracellular matrix. Thus, the use of a cell-based assay to quantify non-specific pinocytosis was expected to be a human F SQ information is provided.

[0223] A set of therapeutic mAbs with clinical pharmacokinetic (PK) data was obtained, of which 17 had an estimated value of target-independent clearance (CL ind ). The mAbs were binned into the following three groups: (1) CL ind < 4.5 mL / kg / d, (2) CL ind > 4.5 mL / kg / d or (3) no CL ind estimate was obtained. A CL ind of 4.5 mL / kg / d translates to a terminal half-life of approximately 10 days in a 80 kg individual in humans.

[0224] As described in Examples 1-3 above, antibodies were analyzed using the CHO-K1 assay to obtain the cellular antibody binding capacity (ABC), and a range of behaviors were observed, as shown in Figure 21A An assay threshold was then generated to better interpret the data. The "low risk" group contains mAbs with a low risk of high CL ind and / or low F SQ due to non-specific pinocytosis. It was defined as all mAbs with an ABC SQ higher than 50% and / or a CL ind < 4.5 mL / kg / d. The "medium risk" group contains mAbs that can or can not exhibit high CL ind and / or low F SQ , and was set two standard deviations above the mean ABC directly mentioned above (i.e. mean + 2 SD). The "high risk" group highlights mAbs with significant non-specific pinocytosis, which can be detrimental to their disposition in humans.

[0225] The mAbs were then grouped by their performance in the CHO-K1 assay (i.e. low risk, medium risk or high risk), and plotted against their respective CL ind values. Figure 21B It was shown that the increased risk defined via higher non-specific pinocytosis rates not only indicates a higher CL ind after intravenous administration in humans, but also indicates a decreased F SQ It is noteworthy that each mAb characterized as high risk exhibited a CL ind higher than 4.5 mL / kg / d, a F SQor both.

[0226] Example 10: Non-specific cellular endocytosis can identify therapeutic proteins with high target-independent clearance in a wide range of structural modalities

[0227] Multispecific engineered proteins (MS-ePRs) are protein therapeutics that can simultaneously engage two or more different targets. These compounds have provided new mechanisms to treat a variety of diseases that were previously inaccessible with monoclonal antibodies (mAbs). They span a wide range of molecular formats that can be tailored for their intended therapeutic use, such as eshaies R., Nature 580(7803): 329-338 (2020); Labrijn et al., Nat. Rev. Drug Discov. 18(8):585-608 (2019); Husain and Ellerman, BioDrugs 32(5):441-464 (2018); Spiess et al., Mol. Immunol. 67(2 Part A): 95-106 (2015); Dickopf et al., Comput. Struct. Biotechnol. J. 18: 1221-1227 (2020). MS-ePRs can also include Fc regions or utilize an immunoglobulin G scaffold (mAb-ePRs) to reduce clearance (CL) and decrease dosing frequency (Deshaies R., Nature 580(7803): 329-338 (2020); Labrijn et al., Nat. Rev. Drug Discov. 18(8):585-608 (2019); Husain and Ellerman, BioDrugs 32(5):441-464 (2018); Spiess et al., Mol. Immunol. 67(2 Part A): 95-106 (2015). The Fc portion facilitates interaction with FcRn, which recycles internalized immunoglobulin G, albumin, and Fc / albumin fusion proteins via intracellular pH-dependent binding to increase serum half-life (Challa et al., Curr. Top. Microbiol. Immunol. 382:249-72 (2014).

[0228] The plasticity of MS-ePRs not only broadens the therapeutic landscape but can also bring development hurdles. Structural arrangements that are ideal for efficacy can also be challenging in terms of synthesis. Certain engineered designs can be more immunogenic or encounter stability issues in vivo. Additionally, mAb-ePR drug disposition is much more complex compared to mAbs (Deshaies R., Nature 580(7803): 329-338 (2020); Husain and Ellerman, BioDrugs 32(5): 441-464 (2018)). Target-mediated kinetics play a key role in the overall pharmacokinetics (PK) profile of mAbs. Target engagement can lead to target-mediated drug disposition (TMDD), whereby mAb CL is closely related to its target biology, leading to dose-dependent changes in mAb disposition (Ovacik and Lin, Clin. Transl. Sci. 11(6): 540-552 (2018); Ryman and Meibohm, CPT Pharmacometrics Syst. Pharmacol. 6(9): 576-588 (2017); Peletier and Gabrielsson, J. Pharmacokinet. Pharmacodyn. 39(5): 429-51 (2012)). The extent of TMDD inherently grows with mAb-ePRs, as there will be at least two separate targets, which can be further complicated by different sites of expression and / or different binding affinities to the mAb-ePR (Deshaies R., Nature 580(7803): 329-338 (2020)).

