Cellular assays for quantifying target-independent clearance of therapeutic molecules

Abstract

This disclosure relates to in vitro cell-based methods useful for predicting the in vivo pharmacokinetic properties of candidate therapeutic proteins. In particular, the cell-based assays described herein are useful for predicting the in vivo non-target-dependent clearance and subcutaneous bioavailability of candidate therapeutic proteins.

Description

[Technical Field]

[0001] The interests under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. 63 / 523,868, filed on 28 June 2023, and U.S. Provisional Patent Application No. 63 / 547,184, filed on 3 November 2023, are asserted herein, and their disclosures are incorporated herein by reference.

[0002] This disclosure relates to a cell-based method for predicting in vivo target-independent clearance of biomolecules.

[0003] Integration by referencing electronically submitted documents The nucleotide / amino acid sequence listing submitted concurrently with this specification is incorporated herein by reference in its entirety and identified as follows: an 8-kilobyte XML document named "10587-WO01-SEC_SequenceListing.xml" created on 27 June 2024. [Background technology]

[0004] The pharmacokinetics (PK) of monoclonal antibodies (mAbs) and multispecific antibodies are determined by both target-dependent clearance (CL) and target-independent clearance (CL) pathways. ind ) is a first-order linear dynamics (i.e., (dA / dt) / [drug]=CL ind It is mathematically described by ). Mechanistically, CL indThe rate of nonspecific endocytosis arises from the interaction of at least two competing processes, with nonspecific endocytosis being balanced by intracellular salvage / recycling via neonatal Fc receptors (FcRn) that return the drug to the systemic circulation (Ovacik, M. and Lin, K. (2018) Tutorial on Monoclonal Antibody Pharmacokinetics and Its Considerations in Early Development. Clin Transl Sci 11, 540-552; Ryman, JT, 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, GML Target-Driven Pharmacokinetics of Biotherapeutics. in Pharmaceutical Sciences). (Encyclopedia.pp1-12). Nonspecific endocytosis of therapeutic proteins can be driven by fluid-phase uptake and nonspecific adsorption internalization. Fluid-phase uptake is a mechanism in which solutes are internalized together with extracellular fluid to a degree directly proportional to the solute concentration (Steinman, et al. (1983) Endocytosis and the recycling 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 J210,1-13). Non-specific adsorptive endocytosis occurs when proteins interact non-specifically with the cell membrane, for example, due to charge-based attraction, and leads to their internalization. If the fluid phase is constitutive, and non-specific adsorption is protein-dependent and can be influenced by factors including local charge, then it is likely to be cell-type specific (Lloyd and Williams (1984) Non-specific adsorptive pinocytosis. Biochem. Soc. Trans 12(3):527-28). FcRn is the alpha chain of a non-covalent heterodimer complex with β2-microglobulin (β2m) that is mainly localized 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 115, 2389-2397; Simister, NE, and Mostov, KE (1989) An Fc receptor structurally related to MHC class I antigens. 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 62, 93-105; Roberts, et al. (1990) Isolation and characterization of the Fc receptor from the fetal yolk sac of the rat.J Cell Biol111,1867-1876;Dickinson,et al.(1999) Bidirectional FcRn-dependent IgG transport in a polarized human intestinal epithelial cell line.J Clin Invest104,903-911;D'Hooghe,et al.(2017)Cell surface dynamics and cellular distribution of endogenous FcRn.PLoS One 12,e0182695). FcRn shows increased affinity for its endogenous ligands, albumin and immunoglobulin G (IgG), at acidic pH levels (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 3, 610; Ober, et al. (2001) Differences in promiscuity for antibody-FcRn interactions across species: applications for therapeutic antibodies.Int Immunol13,1551-1559;Martin,et al. (2001)Crystal structure at 2.8 A of an FcRn / heterodimeric Fc complex: mechanism of pH-dependent binding. Mol Cell 7, 867-877), therefore, these proteins are selectively retained by intracellular transport toward the plasma membrane away from lysosomal degradation, promoting active recycling (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 USA101,11076-11081;Kim,et al. (2004)Net absorption of IgG via FcRn-mediated transcytosis across rat alveolar epithelial cell monolayers.Am J Physiol Lung Cell Mol Physiol287,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 Chem292,13312-13322;Bern, et al.(2020) An engineered human albumin enhances half-life and transmucosal delivery when fused to protein-based biologics. Sci Transl Med12; Strohl, WR (2015) Fusion Proteins for Half-Life Extension of Biologics as a Strategy to Make Biobetters. BioDrugs29, 215-239). This salvage mechanism extends the serum half-life of FcRn ligands compared to similarly sized proteins that do not possess FcRn binding ability (Chaudhury, et al. (2003) The major histocompatibility complex-related Fc receptor for IgG(FcRn) binds albumin and prolongs its lifespan. J Exp Med197, 315-322). In contrast, the target-mediated CL process is nonlinear and may dominate overall mAb CL depending on the 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, JT, 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, GML Target-Driven Pharmacokinetics of Biotherapeutics).(in Pharmaceutical Sciences Encyclopedia, pp. 1-12; and Peletier, LA, and Gabrielsson, J. (2012) Dynamics of target-mediated drug disposition: characteristic profiles and parameter identification. J Pharmacokinet Pharmacodyn 39, 429-451). Target-mediated drug delivery (TMDD) is driven by high-affinity mAb-target binding on the cell surface and 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 Sci11,540-552;Ryman,JT,and Meibohm,B.(2017)Pharmacokinetics of Monoclonal Antibodies.CPT Pharmacometrics Syst Pharmacol6,576-588;Huisinga,et al., Target-Driven Pharmacokinetics of Biotherapeutics.in Pharmaceutical Sciences Encyclopedia.pp 1-15, and Meno-Tetang, GMLTarget-Driven Pharmacokinetics of Biotherapeutics.in Pharmaceutical Sciences Encyclopedia.pp 1-12; and Peletier, LA, and Gabrielsson, J. (2012) Dynamics of target-mediated drug disposition:characteristic profiles and parameter identification.J Pharmacokinet Pharmacodyn 39, 429-451).

[0005] mAb CL ind Variations in FcRn binding can occur for several reasons, even if the target specificity overlaps. For example, altered FcRn binding due to differences in the physicochemical properties of the variable region or other nonspecific endocytosis mechanisms can broadly affect the PK profile of candidate therapeutics (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). Therefore, a key goal in the field of large molecule PK is the ability to predict in vivo PK parameters of early preclinical mAbs by in vitro experimental measures to define in vitro-to-in vivo correlations. indRecent methodologies for classifying these interactions encompass a large number of biophysical interaction assays, including both specific and nonspecific binding. For example, proxies of nonspecific endocytosis, including heparin chromatography and baculovirus particle binding, are commonly used (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 USA 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).The evaluation of Fc-FcRn interactions is typically achieved 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, DE, and Bjorkman, PJ (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). Inferences drawn from the application of these techniques suggest that both nonspecific uptake and FcRn affinity are related to mAb CL. ind It affects CL ind This should be taken into consideration in order to make predictions successful (Grevys, et al. (2022) Antibody variable sequences have a pronounced effect on cellular transport and plasma half-life. iScience 25, 103746).

[0006] FcRn function can also be directly investigated using cells that endogenously or ectopically express human FcRn (hFcRn) and human β2m (hβ2m), and several reports highlight the usefulness of cell-based platforms for testing FcRn transport dynamics 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 Biol111,1867-1876;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 Immunol172,2021-2029;Ober,et al. (2004)Exocytosis of IgG as mediated by the receptor,FcRn:an analysis at the single-molecule level.Proc Natl Acad Sci USA101,11076-11081;Kim,et al. (2004)Net absorption of IgG via FcRn-mediated transcytosis across rat alveolar epithelial cell monolayers.Am J Physiol Lung Cell Mol Physiol287,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 Chem292,13312-13322;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 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 185,673-684;Tesar,et al. (2006)Ligand valency affects transcytosis,recycling and intracellular trafficking mediated by the neonatal Fc receptor.Traffic 7,1127-1142;Claypool,et al.(2004)Bidirectional transepithelial IgG transport by a strongly polarized basolateral membrane Fcgamma-receptor.Mol Biol Cell 15,1746-1759;Goebl,et al.(2008)Neonatal Fc receptor mediates internalization of Fc in transfected human endothelial cells.Mol Biol 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 USA 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 9,781-791; Grevys, et al. (2018) A human endothelial cell-based recycling assay for screening of FcRn targeted molecules. Nat Commun 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 5,832). Using refined in vitro experimental strategies, CL of mAbs. indThis can be evaluated, and several published studies currently demonstrate the advantages of cell-based approaches to provide further biological context not gathered 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 IgG1 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 subsequent intracellular trafficking that expand mechanistic understanding of the processes driving cellular internalization, endosomal FcRn binding, and CL of mAbs. ind This includes direct measurements of subsequent intracellular trafficking that expand mechanistic understanding of the processes driving cellular internalization, endosomal FcRn binding, and CL of mAbs. (Chung, et al. (2019) An in vitro FcRn-dependent transcytosis assay as a screening tool for predictive asses 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 capabilities remain insufficient. Biophysical assays alone, with their limited scope for identifying physicochemical determinants of mAb PKs, often fail to capture the biological framework and are frequently based on subjective assay cutpoints. Combining readouts from multiple techniques can provide more robust interpretations 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 integrate multiple biological processes and physicochemical aspects into endpoints that can facilitate a deeper understanding of mAb PK behavior. However, limitations still exist. For example, one study demonstrated a strong correlation between FcRn-mediated transcytosis and mAb CL, thereby showing that higher transcytosis indicated elevated CL in vivo. The underlying mechanistic basis for this trend remains uninvestigated, and the authors emphasized that this assay cannot successfully infer the clearance of manipulated FcRn-affinity-enhanced mAbs (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). Another study demonstrated that the degree of FcRn recycling is highly dependent on the amount of mAb internalized by specific or nonspecific processes, and that the charge profile of the Fv region is a significant 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, this test was limited in scope by the number of compounds tested.

[0008] This disclosure aims to overcome this problem and other deficiencies in the art. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Ovacik, M., and Lin, K. (2018) Tutorial on Monoclonal Antibody Pharmacokinetics and Its Considerations in Early Development. Clin Transl Sci 11,540-552 [Non-Patent Document 2] Ryman, JT, and Meibohm, B. (2017) Pharmacokinetics of Monoclonal Antibodies. CPT Pharmacometrics Syst Pharmacol 6,576-588 [Non-Patent Document 3] Huisinga,et al.,Target-Driven Pharmacokinetics of Biotherapeutics.in Pharmaceutical Sciences Encyclopedia.pp 1-15 [Non-Patent Document 4] Meno-Tetang,GMLTarget-Driven Pharmacokinetics of Biotherapeutics.in Pharmaceutical Sciences Encyclopedia.pp 1-12 [Non-Patent Document 5] Steinman, et al. (1983) Endocytosis and the recycling of plasma membrane. J Cell Biol 96,1-27 [Non-Patent Document 6] Besterman, JM, and Low, RB (1983) Endocytosis: a review of mechanisms and plasma membrane dynamics.Biochem J 210,1-13 [Non-Patent Document 7] Lloyd and Williams(1984)Non-specific adsorptive pinocytosis.Biochem.Soc.Trans.12(3):527-28 [Non-Patent Document 8] 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 [Non-Patent Document 9] Simister, NE, and Mostov, KE (1989) An Fc receptor structurally related to MHC class I antigens. Nature 337, 184-187 [Non-Patent Document 10] 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 [Non-Patent Document 11] 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 [Non-Patent Document 12] Dickinson, et al. (1999) Bidirectional FcRn-dependent IgG transport in a polarized human intestinal epithelial cell line.J Clin Invest 104,903-911 [Non-Patent Document 13] D'Hooghe,et al.(2017)Cell surface dynamics and cellular distribution of endogenous FcRn.PLoS One 12,e0182695 [Non-Patent Document 14] 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 [Non-Patent Document 15] 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 [Non-Patent Document 16] Andersen,et al.(2012)Structure-based mutagenesis reveals the albumin-binding site of the neonatal Fc receptor.Nat Commun 3,610 [Non-Patent Document 17] Ober,et al.(2001)Differences in promiscuity for antibody-FcRn interactions across species:implications for therapeutic antibodies.Int Immunol 13,1551-1559 [Non-Patent Document 18] Martin,et al.(2001)Crystal structure at 2.8 A of an FcRn / heterodimeric Fc complex:mechanism of pH-dependent binding.Mol Cell 7,867-877 [Non-Patent Document 19] 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 [Non-Patent Document 20] Ober,et al.(2004)Exocytosis of IgG as mediated by the receptor,FcRn:an analysis at the single-molecule level.Proc Natl Acad Sci USA 101,11076-11081 [Non-Patent Document 21] 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 [Non-Patent Document 22] 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 [Non-Patent Document 23] Bern,et al.(2020)An engineered human albumin enhances half-life and transmucosal delivery when fused to protein-based biologics.Sci Transl Med 12 [Non-Patent Document 24] Strohl, WR (2015) Fusion Proteins for Half-Life Extension of Biologics as a Strategy to Make Biobetters.BioDrugs 29,215-239 [Non-Patent Document 25] Peletier, LA, and Gabrielsson, J. (2012) Dynamics of target-mediated drug disposition: characteristic profiles and parameter identification. J Pharmacokinet Pharmacodyn 39,429-451 [Non-Patent Document 26] Kelly,et al.(2016)Target-independent variable region mediated effects on antibody clearance can be FcRn independent.MAbs 8,1269-1275 [Non-Patent Document 27] 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

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[0010] A first aspect of this disclosure relates to in vivo non-target-mediated clearance (CL) of biomolecules. indThe present invention relates to a method for predicting in vivo non-target-mediated clearance of biomolecules. The method comprises providing a cell preparation in which the cells of the preparation do not express the target of a biomolecule. The method further comprises incubating the cell preparation in a culture medium containing biomolecules under conditions that mimic physiological conditions in vivo, and determining the amount of biomolecules taken up by the cells of the preparation after incubation. The method further comprises predicting in vivo non-target-mediated clearance of biomolecules based on the said determination.

[0011] Another aspect of the present disclosure relates to a method for predicting in vivo non-target-mediated clearance of FcRn interacting molecules. The method includes providing a first cell preparation in which the cells of the first preparation do not express human neonatal Fc receptor (hFcRn), and providing a second cell preparation in which the cells of the second preparation express a heterodimer of hFcRn and human β-2-microglobulin (hβ2m). The cells of the first and second cell preparations do not express targets of FcRn interacting molecules. The method further includes subjecting the first and second cell preparations to first and second incubation periods, the first incubation period comprising incubating the cell preparations with a medium containing FcRn interacting molecules under acidic and / or non-acidic conditions. The second incubation period comprises incubating the cell preparations after the first incubation with a medium lacking FcRn interacting molecules under non-acidic conditions. The method further includes determining the amount of FcRn interacting molecules taken up by cells in the first and second preparations after the first incubation period and / or second incubation period, and measuring the amount of FcRn interacting molecules in the culture medium after the second incubation period. The method further includes quantifying nonspecific endocytosis and FcRn recycling of FcRn interacting molecules based on the determination and measurement steps. In vivo nontarget-mediated clearance of FcRn interacting molecules is predicted based on the quantification step.

[0012] Another aspect of this disclosure relates to a method for predicting the subcutaneous bioavailability of a biomolecule based on the degree of nonspecific endocytosis of the biomolecule predicted based on the method disclosed herein.

[0013] The in vivo clearance mechanisms of biomolecules encompass both target-mediated and target-independent processes. Two distinct determinants of biomolecular clearance, separate from target-mediated influences, are pH-dependent recycling by FcRn and nonspecific endocytosis, each exhibiting intermolecular differences in rate and degree. While approaches to quantify these dynamics have demonstrated strong utility in establishing in vitro-in vivo correlations, they are limited mechanistically or by the number of analytes.

[0014] Several biophysical techniques, including baculovirus particle conjugation and heparin chromatography, are typically used to test the potential for biomolecules such as mAbs to undergo nonspecific endocytosis. However, since these methods do not provide insight into specific rates of cellular turnover, a reproducible and robust mammalian cell-based method for quantifying biomolecular nonspecific endocytosis is described herein. Data generated by this cell-based method can be used to identify total pharmacokinetic (PK) responsibility during preclinical drug development and to inform mechanistic PK models for human translation.

[0015] Functional cell-based FcRn recycling assays using mammalian cells are also disclosed herein. A series of pH-dependent internalization assays using model antibodies confirmed the proper function of the human FcRn complex in this assay. Furthermore, nonspecific endocytosis was observed to be the primary endocytosis pathway of the tested antibodies in the absence of the receptor. These cell assays were applied to evaluate FcRn and nonspecific interactions in a set of clinical antibodies, multispecific antibodies, and Fc fusion proteins with a range of PK behaviors. The results demonstrate that nonspecific endocytosis rates, pH-dependent nonspecific interactions, and engagement with FcRn all contribute to the overall recycling efficiency of these therapeutic molecules.

[0016] To highlight the predicted volume of the assay described herein, in humans exceeding 5 mL / kg / day, CL ind All antibodies possessing this characteristic were successfully identified by this assay. These results indicate that the combination of cell assays provides overall in vivo recycling efficiency and CL of biomolecules. ind This demonstrates that it is possible to identify the individual mechanisms underlying these processes. [Brief explanation of the drawing]