[0229] Target-independent mechanisms of mAb CL are also important for mAb-ePRs, including FcRn-mediated recycling in non-targeted cell types and non-specific adsorptive endocytosis, as described above. Complex structures that can arise from protein engineering can lead to worse interactions with FcRn or higher rates of non-specific adsorptive uptake by non-target cell types, which can lead to rapid target-independent CL of mAb-ePRs (CL ind)(Datta-Mannan, Antibodies (Basel) 11(1): 2 (2021); Datta-Mannan et al., Biochemistry 58(28): 3116-3132 (2019)). However, direct cellular measurements of mAb-ePR non-specific transcytosis have not been reported, especially for a large number of proteins with a wide range of structural features and preclinical CL ind values.

[0230] To address this limitation, two distinct protein panels were generated and their rates of non-specific transcytosis quantified by obtaining ABC values at pH 7.4 and / or pH 5.8 in CHO-K1 cells. The first protein panel contained 48 mAb-ePRs produced on a stable, effector function-free Fc backbone patented to Amgen, Inc. (Liu et al., J. Biol. Chem. 295(5): 1876-1883 (2017)). These proteins had a variety of molecular forms that contained different targeted valencies, structural arrangements of binding moieties, and a wide range of CL ind values in Tg32 human FcRn transgenic mice after a single intravenous dose. Figure 22A ABC values were calculated as described in Figure 3. Using the same numerical thresholds and methods as outlined above and shown in Figure 21, mAb-ePRs were placed in a low-risk bin, a medium-risk bin, or a high-risk bin based on their ABCs. Consistent with what was observed for the clinical mAb panel, in Tg32 mice, 94% of mAb-ePRs placed in the high-risk bin (i.e., that exhibited high non-specific transcytosis quantified via their ABCs) and 92% of mAb-ePRs in the medium-risk group exhibited CL ind values higher than 0.5 mL / kg / h. Additionally, the high-risk group was able to successfully identify mAb-ePRs with the highest CL ind values. Figure 22B

[0231] The second panel of proteins consisted of five individual protein structures bivalently fused to a human Fc domain, which make a total of 28 different Fc-fusion proteins that bear no resemblance to human IgG or typical mAb-ePRs. The primary goal of this panel was to determine whether non-specific transcytosis could be used to describe CL ind values for any protein (not just proteins built on IgG or corresponding scaffolds). The Fc-fusion panel exhibited a range of CL ind values after a single intravenous dose at 2 mg / kg in C57BL / 6 wild-type mice. Figure 23A ​CHO-K1 ABC values ​​were obtained at both pH 5.8 and pH 7.4, and these values ​​were then compared with their CL values. ind Drawing ( Figure 23B and Figure 23C pH 7.4 was used to directly measure nonspecific endocytosis in mammalian cells. A pH 5.8 condition simulated the endosomal environment to quantify the positively charged and nonspecific pH-dependent changes expected to occur during intracellular transport due to endosome acidification, as discussed above. Using the same ABC cutoff values ​​for mAb and mAb-ePR proteomes as described above, proteins were placed in low-risk, medium-risk, and high-risk groups based on the extent of nonspecific endocytosis (calculated using the ABC method). Ninety-four percent of the proteins in the high-risk chamber had a CL greater than 3 mL / kg / h. ind Similar to mAb-ePR, it successfully captured data with the highest CL. ind Proteins.

[0232] The use of endosome-nonspecific pH 5.8 as a substitute measure was evaluated in identifying high-risk CL. ind Usefulness in proteins. All proteins exhibiting high nonspecific endocytosis at both pH 5.8 and pH 7.4 showed high CL. ind ( Figure 23B , Figure 23C Then, we filtered proteins by only examining the pH 5.8 ABC value of Fc-fusion proteins that had low or medium risk ABC at pH 7.4. Figure 23E Of these, three proteins showed a significant increase in non-specific behavior at lower pH, with two of them exhibiting very high CL values ​​in wild-type mice. ind ( Figure 23F This means that pH 5.8 conditions can be used to further identify increased Cl-. ind Treatment modalities that increase risk.