[0017] [Figure 1A] Figures 1A and 1C show the serum concentration-time profiles of ASA and anti-IL-4Rα mAb in wild-type mice. Data were obtained from separate studies using different intravenous bolus doses for ASA (3 mg / kg) (Figure 1A) and anti-IL-4Rα mAb (1 mg / kg) (Figure 1B). Figure 1C shows the predicted values ​​for Figures 1A and 1B obtained by computational fitting using a two-compartment pharmacokinetic model with linear exclusion from the central compartment (CL) and distribution between the vascular and non-vascular compartments (CLD). Parameter estimates are reported in Table 1: C1 and V1, serum concentration and volume of the central compartment; C2 and V2, serum concentration and volume of the non-vascular compartment; ASA, anti-streptavidin antibody. [Figure 1B]Figures 1A and 1C show the serum concentration-time profiles of ASA and anti-IL-4Rα mAb in wild-type mice. Data were obtained from separate studies using different intravenous bolus doses for ASA (3 mg / kg) (Figure 1A) and anti-IL-4Rα mAb (1 mg / kg) (Figure 1B). Figure 1C shows the predicted values ​​for Figures 1A and 1B obtained by computational fitting using a two-compartment pharmacokinetic model with linear exclusion from the central compartment (CL) and distribution between the vascular and non-vascular compartments (CLD). Parameter estimates are reported in Table 1: C1 and V1, serum concentration and volume of the central compartment; C2 and V2, serum concentration and volume of the non-vascular compartment; ASA, anti-streptavidin antibody. [Figure 1C] Figures 1A and 1C show the serum concentration-time profiles of ASA and anti-IL-4Rα mAb in wild-type mice. Data were obtained from separate studies using different intravenous bolus doses for ASA (3 mg / kg) (Figure 1A) and anti-IL-4Rα mAb (1 mg / kg) (Figure 1B). Figure 1C shows the predicted values ​​for Figures 1A and 1B obtained by computational fitting using a two-compartment pharmacokinetic model with linear exclusion from the central compartment (CL) and distribution between the vascular and non-vascular compartments (CLD). Parameter estimates are reported in Table 1: C1 and V1, serum concentration and volume of the central compartment; C2 and V2, serum concentration and volume of the non-vascular compartment; ASA, anti-streptavidin antibody. [Figure 2A]Figures 2A–2D show the internalization dynamics of anti-IL-4Rα mAb and ASA in CHO-K1 cells by flow cytometry and high-concentration confocal microscopy. After uptake studies, cells were washed, trypsinized, stained with live / dead dyes, fixed, permeabilized, stained with Alexa Fluor 647-conjugated anti-human Fc mouse IgG, washed, and analyzed by flow cytometry. A representative gating strategy is shown in Figure 2A. For each sample, the median fluorescence intensity of a single live-cell event was obtained. CHO-K1 cells were incubated with 100 μg / mL of anti-IL-4Rα mAb or ASA at 37°C or 4°C for escalation periods. Biphasic, time-dependent (Figure 2B) and linear, concentration-dependent (Figure 2C) internalization was observed for anti-IL-4Rα mAb, but only under 37°C conditions. In contrast, ASA endocytosis was negligible at 37°C. Compared to the untreated control, minimal cell surface binding was detected for anti-IL-4Rα mAb and ASA at 4°C. Figure 2D is a panel of confocal microscopy images of CHO-K1 cells after incubation and similar post-experimental treatment using anti-IL-4Rα mAb (Figure 2D, right image) or ASA (Figure 2D, center image). Untreated cells are shown in the left panel of Figure 2D. Cells were stained with CellMask Blue and Hoechst to visualize whole cells (cytoplasm and nucleus) and fluorescently labeled anti-Fc antibody (red). These images demonstrate the usefulness of confocal microscopy as an alternative to flow cytometry for high-throughput quantification of therapeutic protein endogenization dynamics. ASA, anti-streptavidin antibody; CHO-K1, Chinese hamster ovary cells. [Figure 2B]Figures 2A–2D show the internalization dynamics of anti-IL-4Rα mAb and ASA in CHO-K1 cells by flow cytometry and high-concentration confocal microscopy. After uptake studies, cells were washed, trypsinized, stained with live / dead dyes, fixed, permeabilized, stained with Alexa Fluor 647-conjugated anti-human Fc mouse IgG, washed, and analyzed by flow cytometry. A representative gating strategy is shown in Figure 2A. For each sample, the median fluorescence intensity of a single live-cell event was obtained. CHO-K1 cells were incubated with 100 μg / mL of anti-IL-4Rα mAb or ASA at 37°C or 4°C for escalation periods. Biphasic, time-dependent (Figure 2B) and linear, concentration-dependent (Figure 2C) internalization was observed for anti-IL-4Rα mAb, but only under 37°C conditions. In contrast, ASA endocytosis was negligible at 37°C. Compared to the untreated control, minimal cell surface binding was detected for anti-IL-4Rα mAb and ASA at 4°C. Figure 2D is a panel of confocal microscopy images of CHO-K1 cells after incubation and similar post-experimental treatment using anti-IL-4Rα mAb (Figure 2D, right image) or ASA (Figure 2D, center image). Untreated cells are shown in the left panel of Figure 2D. Cells were stained with CellMask Blue and Hoechst to visualize whole cells (cytoplasm and nucleus) and fluorescently labeled anti-Fc antibody (red). These images demonstrate the usefulness of confocal microscopy as an alternative to flow cytometry for high-throughput quantification of therapeutic protein endogenization dynamics. ASA, anti-streptavidin antibody; CHO-K1, Chinese hamster ovary cells. [Figure 2C]Figures 2A–2D show the internalization dynamics of anti-IL-4Rα mAb and ASA in CHO-K1 cells by flow cytometry and high-concentration confocal microscopy. After uptake studies, cells were washed, trypsinized, stained with live / dead dyes, fixed, permeabilized, stained with Alexa Fluor 647-conjugated anti-human Fc mouse IgG, washed, and analyzed by flow cytometry. A representative gating strategy is shown in Figure 2A. For each sample, the median fluorescence intensity of a single live-cell event was obtained. CHO-K1 cells were incubated with 100 μg / mL of anti-IL-4Rα mAb or ASA at 37°C or 4°C for escalation periods. Biphasic, time-dependent (Figure 2B) and linear, concentration-dependent (Figure 2C) internalization was observed for anti-IL-4Rα mAb, but only under 37°C conditions. In contrast, ASA endocytosis was negligible at 37°C. Compared to the untreated control, minimal cell surface binding was detected for anti-IL-4Rα mAb and ASA at 4°C. Figure 2D is a panel of confocal microscopy images of CHO-K1 cells after incubation and similar post-experimental treatment using anti-IL-4Rα mAb (Figure 2D, right image) or ASA (Figure 2D, center image). Untreated cells are shown in the left panel of Figure 2D. Cells were stained with CellMask Blue and Hoechst to visualize whole cells (cytoplasm and nucleus) and fluorescently labeled anti-Fc antibody (red). These images demonstrate the usefulness of confocal microscopy as an alternative to flow cytometry for high-throughput quantification of therapeutic protein endogenization dynamics. ASA, anti-streptavidin antibody; CHO-K1, Chinese hamster ovary cells. [Figure 2D]Figures 2A–2D show the internalization dynamics of anti-IL-4Rα mAb and ASA in CHO-K1 cells by flow cytometry and high-concentration confocal microscopy. After uptake studies, cells were washed, trypsinized, stained with live / dead dyes, fixed, permeabilized, stained with Alexa Fluor 647-conjugated anti-human Fc mouse IgG, washed, and analyzed by flow cytometry. A representative gating strategy is shown in Figure 2A. For each sample, the median fluorescence intensity of a single live-cell event was obtained. CHO-K1 cells were incubated with 100 μg / mL of anti-IL-4Rα mAb or ASA at 37°C or 4°C for escalation periods. Biphasic, time-dependent (Figure 2B) and linear, concentration-dependent (Figure 2C) internalization was observed for anti-IL-4Rα mAb, but only under 37°C conditions. In contrast, ASA endocytosis was negligible at 37°C. Compared to the untreated control, minimal cell surface binding was detected for anti-IL-4Rα mAb and ASA at 4°C. Figure 2D is a panel of confocal microscopy images of CHO-K1 cells after incubation and similar post-experimental treatment using anti-IL-4Rα mAb (Figure 2D, right image) or ASA (Figure 2D, center image). Untreated cells are shown in the left panel of Figure 2D. Cells were stained with CellMask Blue and Hoechst to visualize whole cells (cytoplasm and nucleus) and fluorescently labeled anti-Fc antibody (red). These images demonstrate the usefulness of confocal microscopy as an alternative to flow cytometry for high-throughput quantification of therapeutic protein endogenization dynamics. ASA, anti-streptavidin antibody; CHO-K1, Chinese hamster ovary cells. [Figure 3A]Figures 3A–3D provide an overview of the development of a quantitative endocytosis assay using flow cytometry. Figure 3A shows histograms of five bead populations superimposed when stained with 5 or 10 μg / mL anti-human Fc mouse IgG1 conjugated with Alexa Fluor 647. Also shown is a plot of median fluorescence intensity against antibody binding capacity (ABC), indicating that both staining conditions yielded linear curves with an r² value of 0.999. Figure 3B is a graph showing assay reproducibility, evaluated by performing uptake tests in CHO-K1 cells using 100 μg / mL anti-IL-4Rα mAb, or ASA incubated at 37°C for 60 minutes over several different experimental days. Mean anti-IL-4Rα mAb ABC: 40,839 (SD 6614, CV 16.2%); Mean ASA ABC: 921 (SD 315, CV 34.2%). N=20 per sample group. Figure 3C shows representative histograms of a single ASA and untreated sample with a gate incorporating 99% of the untreated population, corresponding to the "negative" signal. ASA endocytosis in the experimental setup was very low, with approximately 92% of ASA signals located within the "negative" gate. Figure 3D is an illustrative overview of the quantitative endocytosis methodology. ASA, anti-streptavidin antibody; IgG1, immunoglobulin G1; CHO-K1, Chinese hamster ovary cells; SD, standard deviation; CV, coefficient of variation. [Figure 3B]Figures 3A–3D provide an overview of the development of a quantitative endocytosis assay using flow cytometry. Figure 3A shows histograms of five bead populations superimposed when stained with 5 or 10 μg / mL anti-human Fc mouse IgG1 conjugated with Alexa Fluor 647. Also shown is a plot of median fluorescence intensity against antibody binding capacity (ABC), indicating that both staining conditions yielded linear curves with an r² value of 0.999. Figure 3B is a graph showing assay reproducibility, evaluated by performing uptake tests in CHO-K1 cells using 100 μg / mL anti-IL-4Rα mAb, or ASA incubated at 37°C for 60 minutes over several different experimental days. Mean anti-IL-4Rα mAb ABC: 40,839 (SD 6614, CV 16.2%); Mean ASA ABC: 921 (SD 315, CV 34.2%). N=20 per sample group. Figure 3C shows representative histograms of a single ASA and untreated sample with a gate incorporating 99% of the untreated population, corresponding to the "negative" signal. ASA endocytosis in the experimental setup was very low, with approximately 92% of ASA signals located within the "negative" gate. Figure 3D is an illustrative overview of the quantitative endocytosis methodology. ASA, anti-streptavidin antibody; IgG1, immunoglobulin G1; CHO-K1, Chinese hamster ovary cells; SD, standard deviation; CV, coefficient of variation. [Figure 3C]Figures 3A–3D provide an overview of the development of a quantitative endocytosis assay using flow cytometry. Figure 3A shows histograms of five bead populations superimposed when stained with 5 or 10 μg / mL anti-human Fc mouse IgG1 conjugated with Alexa Fluor 647. Also shown is a plot of median fluorescence intensity against antibody binding capacity (ABC), indicating that both staining conditions yielded linear curves with an r² value of 0.999. Figure 3B is a graph showing assay reproducibility, evaluated by performing uptake tests in CHO-K1 cells using 100 μg / mL anti-IL-4Rα mAb, or ASA incubated at 37°C for 60 minutes over several different experimental days. Mean anti-IL-4Rα mAb ABC: 40,839 (SD 6614, CV 16.2%); Mean ASA ABC: 921 (SD 315, CV 34.2%). N=20 per sample group. Figure 3C shows representative histograms of a single ASA and untreated sample with a gate incorporating 99% of the untreated population, corresponding to the "negative" signal. ASA endocytosis in the experimental setup was very low, with approximately 92% of ASA signals located within the "negative" gate. Figure 3D is an illustrative overview of the quantitative endocytosis methodology. ASA, anti-streptavidin antibody; IgG1, immunoglobulin G1; CHO-K1, Chinese hamster ovary cells; SD, standard deviation; CV, coefficient of variation. [Figure 3D]Figures 3A–3D provide an overview of the development of a quantitative endocytosis assay using flow cytometry. Figure 3A shows histograms of five bead populations superimposed when stained with 5 or 10 μg / mL anti-human Fc mouse IgG1 conjugated with Alexa Fluor 647. Also shown is a plot of median fluorescence intensity against antibody binding capacity (ABC), indicating that both staining conditions yielded linear curves with an r² value of 0.999. Figure 3B is a graph showing assay reproducibility, evaluated by performing uptake tests in CHO-K1 cells using 100 μg / mL anti-IL-4Rα mAb, or ASA incubated at 37°C for 60 minutes over several different experimental days. Mean anti-IL-4Rα mAb ABC: 40,839 (SD 6614, CV 16.2%); Mean ASA ABC: 921 (SD 315, CV 34.2%). N=20 per sample group. Figure 3C shows representative histograms of a single ASA and untreated sample with a gate incorporating 99% of the untreated population, corresponding to the "negative" signal. ASA endocytosis in the experimental setup was very low, with approximately 92% of ASA signals located within the "negative" gate. Figure 3D is an illustrative overview of the quantitative endocytosis methodology. ASA, anti-streptavidin antibody; IgG1, immunoglobulin G1; CHO-K1, Chinese hamster ovary cells; SD, standard deviation; CV, coefficient of variation. [Figure 4A]Figures 4A and 4C show the surface charge distribution on anti-IL-4Rα mAb (Figure 4A) and ASA (Figure 4B), demonstrating that nonspecific endocytosis can be reduced by removing exposed positive charges. Figures 4A and 4B include an Fv model ribbon representation (dark blue-green heavy chain, light blue-green light chain) showing heavy chain CDRs in orange (CDR1, CDR2) and red (CDR3). Light chain CDRs are shown in purple. Hydrophobic patches are shown in green. Negative and positive patches are shown in red and blue, respectively. The sites of targeted mutations are indicated. Anti-IL-4Rα mAb (Figure 4A) had more positive patches than ASA (Figure 4B), and had two positive patches on the heavy chain CDR. Various point mutations were performed on anti-IL-4Rα mAb CDRs and Fv to reduce the surface positive charge within the identified charge patches. The graph in Figure 4C shows the substantial reduction in nonspecific endocytosis measured for all anti-IL-4Rα mAb variants. CDR, complementarity-determining region; WT, wild-type anti-IL-4Rα mAb; LC, light chain; CDR H1, first complementarity-determining region of the heavy chain; CDR H3, third complementarity-determining region of the heavy chain. [Figure 4B]Figures 4A and 4C show the surface charge distribution on anti-IL-4Rα mAb (Figure 4A) and ASA (Figure 4B), demonstrating that nonspecific endocytosis can be reduced by removing exposed positive charges. Figures 4A and 4B include an Fv model ribbon representation (dark blue-green heavy chain, light blue-green light chain) showing heavy chain CDRs in orange (CDR1, CDR2) and red (CDR3). Light chain CDRs are shown in purple. Hydrophobic patches are shown in green. Negative and positive patches are shown in red and blue, respectively. The sites of targeted mutations are indicated. Anti-IL-4Rα mAb (Figure 4A) had more positive patches than ASA (Figure 4B), and had two positive patches on the heavy chain CDR. Various point mutations were performed on anti-IL-4Rα mAb CDRs and Fv to reduce the surface positive charge within the identified charge patches. The graph in Figure 4C shows the substantial reduction in nonspecific endocytosis measured for all anti-IL-4Rα mAb variants. CDR, complementarity-determining region; WT, wild-type anti-IL-4Rα mAb; LC, light chain; CDR H1, first complementarity-determining region of the heavy chain; CDR H3, third complementarity-determining region of the heavy chain. [Figure 4C]Figures 4A and 4C show the surface charge distribution on anti-IL-4Rα mAb (Figure 4A) and ASA (Figure 4B), demonstrating that nonspecific endocytosis can be reduced by removing exposed positive charges. Figures 4A and 4B include an Fv model ribbon representation (dark blue-green heavy chain, light blue-green light chain) showing heavy chain CDRs in orange (CDR1, CDR2) and red (CDR3). Light chain CDRs are shown in purple. Hydrophobic patches are shown in green. Negative and positive patches are shown in red and blue, respectively. The sites of targeted mutations are indicated. Anti-IL-4Rα mAb (Figure 4A) had more positive patches than ASA (Figure 4B), and had two positive patches on the heavy chain CDR. Various point mutations were performed on anti-IL-4Rα mAb CDRs and Fv to reduce the surface positive charge within the identified charge patches. The graph in Figure 4C shows the substantial reduction in nonspecific endocytosis measured for all anti-IL-4Rα mAb variants. CDR, complementarity-determining region; WT, wild-type anti-IL-4Rα mAb; LC, light chain; CDR H1, first complementarity-determining region of the heavy chain; CDR H3, third complementarity-determining region of the heavy chain. [Figure 5] Figure 5 is a panel of serum concentration-time profiles for a second set of preclinical antibodies, all binding to the same target (mAb B1-B5), after a single intravenous bolus administration in wild-type mice. The data were fitted to mean serum concentrations using a two-compartment computational model, as graphed in Figure 1. [Figure 6A]Figures 6A–6D demonstrate that nonspecific endocytosis is conserved across different species and cell types. Using the mAb panel from Figure 5, which contains various CLs in wild-type mice, uptake was performed in either CHO-K1 (Chinese hamster (Cricetulus griseus), ovarian epithelial-like cells; Figure 6A) or Vero cells (savanna monkey (Cercopithecus aethiops), renal epithelial cells; Figure 6B). Uptake ranges independent of pI were observed. These results also demonstrate the superior sensitivity of the endocytosis assay performed at 37°C compared to cell surface binding only (i.e., the 4°C group). Nonspecific endocytosis to either CHO-K1 or Vero cells strongly correlated with CL, as shown in Figure 6D, indicating that this attribute is a key CLind factor for these mAbs. Furthermore, the results for Vero cells and CHO-K1 cells strongly correspond to each other (Figure 6C), supporting nonspecific endocytosis as a mechanism of mAb internalization. All data