[0233] Results from mAb-ePR and Fc-fusion proteins demonstrate that high nonspecific endocytosis provides a pathway to high CL. ind Strongly correlated in vitro measurements. The data presented in this paper further demonstrate the ability of the described cell-based endocytosis assay to identify excessive CL in rodents. ind The ability to quantify structurally unrelated proteins. Furthermore, the described method provides a unique and novel ability to quantify pH-dependent changes in the nonspecific behavior of biopharmaceuticals. However, assessment of nonspecific endocytosis alone is insufficient to successfully identify proteins with elevated CL. ind All compounds. This was observed in the low-risk and medium-risk groups, which contained CL levels that are elevated in rodents.ind of several constructs. As detailed above, there are additional factors driving mAb, mAb-ePR, and any other biologic modalities ind of FcRn interaction. Therefore, the combination of cell-based readouts for non-specific endocytosis, pH-dependent changes of non-specific / charge, and engagement with FcRn greatly increases the ability to identify high-risk biologies that interact with FcRn and guide protein engineering efforts to improve the PK attributes of preclinical candidates.

[0234] Each of the references cited herein is hereby incorporated by reference in its entirety and for all purposes to the extent that it teaches what is not inconsistent with the present disclosure.

[0235] The present application is not to be limited in scope by the specific embodiments described herein, which are intended as single illustrations of individual embodiments of the application. Functionally equivalent methods and components are within the scope of the present application. Indeed, various modifications of the application, in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

Claims

1. A method of predicting in vivo non-target mediated clearance of a biomolecule, the method comprising: providing a cell preparation, wherein the cells of the preparation do not express a target of the biomolecule; incubating the cell preparation with a medium containing the biomolecule under conditions that mimic in vivo physiological conditions; determining the amount of the biomolecule taken up by the cells of the preparation following the incubation; and predicting in vivo non-target mediated clearance of the biomolecule based on the determining.

2. The method of claim 1, wherein the medium has a pH of about 7.0 to about 8.0 during the incubation.

3. The method of claim 1, wherein the medium has a pH of about 5.6 to about 7.0 during the incubation.

4. The method of claim 1, wherein the incubation is performed at 37 °C for about 60 minutes.

5. The method of claim 1, wherein the determining comprises: permeabilizing the cells of the preparation following the incubation; detecting the amount of biomolecule taken up by the cells of the preparation; and quantifying the amount of biomolecule taken up by the cells of the preparation based on the detecting, wherein predicting the in vivo non-target mediated clearance of the biomolecule is based on the quantifying.

6. The method of claim 5, further comprising: incubating the permeabilized cells with a detectable moiety that binds to the biomolecule following the permeabilization, wherein the amount of detectable moiety bound to the biomolecule is detected.

7. The method of claim 5, wherein the quantifying comprises: comparing the amount of biomolecule taken up by the cells detected to one or more quantitative reference values.

8. The method of claim 7, wherein the one or more quantitative reference values comprise a calibration curve of biomolecule binding capacity.

9. The method of claim 6, wherein the detectable moiety is an anti-Fc antibody coupled to a label.

10. The method of claim 5, wherein the detecting is performed using flow cytometry, immunoassay, or microscopy.

11. The method of claim 1, wherein the biomolecule is a human antibody, an epitope binding fragment of a human antibody, or a human antibody derivative.

12. The method of claim 1, wherein the biomolecule is a multispecific antibody.

13. The method of claim 1, wherein the biomolecule is a recombinant protein or a fusion protein.

14. The method of claim 1, wherein the cells of the preparation do not express an Fc receptor.

15. The method of claim 1, wherein the cells of the preparation do not express a human neonatal Fc receptor (hFcRn) and human β2m (hβ2m) complex.

16. The method of claim 1, wherein the cell preparation is a preparation of cells selected from the group consisting of Chinese hamster ovary (CHO) cells, Madin-Darby canine kidney (MDCK) cells, Vero cells, HUVEC, HEK293 cells, and primary endothelial cells.