are plotted as mean ± SD, with n=3-4 per group. CL, clearance; CHO-K1, Chinese hamster ovary cells; CLind, target-independent clearance. [Figure 6B]Figures 6A–6D demonstrate that nonspecific endocytosis is conserved across different species and cell types. Using the mAb panel from Figure 5, which contains various CLs in wild-type mice, uptake was performed in either CHO-K1 (Chinese hamster (Cricetulus griseus), ovarian epithelial-like cells; Figure 6A) or Vero cells (savanna monkey (Cercopithecus aethiops), renal epithelial cells; Figure 6B). Uptake ranges independent of pI were observed. These results also demonstrate the superior sensitivity of the endocytosis assay performed at 37°C compared to cell surface binding only (i.e., the 4°C group). Nonspecific endocytosis to either CHO-K1 or Vero cells strongly correlated with CL, as shown in Figure 6D, indicating that this attribute is a key CLind factor for these mAbs. Furthermore, the results for Vero cells and CHO-K1 cells strongly correspond to each other (Figure 6C), supporting nonspecific endocytosis as a mechanism of mAb internalization. All data are plotted as mean ± SD, with n=3-4 per group. CL, clearance; CHO-K1, Chinese hamster ovary cells; CLind, target-independent clearance. [Figure 6C]Figures 6A–6D demonstrate that nonspecific endocytosis is conserved across different species and cell types. Using the mAb panel from Figure 5, which contains various CLs in wild-type mice, uptake was performed in either CHO-K1 (Chinese hamster (Cricetulus griseus), ovarian epithelial-like cells; Figure 6A) or Vero cells (savanna monkey (Cercopithecus aethiops), renal epithelial cells; Figure 6B). Uptake ranges independent of pI were observed. These results also demonstrate the superior sensitivity of the endocytosis assay performed at 37°C compared to cell surface binding only (i.e., the 4°C group). Nonspecific endocytosis to either CHO-K1 or Vero cells strongly correlated with CL, as shown in Figure 6D, indicating that this attribute is a key CLind factor for these mAbs. Furthermore, the results for Vero cells and CHO-K1 cells strongly correspond to each other (Figure 6C), supporting nonspecific endocytosis as a mechanism of mAb internalization. All data are plotted as mean ± SD, with n=3-4 per group. CL, clearance; CHO-K1, Chinese hamster ovary cells; CLind, target-independent clearance. [Figure 6D]Figures 6A–6D demonstrate that nonspecific endocytosis is conserved across different species and cell types. Using the mAb panel from Figure 5, which contains various CLs in wild-type mice, uptake was performed in either CHO-K1 (Chinese hamster (Cricetulus griseus), ovarian epithelial-like cells; Figure 6A) or Vero cells (savanna monkey (Cercopithecus aethiops), renal epithelial cells; Figure 6B). Uptake ranges independent of pI were observed. These results also demonstrate the superior sensitivity of the endocytosis assay performed at 37°C compared to cell surface binding only (i.e., the 4°C group). Nonspecific endocytosis to either CHO-K1 or Vero cells strongly correlated with CL, as shown in Figure 6D, indicating that this attribute is a key CLind factor for these mAbs. Furthermore, the results for Vero cells and CHO-K1 cells strongly correspond to each other (Figure 6C), supporting nonspecific endocytosis as a mechanism of mAb internalization. All data are plotted as mean ± SD, with n=3-4 per group. CL, clearance; CHO-K1, Chinese hamster ovary cells; CLind, target-independent clearance. [Figure 7A]Figures 7A–7C show the co-expression of hFcRn-GFP and hβ2m in stably transfected MDCK II cells. Maximum projection of confocal images after anti-FcRn immunofluorescence staining confirmed hFcRn-GFP (green, yellow) expression in selected hFcRn-GFP / hβ2m MDCK II cells (Figure 7A). Cells were stained with rabbit anti-FcRn antibody using a goat anti-rabbit secondary antibody conjugated with Alexa Fluor 594 (yellow). Parental MDCK II cells showed positive nuclear staining only (Hoechst, blue). The upper (i) and lower (ii) images in Figure 7A show the presence and absence of the indicated GFP channel, respectively. Scale bar, 20 μm. Figure 7B shows co-staining of parental and hFcRn-GFP / hβ2m MDCK II cells with targeted anti-hFcRn and anti-hβ2m antibodies or corresponding isotype controls, demonstrating robust transgene co-expression in transfected and sorted hFcRn-GFP / hβ2m MDCK II cells. Cells were stained either with or without fixation / permeabilization (whole) or without (cell surface). The population of GFP-hFcRn-GFP / hβ2m MDCK II cells was consistently observed visually (as seen in Figure 7A) and by flow cytometry under maintained selective pressure. As determined by flow cytometry, it constituted less than 5% of the total population. Therefore, all subsequent analyses using fluorescence detection, including those shown in the current figure, were performed on GFP+hFcRn-GFP / hβ2m MDCK II cells. The median fluorescence intensities of hFcRn and hβ2m signals from hFcRn-GFP / hβ2m MDCK II cells in Figure 7B, as shown in the graph in Figure 7C, indicated that the majority of hFcRn-GFP and hβ2m were intracellular. 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. [Figure 7B]Figures 7A–7C show the co-expression of hFcRn-GFP and hβ2m in stably transfected MDCK II cells. Maximum projection of confocal images after anti-FcRn immunofluorescence staining confirmed hFcRn-GFP (green, yellow) expression in selected hFcRn-GFP / hβ2m MDCK II cells (Figure 7A). Cells were stained with rabbit anti-FcRn antibody using a goat anti-rabbit secondary antibody conjugated with Alexa Fluor 594 (yellow). Parental MDCK II cells showed positive nuclear staining only (Hoechst, blue). The upper (i) and lower (ii) images in Figure 7A show the presence and absence of the indicated GFP channel, respectively. Scale bar, 20 μm. Figure 7B shows co-staining of parental and hFcRn-GFP / hβ2m MDCK II cells with targeted anti-hFcRn and anti-hβ2m antibodies or corresponding isotype controls, demonstrating robust transgene co-expression in transfected and sorted hFcRn-GFP / hβ2m MDCK II cells. Cells were stained either with or without fixation / permeabilization (whole) or without (cell surface). The population of GFP-hFcRn-GFP / hβ2m MDCK II cells was consistently observed visually (as seen in Figure 7A) and by flow cytometry under maintained selective pressure. As determined by flow cytometry, it constituted less than 5% of the total population. Therefore, all subsequent analyses using fluorescence detection, including those shown in the current figure, were performed on GFP+hFcRn-GFP / hβ2m MDCK II cells. The median fluorescence intensities of hFcRn and hβ2m signals from hFcRn-GFP / hβ2m MDCK II cells in Figure 7B, as shown in the graph in Figure 7C, indicated that the majority of hFcRn-GFP and hβ2m were intracellular. 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. [Figure 7C]Figures 7A–7C show the co-expression of hFcRn-GFP and hβ2m in stably transfected MDCK II cells. Maximum projection of confocal images after anti-FcRn immunofluorescence staining confirmed hFcRn-GFP (green, yellow) expression in selected hFcRn-GFP / hβ2m MDCK II cells (Figure 7A). Cells were stained with rabbit anti-FcRn antibody using a goat anti-rabbit secondary antibody conjugated with Alexa Fluor 594 (yellow). Parental MDCK II cells showed positive nuclear staining only (Hoechst, blue). The upper (i) and lower (ii) images in Figure 7A show the presence and absence of the indicated GFP channel, respectively. Scale bar, 20 μm. Figure 7B shows co-staining of parental and hFcRn-GFP / hβ2m MDCK II cells with targeted anti-hFcRn and anti-hβ2m antibodies or corresponding isotype controls, demonstrating robust transgene co-expression in transfected and sorted hFcRn-GFP / hβ2m MDCK II cells. Cells were stained either with or without fixation / permeabilization (whole) or without (cell surface). The population of GFP-hFcRn-GFP / hβ2m MDCK II cells was consistently observed visually (as seen in Figure 7A) and by flow cytometry under maintained selective pressure. As determined by flow cytometry, it constituted less than 5% of the total population. Therefore, all subsequent analyses using fluorescence detection, including those shown in the current figure, were performed on GFP+hFcRn-GFP / hβ2m MDCK II cells. The median fluorescence intensities of hFcRn and hβ2m signals from hFcRn-GFP / hβ2m MDCK II cells in Figure 7B, as shown in the graph in Figure 7C, indicated that the majority of hFcRn-GFP and hβ2m were intracellular. 