17. A method of predicting in vivo non-target mediated clearance of an FcRn interacting molecule, the method comprising: providing a cell preparation, wherein the cells of the preparation do not express a target of the biomolecule; incubating the cell preparation with a medium containing the biomolecule under conditions that mimic in vivo physiological conditions; determining the amount of the biomolecule taken up by the cells of the preparation following the incubation; and predicting in vivo non-target mediated clearance of the biomolecule based on the determining. providing a first cell preparation, wherein the cells of the first preparation do not express hFcRn; providing a second cell preparation, wherein the cells of the second preparation express a heterodimer of human neonatal Fc receptor (hFcRn) and human β2m (hβ2m), and wherein the cells of the first and second preparations do not express a binding target of the FcRn interacting molecule; subjecting the first and second cell preparations to a first incubation period and a second incubation period, wherein the first incubation period comprises incubating the cell preparations with media containing the FcRn interacting molecule under acidic and / or non-acidic conditions, and wherein the second incubation period comprises incubating the cell preparations with media lacking the FcRn interacting molecule under non-acidic conditions following the first incubation; determining the amount of the FcRn interacting molecule taken up by the cells of the first and second preparations following the first and / or second incubation period; measuring the amount of the FcRn interacting molecule in the media from the second incubation period collected at the end of the second incubation period; quantifying non-specific pinocytosis and FcRn recycling of the FcRn interacting molecule based on the determining and the measuring, respectively; and predicting in vivo non-target mediated clearance of the FcRn interacting molecule based on the quantifying.

18. The method of claim 17, wherein the first incubation comprises incubating the first and second cell preparations with media containing the FcRn interacting molecule under acidic and non-acidic conditions.

19. The method of claim 17, wherein the first incubation comprises incubating the first and second cell preparations with media containing the FcRn interacting molecule under acidic or non-acidic conditions.

20. The method of any one of claims 17-19, wherein the non-acidic conditions comprise a media pH of 7.0 to 8.

0.

21. The method of any one of claims 17-19, wherein the acidic conditions comprise a media pH of 5.6 to 6.

9.

22. The method of claim 17, wherein the first incubation period comprises incubating the first and second cell preparations with media containing the FcRn interacting molecule at 37°C for about 60 to about 120 minutes.

23. The method of claim 17, wherein the first incubation period comprises incubating the first and second cell preparations with media comprising a concentration of FcRn interacting molecule that does not saturate FcRn binding occupancy of the cells of the second cell preparation.

24. The method of claim 23, wherein the concentration of FcRn interacting molecule in the media achieves between 25%-90% FcRn binding occupancy of the cells of the second cell preparation.

25. The method of claim 17, wherein the second incubation period comprises incubating the first cell preparation and the second cell preparation with media lacking the FcRn- interacting molecule at 37 °C for about 3 hours to about 5 hours.

26. The method of claim 17, wherein the determining comprises: permeabilizing cells of the first cell preparation and the second cell preparation after the first incubation period and / or the second incubation period; detecting an amount of FcRn-interacting molecule taken up by cells of each preparation; and quantifying the amount of FcRn-interacting molecule taken up by cells of each preparation based on the detecting, wherein the predicting the in vivo non-target mediated clearance of the FcRn-interacting molecule is based on the quantifying.

27. The method of claim 26, further comprising: after the permeabilizing, contacting the permeabilized cells with a detectable moiety that binds the FcRn-interacting molecule, wherein an amount of detectable moiety bound to the FcRn-interacting molecule is detected.

28. The method of claim 26, wherein the quantifying comprises: comparing the detected amount of FcRn-interacting molecule taken up by the cells to one or more quantitative reference values.

29. The method of claim 28, wherein the one or more quantitative reference values comprise a calibration curve of FcRn-interacting molecule binding capacity.

30. The method of claim 27, wherein the detectable moiety is an anti-Fc antibody coupled to a label.

31. The method of claim 26, wherein the detecting is performed using flow cytometry, immunoassay, or microscopy.

32. The method of claim 17, wherein the FcRn-interacting molecule comprises a fragment crystallizable (Fc) region.

33. The method of claim 32, wherein the Fc region of the FcRn-interacting molecule has been engineered for enhanced binding to FcRn relative to a corresponding non-engineered Fc region.

34. The method of claim 32, wherein the FcRn-interacting molecule comprises a human antibody, an antigen-binding fragment of a human antibody comprising an Fc region, or a human antibody derivative comprising an Fc region.

35. The method of claim 17, wherein the FcRn-interacting molecule is an Fc fusion protein or an albumin fusion protein.

36. The method of claim 17, wherein the first cell preparation and the second cell preparation are selected from the group consisting of Chinese hamster ovary (CHO) cells, Madin-Darby canine kidney (MDCK) cells, Vero cells, HUVECs, HEK293 cells, and primary endothelial cells.

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