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. [Figure 8A]Figures 8A-8C demonstrate the internalization dynamics of ASAWT-DL650 and HSA-DL650 in hFcRn-GFP / hβ2m and parental MDCK II cells. To confirm the function of hFcRn-GFP / hβ2m, time-dependent endocytosis studies were performed using 10 or 100 μg / mL of ASAWT-DL650 at pH 5.8, 7.4, or 8.0 (Figure 8A). Representative histograms of the time-dependent uptake results of 100 μg / mL of ASAWT-DL650 at pH 5.8 are shown (Figure 8A, upper panel). hFcRn-GFP / hβ2m MDCK II cells showed increased ASAWT-DL650 endocytosis with decreasing pH, which was time- and concentration-dependent (Figure 8A, lower left graph). No difference was observed in the ASAWT-DL650 endocytosis rate in parent MDCK II cells across the tested pH conditions (Figure 8A, bottom right graph). FcRn-mediated concentration-dependent endocytosis studies using ASAWT-DL650 (Figure 8B) or HSA-DL650 (Figure 8C) were performed in FcRn-GFP / β2m MDCK II cells at pH 5.8 for 20 minutes, with or without a 50 mg / mL unlabeled subject as a competitive inhibitor. To obtain estimates of the ASAWT-DL650 and HSA-DL650 concentrations achieving half the maximum uptake rate (Km) and the maximum rate (Vmax) within each experimental system, specific internalization data expressed as rate per minute were applied to the Michaelis-Menten equation (see Figures 8B(ii) and 8C(ii)). The insets (iii) at the bottom of Figures 8B and 8C show the results of tests performed in the presence of excess unlabeled protein (Figures 8B(i) and 8C(i)), demonstrating linear concentration-dependent uptake. Linear regression was used to fit the data and support the observation of nonspecific endocytosis in the absence of receptor-mediated internalization. N=3-4 / study, mean ± SD. MeFI, median fluorescence intensity; human serum albumin fluorescently conjugated with HSA-DL650 and DyLight 650. [Figure 8B]Figures 8A-8C demonstrate the internalization dynamics of ASAWT-DL650 and HSA-DL650 in hFcRn-GFP / hβ2m and parental MDCK II cells. To confirm the function of hFcRn-GFP / hβ2m, time-dependent endocytosis studies were performed using 10 or 100 μg / mL of ASAWT-DL650 at pH 5.8, 7.4, or 8.0 (Figure 8A). Representative histograms of the time-dependent uptake results of 100 μg / mL of ASAWT-DL650 at pH 5.8 are shown (Figure 8A, upper panel). hFcRn-GFP / hβ2m MDCK II cells showed increased ASAWT-DL650 endocytosis with decreasing pH, which was time- and concentration-dependent (Figure 8A, lower left graph). No difference was observed in the ASAWT-DL650 endocytosis rate in parent MDCK II cells across the tested pH conditions (Figure 8A, bottom right graph). FcRn-mediated concentration-dependent endocytosis studies using ASAWT-DL650 (Figure 8B) or HSA-DL650 (Figure 8C) were performed in FcRn-GFP / β2m MDCK II cells at pH 5.8 for 20 minutes, with or without a 50 mg / mL unlabeled subject as a competitive inhibitor. To obtain estimates of the ASAWT-DL650 and HSA-DL650 concentrations achieving half the maximum uptake rate (Km) and the maximum rate (Vmax) within each experimental system, specific internalization data expressed as rate per minute were applied to the Michaelis-Menten equation (see Figures 8B(ii) and 8C(ii)). The insets (iii) at the bottom of Figures 8B and 8C show the results of tests performed in the presence of excess unlabeled protein (Figures 8B(i) and 8C(i)), demonstrating linear concentration-dependent uptake. Linear regression was used to fit the data and support the observation of nonspecific endocytosis in the absence of receptor-mediated internalization. N=3-4 / study, mean ± SD. MeFI, median fluorescence intensity; human serum albumin fluorescently conjugated with HSA-DL650 and DyLight 650. [Figure 8C]Figures 8A-8C demonstrate the internalization dynamics of ASAWT-DL650 and HSA-DL650 in hFcRn-GFP / hβ2m and parental MDCK II cells. To confirm the function of hFcRn-GFP / hβ2m, time-dependent endocytosis studies were performed using 10 or 100 μg / mL of ASAWT-DL650 at pH 5.8, 7.4, or 8.0 (Figure 8A). Representative histograms of the time-dependent uptake results of 100 μg / mL of ASAWT-DL650 at pH 5.8 are shown (Figure 8A, upper panel). hFcRn-GFP / hβ2m MDCK II cells showed increased ASAWT-DL650 endocytosis with decreasing pH, which was time- and concentration-dependent (Figure 8A, lower left graph). No difference was observed in the ASAWT-DL650 endocytosis rate in parent MDCK II cells across the tested pH conditions (Figure 8A, bottom right graph). FcRn-mediated concentration-dependent endocytosis studies using ASAWT-DL650 (Figure 8B) or HSA-DL650 (Figure 8C) were performed in FcRn-GFP / β2m MDCK II cells at pH 5.8 for 20 minutes, with or without a 50 mg / mL unlabeled subject as a competitive inhibitor. To obtain estimates of the ASAWT-DL650 and HSA-DL650 concentrations achieving half the maximum uptake rate (Km) and the maximum rate (Vmax) within each experimental system, specific internalization data expressed as rate per minute were applied to the Michaelis-Menten equation (see Figures 8B(ii) and 8C(ii)). The insets (iii) at the bottom of Figures 8B and 8C show the results of tests performed in the presence of excess unlabeled protein (Figures 8B(i) and 8C(i)), demonstrating linear concentration-dependent uptake. Linear regression was used to fit the data and support the observation of nonspecific endocytosis in the absence of receptor-mediated internalization. N=3-4 / study, mean ± SD. MeFI, median fluorescence intensity; human serum albumin fluorescently conjugated with HSA-DL650 and DyLight 650. [Figure 9A]Figures 9A–9C show the intracellular localization of hFcRn-GFP in hFcRn-GFP / hβ2m-MDCK II cells after time-dependent incubation with ASAWT-DL650. Human FcRn-GFP / hβ2m-MDCK II cells were lysosome-labeled by loading with 10 kDa Texas Red dextran (TR-dextran) the day before the uptake test. On the imaging day, the cells were incubated with 10 or 100 μg / mL ASAWT-DL650 at pH 5.8 or 7.4 at the above time points. Figure 9A shows a panel of representative images of hFcRn-GFP / hβ2m-MDCK II cells treated with 100 μg / mL ASAWT-DL650 at pH 5.8. Hoechst was used as the nuclear stain. Arrows indicate GFP- cells lacking hFcRn-GFP. These findings provide further confirmation of appropriate transgene function, as the GFP- population consistently tested negative for ASAWT-DL650 under the tested conditions. Mean fluorescence intensity (MFI) of intracellular ASAWT-DL650 was obtained for GFP+ cells (Figure 9B), which showed time-dependent, concentration-dependent, and pH-dependent results consistent with observations using flow cytometry. ****p<0.0001 indicates treated vs. untreated control MFI after two-way ANOVA by Tukey's multiple comparison. N=3 per group, mean ±SD. Figure 9C shows Pearson correlation coefficient analysis for lysosomal intracellular signals of hFcRn-GFP, ASAWT-DL650, and ASAWT-DL650, which resulted in positive co-localization only between hFcRn-GFP and ASAWT-DL650 during the testing period. One-way ANOVA was performed on the *, ***, and ****hFcRn groups against the ASAWT group, followed by Dunnett's multiple comparisons at 15 minutes, resulting in p<0.05, <0.001, and <0.0001. N=3 per group, mean ± SD. [Figure 9B]Figures 9A–9C show the intracellular localization of hFcRn-GFP in hFcRn-GFP / hβ2m-MDCK II cells after time-dependent incubation with ASAWT-DL650. Human FcRn-GFP / hβ2m-MDCK II cells were lysosome-labeled by loading with 10 kDa Texas Red dextran (TR-dextran) the day before the uptake test. On the imaging day, the cells were incubated with 10 or 100 μg / mL ASAWT-DL650 at pH 5.8 or 7.4 at the above time points. Figure 9A shows a panel of representative images of hFcRn-GFP / hβ2m-MDCK II cells treated with 100 μg / mL ASAWT-DL650 at pH 5.8. Hoechst was used as the nuclear stain. Arrows indicate GFP- cells lacking hFcRn-GFP. These findings provide further confirmation of appropriate transgene function, as the GFP- population consistently tested negative for ASAWT-DL650 under the tested conditions. Mean fluorescence intensity (MFI) of intracellular ASAWT-DL650 was obtained for GFP+ cells (Figure 9B), which showed time-dependent, concentration-dependent, and pH-dependent results consistent with observations using flow cytometry. ****p<0.0001 indicates treated vs. untreated control MFI after two-way ANOVA by Tukey's multiple comparison. N=3 per group, mean ±SD. Figure 9C shows Pearson correlation coefficient analysis for lysosomal intracellular signals of hFcRn-GFP, ASAWT-DL650, and ASAWT-DL650, which resulted in positive co-localization only between hFcRn-GFP and ASAWT-DL650 during the testing period. One-way ANOVA was performed on the *, ***, and ****hFcRn groups against the ASAWT group, followed by Dunnett's multiple comparisons at 15 minutes, resulting in p<0.05, <0.001, and <0.0001. N=3 per group, mean ± SD. [Figure 9C]Figures 9A–9C show the intracellular localization of hFcRn-GFP in hFcRn-GFP / hβ2m-MDCK II cells after time-dependent incubation with ASAWT-DL650. Human FcRn-GFP / hβ2m-MDCK II cells were lysosome-labeled by loading with 10 kDa Texas Red dextran (TR-dextran) the day before the uptake test. On the imaging day, the cells were incubated with 10 or 100 μg / mL ASAWT-DL650 at pH 5.8 or 7.4 at the above time points. Figure 9A shows a panel of representative images of hFcRn-GFP / hβ2m-MDCK II cells treated with 100 μg / mL ASAWT-DL650 at pH 5.8. Hoechst was used as the nuclear stain. Arrows indicate GFP- cells lacking hFcRn-GFP. These findings provide further confirmation of appropriate transgene function, as the GFP- population consistently tested negative for ASAWT-DL650 under the tested conditions. Mean fluorescence intensity (MFI) of intracellular ASAWT-DL650 was obtained for GFP+ cells (Figure 9B), which showed time-dependent, concentration-dependent, and pH-dependent results consistent with observations using flow cytometry. ****p<0.0001 indicates treated vs. untreated control MFI after two-way ANOVA by Tukey's multiple comparison. N=3 per group, mean ±SD. Figure 9C shows Pearson correlation coefficient analysis for lysosomal intracellular signals of hFcRn-GFP, ASAWT-DL650, and ASAWT-DL650, which resulted in positive co-localization only between hFcRn-GFP and ASAWT-DL650 during the testing period. One-way ANOVA was performed on the *, ***, and ****hFcRn groups against the ASAWT group, followed by Dunnett's multiple comparisons at 15 minutes, resulting in p<0.05, <0.001, and <0.0001. N=3 per group, mean ± SD. [Figure 10A]Figures 10A–10E illustrate the development of a cell-based hFcRn recycling assay as described herein and claimed herein. Figure 10A is a schematic diagram of the experimental workflow. Parental MDCK II cells were included to provide an evaluation of nonspecific mAb interactions. Because negligible intracellular transport can occur under 4°C conditions, a 4-hour recycling phase at this temperature provided confirmation of the active process in the 37°C group, in addition to measuring the total amount of mAbs internalized during the loading phase. The MDCK II hFcRn recycling test was performed on ASAWT or a mutant lacking hFcRn affinity (ASAAAA). Human FcRn-mediated uptake and recycling were found to be higher in ASAWT at pH 5.8 compared to pH 7.4, essentially zero for ASAAAA mAbs (parental MDCK II is no different from hFcRn-GFP / hβ2m-MDCK II), and increased in incubation at 37°C compared to 4°C, which supports active cell transport (Figure 10B, left graph). Furthermore, nonspecific endocytosis was determined to be negligible for all ASA mAbs tested, based on minimal internalization in parental MDCK II at any pH value (Figure 10B, right graph). These findings indicate that mAbs with low nonspecific uptake at pH 7.4 (i.e., low endocytosis in parental MDCK II) exhibit very low hFcRn-mediated recycling, as this is determined by the initial amount of recycled fraction internalized. Therefore, mAbs with favorable PK behavior would be difficult to evaluate using only pH 7.4 loading conditions. Incubation at pH 5.8 provides a means of loading mAbs into cells with sufficient amounts to elucidate hFcRn recycling efficiency. The significance of the differences in hFcRn-GFP / hβ2m-MDCK II cells was measured by two-way ANOVA with Tukey's multiple comparison test, where **** means adjusted p<0.0001. Bar graphs represent mean ± SD, with n=6-8 replicates per group.Figures 10C and 10D are graphs showing the serum concentration-time profiles of ASAWT and ASAAAA after intravenous administration to Tg32 (Figure 10C) or Tg276 (Figure 10D) hFcRn transgenic mouse models, and evaluating their arrangement. ASAAAA had a rapid CL consistent with the absence of hFcRn binding. Mean ± SD, time point, and n=3 per group. As shown in the graph in Figure 10E, the hFcRn recycling score for ASAWT, using results from the pH 5.8 loading stage, was significantly higher compared to ASAAAA. The scoring results are consistent with the in vivo CL determined in Figures 10C and 10D. ***p<0.001 unpaired t-test, mean ± SD. The dashed green, yellow, and red lines represent recycling scores of 100%, 50%, and 25% for ASAWT, respectively. [Figure 10B]Figures 10A–10E illustrate the development of a cell-based hFcRn recycling assay as described herein and claimed herein. Figure 10A is a schematic diagram of the experimental workflow. Parental MDCK II cells were included to provide an evaluation of nonspecific mAb interactions. Because negligible intracellular transport can occur under 4°C conditions, a 4-hour recycling phase at this temperature provided confirmation of the active process in the 37°C group, in addition to measuring the total amount of mAbs internalized during the loading phase. The MDCK II hFcRn recycling test was performed on ASAWT or a mutant lacking hFcRn affinity (ASAAAA). Human FcRn-mediated uptake and recycling were found to be higher in ASAWT at pH 5.8 compared to pH 7.4, essentially zero for ASAAAA mAbs (parental MDCK II is no different from hFcRn-GFP / hβ2m-MDCK II), and increased in incubation at 37°C compared to 4°C, which supports active cell transport (Figure 10B, left graph). Furthermore, nonspecific endocytosis was determined to be negligible for all ASA mAbs tested, based on minimal internalization in parental MDCK II at any pH value (Figure 10B, right graph). These findings indicate that mAbs with low nonspecific uptake at pH 7.4 (i.e., low endocytosis in parental MDCK II) exhibit very low hFcRn-mediated recycling, as this is determined by the initial amount of recycled fraction internalized. Therefore, mAbs with favorable PK behavior would be difficult to evaluate using only pH 7.4 loading conditions. Incubation at pH 5.8 provides a means of loading mAbs into cells with sufficient amounts to elucidate hFcRn recycling efficiency. The significance of the differences in hFcRn-GFP / hβ2m-MDCK II cells was measured by two-way ANOVA with Tukey's multiple comparison test, where **** means adjusted p<0.0001. Bar graphs represent mean ± SD, with n=6-8 replicates per group.Figures 10C and 10D are graphs showing the serum concentration-time profiles of ASAWT and ASAAAA after intravenous administration to Tg32 (Figure 10C) or Tg276 (Figure 10D) hFcRn transgenic mouse models, and evaluating their arrangement. ASAAAA had a rapid CL consistent with the absence of hFcRn binding. Mean ± SD, time point, and n=3 per group. As shown in the graph in Figure 10E, the hFcRn recycling score for ASAWT, using results from the pH 5.8 loading stage, was significantly higher compared to ASAAAA. The scoring results are consistent with the in vivo CL determined in Figures 10C and 10D. ***p<0.001 unpaired t-test, mean ± SD. The dashed green, yellow, and red lines represent recycling scores of 100%, 50%, and 25% for ASAWT, respectively. [Figure 10C]Figures 10A–10E illustrate the development of a cell-based hFcRn recycling assay as described herein and claimed herein. Figure 10A is a schematic diagram of the experimental workflow. Parental MDCK II cells were included to provide an evaluation of nonspecific mAb interactions. Because negligible intracellular transport can occur under 4°C conditions, a 4-hour recycling phase at this temperature provided confirmation of the active process in the 37°C group, in addition to measuring the total amount of mAbs internalized during the loading phase. The MDCK II hFcRn recycling test was performed on ASAWT or a mutant lacking hFcRn affinity (ASAAAA). Human FcRn-mediated uptake and recycling were found to be higher in ASAWT at pH 5.8 compared to pH 7.4, essentially zero for ASAAAA mAbs (parental MDCK II is no different from hFcRn-GFP / hβ2m-MDCK II), and increased in incubation at 37°C compared to 4°C, which supports active cell transport (Figure 10B, left graph). Furthermore, nonspecific endocytosis was determined to be negligible for all ASA mAbs tested, based on minimal internalization in parental MDCK II at any pH value (Figure 10B, right graph). These findings indicate that mAbs with low nonspecific uptake at pH 7.4 (i.e., low endocytosis in parental MDCK II) exhibit very low hFcRn-mediated recycling, as this is determined by the initial amount of recycled fraction internalized. Therefore, mAbs with favorable PK behavior would be difficult to evaluate using only pH 7.4 loading conditions. Incubation at pH 5.8 provides a means of loading mAbs into cells with sufficient amounts to elucidate hFcRn recycling efficiency. The significance of the differences in hFcRn-GFP / hβ2m-MDCK II cells was measured by two-way ANOVA with Tukey's multiple comparison test, where **** means adjusted p<0.0001. Bar graphs represent mean ± SD, with n=6-8 replicates per group.Figures 10C and 10D are graphs showing the serum concentration-time profiles of ASAWT and ASAAAA after intravenous administration to Tg32 (Figure 10C) or Tg276 (Figure 10D) hFcRn transgenic mouse models, and evaluating their arrangement. ASAAAA had a rapid CL consistent with the absence of hFcRn binding. Mean ± SD, time point, and n=3 per group. As shown in the graph in Figure 10E, the hFcRn recycling score for ASAWT, using results from the pH 5.8 loading stage, was significantly higher compared to ASAAAA. The scoring results are consistent with the in vivo CL determined in Figures 10C and 10D. ***p<0.001 unpaired t-test, mean ± SD. The dashed green, yellow, and red lines represent recycling scores of 100%, 50%, and 25% for ASAWT, respectively. [Figure 10D]Figures 10A–10E illustrate the development of a cell-based hFcRn recycling assay as described herein and claimed herein. Figure 10A is a schematic diagram of the experimental workflow. Parental MDCK II cells were included to provide an evaluation of nonspecific mAb interactions. Because negligible intracellular transport can occur under 4°C conditions, a 4-hour recycling phase at this temperature provided confirmation of the active process in the 37°C group, in addition to measuring the total amount of mAbs internalized during the loading phase. The MDCK II hFcRn recycling test was performed on ASAWT or a mutant lacking hFcRn affinity (ASAAAA). Human FcRn-mediated uptake and recycling were found to be higher in ASAWT at pH 5.8 compared to pH 7.4, essentially zero for ASAAAA mAbs (parental MDCK II is no different from hFcRn-GFP / hβ2m-MDCK II), and increased in incubation at 37°C compared to 4°C, which supports active cell transport (Figure 10B, left graph). Furthermore, nonspecific endocytosis was determined to be negligible for all ASA mAbs tested, based on minimal internalization in parental MDCK II at any pH value (Figure 10B, right graph). These findings indicate that mAbs with low nonspecific uptake at pH 7.4 (i.e., low endocytosis in parental MDCK II) exhibit very low hFcRn-mediated recycling, as this is determined by the initial amount of recycled fraction internalized. Therefore, mAbs with favorable PK behavior would be difficult to evaluate using only pH 7.4 loading conditions. Incubation at pH 5.8 provides a means of loading mAbs into cells with sufficient amounts to elucidate hFcRn recycling efficiency. The significance of the differences in hFcRn-GFP / hβ2m-MDCK II cells was measured by two-way ANOVA with Tukey's multiple comparison test, where **** means adjusted p<0.0001. Bar graphs represent mean ± SD, with n=6-8 replicates per group.Figures 10C and 10D are graphs showing the serum concentration-time profiles of ASAWT and ASAAAA after intravenous administration to Tg32 (Figure 10C) or Tg276 (Figure 10D) hFcRn transgenic mouse models, and evaluating their arrangement. ASAAAA had a rapid CL consistent with the absence of hFcRn binding. Mean ± SD, time point, and n=3 per group. As shown in the graph in Figure 10E, the hFcRn recycling score for ASAWT, using results from the pH 5.8 loading stage, was significantly higher compared to ASAAAA. The scoring results are consistent with the in vivo CL determined in Figures 10C and 10D. ***p<0.001 unpaired t-test, mean ± SD. The dashed green, yellow, and red lines represent recycling scores of 100%, 50%, and 25% for ASAWT, respectively. [Figure 10E]Figures 10A–10E illustrate the development of a cell-based hFcRn recycling assay as described herein and claimed herein. Figure 10A is a schematic diagram of the experimental workflow. Parental MDCK II cells were included to provide an evaluation of nonspecific mAb interactions. Because negligible intracellular transport can occur under 4°C conditions, a 4-hour recycling phase at this temperature provided confirmation of the active process in the 37°C group, in addition to measuring the total amount of mAbs internalized during the loading phase. The MDCK II hFcRn recycling test was performed on ASAWT or a mutant lacking hFcRn affinity (ASAAAA). Human FcRn-mediated uptake and recycling were found to be higher in ASAWT at pH 5.8 compared to pH 7.4, essentially zero for ASAAAA mAbs (parental MDCK II is no different from hFcRn-GFP / hβ2m-MDCK II), and increased in incubation at 37°C compared to 4°C, which supports active cell transport (Figure 10B, left graph). Furthermore, nonspecific endocytosis was determined to be negligible for all ASA mAbs tested, based on minimal internalization in parental MDCK II at any pH value (Figure 10B, right graph). These findings indicate that mAbs with low nonspecific uptake at pH 7.4 (i.e., low endocytosis in parental MDCK II) exhibit very low hFcRn-mediated recycling, as this is determined by the initial amount of recycled fraction internalized. Therefore, mAbs with favorable PK behavior would be difficult to evaluate using only pH 7.4 loading conditions. Incubation at pH 5.8 provides a means of loading mAbs into cells with sufficient amounts to elucidate hFcRn recycling efficiency. The significance of the differences in hFcRn-GFP / hβ2m-MDCK II cells was measured by two-way ANOVA with Tukey's multiple comparison test, where **** means adjusted p<0.0001. Bar graphs represent mean ± SD, with n=6-8 replicates per group.Figures 10C and 10D are graphs showing the serum concentration-time profiles of ASAWT and ASAAAA after intravenous administration to Tg32 (Figure 10C) or Tg276 (Figure 10D) hFcRn transgenic mouse models, and evaluating their arrangement. ASAAAA had a rapid CL consistent with the absence of hFcRn binding. Mean ± SD, time point, and n=3 per group. As shown in the graph in Figure 10E, the hFcRn recycling score for ASAWT, using results from the pH 5.8 loading stage, was significantly higher compared to ASAAAA. The scoring results are consistent with the in vivo CL determined in Figures 10C and 10D. ***p<0.001 unpaired t-test, mean ± SD. The dashed green, yellow, and red lines represent recycling scores of 100%, 50%, and 25% for ASAWT, respectively. [Figure 11A]Figures 11A–11D show that low hFcRn recycling scores correspond to high CLind in humans. Figure 11A shows a representative dataset of mAb1 from hFcRn recycling tests in transfected MDCK II cells and parental MDCK II cells, encompassing all conditions used to obtain hFcRn recycling scores. The amount internalized during the loading phase was obtained via the remaining 4°C group, as negligible intracellular transport is expected at this temperature. mAb1 reference showed pH-dependent uptake in hFcRn-GFP / hβ2m, which was consistent with the degree of recycling at matched pH values. Furthermore, low nonspecific uptake was measured in parental MDCK II cells. As shown in Figure 11B, hFcRn recycling scores (FREMS) were obtained for eight mAbs and plotted against their human CLind values. Low recycling scores indicated high nonspecific uptake and / or inefficient hFcRn-mediated recycling, with values ​​below 0.25 strongly indicating rapid CLind in vivo. The pH 5.8 loading condition was used for scoring because the amount of internalized mAbs with CLind less than 5 mL / d / kg was very low at pH 7.4. MAb1 served as a reference mAb for a complete mAb panel comparison. *, **, ***FcRn recycling scores were compared with mAb1 using Dunnett's multiple comparison one-way ANOVA, with p<0.05, 0.01, and 0.001. The green, yellow, and red dashed lines represent the 100%, 50%, and 25% recycling scores for mAb1, respectively. The loading period was generated from parental MDCK II cells at 4°C (showing the amount internalized) and showed significantly higher anti-IL-4Rα mAb uptake at pH 7.4 compared to mAb1 (Figure 11C) (p<0.0001, one-way ANOVA with Dunnett's multiple comparison). At pH 5.8, both anti-IL-4Rα mAb (††††, p<0.0001) and mAb9 (†††, p<0.001) showed significantly higher nonspecific uptake compared to mAb1 (one-way ANOVA with Dunnett's multiple comparisons). mAb9 and mAb1 also showed a significant increase in nonspecific uptake at pH 7.4 compared to pH 5.8 (*, **** after multiple t-tests, p<0.05, 0.0001).Nonspecific endocytosis of the mAb panel was measured by incubating CHO-K1 cells with 100 μg / mL of mAb at either pH 5.8 or 7.4 for 60 minutes at 37°C, followed by flow cytometry of fixed and permeabilized cells using anti-human Fc detection (Figure 11D). Only anti-IL-4Rα mAb (p<0.0001) and mAb9 (p<0.01) showed significantly higher nonspecific uptake at pH 7.4 compared to ASAWT (standard one-way ANOVA with Dunnett's multiple comparison test). Furthermore, the degree of internalization of anti-IL-4Rα mAb at pH 7.4 was dramatically higher than that of mAb9. At pH 5.8, all mAbs showed significantly higher nonspecific endocytosis to CHO-K1 cells compared to the pH 7.4 condition, with mAb9 showing the greatest change (multiple unpaired t-tests, p < 0.05). In summary, these results demonstrate that the tested mAbs showed a significantly increased tendency towards nonspecific behavior at pH 5.8 compared to pH 7.4. [Figure 11B]Figures 11A–11D show that low hFcRn recycling scores correspond to high CLind in humans. Figure 11A shows a representative dataset of mAb1 from hFcRn recycling tests in transfected MDCK II cells and parental MDCK II cells, encompassing all conditions used to obtain hFcRn recycling scores. The amount internalized during the loading phase was obtained via the remaining 4°C group, as negligible intracellular transport is expected at this temperature. mAb1 reference showed pH-dependent uptake in hFcRn-GFP / hβ2m, which was consistent with the degree of recycling at matched pH values. Furthermore, low nonspecific uptake was measured in parental MDCK II cells. As shown in Figure 11B, hFcRn recycling scores (FREMS) were obtained for eight mAbs and plotted against their human CLind values. Low recycling scores indicated high nonspecific uptake and / or inefficient hFcRn-mediated recycling, with values ​​below 0.25 strongly indicating rapid CLind in vivo. The pH 5.8 loading condition was used for scoring because the amount of internalized mAbs with CLind less than 5 mL / d / kg was very low at pH 7.4. MAb1 served as a reference mAb for a complete mAb panel comparison. *, **, ***FcRn recycling scores were compared with mAb1 using Dunnett's multiple comparison one-way ANOVA, with p<0.05, 0.01, and 0.001. The green, yellow, and red dashed lines represent the 100%, 50%, and 25% recycling scores for mAb1, respectively. The loading period was generated from parental MDCK II cells at 4°C (showing the amount internalized) and showed significantly higher anti-IL-4Rα mAb uptake at pH 7.4 compared to mAb1 (Figure 11C) (p<0.0001, one-way ANOVA with Dunnett's multiple comparison). At pH 5.8, both anti-IL-4Rα mAb (††††, p<0.0001) and mAb9 (†††, p<0.001) showed significantly higher nonspecific uptake compared to mAb1 (one-way ANOVA with Dunnett's multiple comparisons). mAb9 and mAb1 also showed a significant increase in nonspecific uptake at pH 7.4 compared to pH 5.8 (*, **** after multiple t-tests, p<0.05, 0.0001).Nonspecific endocytosis of the mAb panel was measured by incubating CHO-K1 cells with 100 μg / mL of mAb at either pH 5.8 or 7.4 for 60 minutes at 37°C, followed by flow cytometry of fixed and permeabilized cells using anti-human Fc detection (Figure 11D). Only anti-IL-4Rα mAb (p<0.0001) and mAb9 (p<0.01) showed significantly higher nonspecific uptake at pH 7.4 compared to ASAWT (standard one-way ANOVA with Dunnett's multiple comparison test). Furthermore, the degree of internalization of anti-IL-4Rα mAb at pH 7.4 was dramatically higher than that of mAb9. At pH 5.8, all mAbs showed significantly higher nonspecific endocytosis to CHO-K1 cells compared to the pH 7.4 condition, with mAb9 showing the greatest change (multiple unpaired t-tests, p < 0.05). In summary, these results demonstrate that the tested mAbs showed a significantly increased tendency towards nonspecific behavior at pH 5.8 compared to pH 7.4. [Figure 11C]Figures 11A–11D show that low hFcRn recycling scores correspond to high CLind in humans. Figure 11A shows a representative dataset of mAb1 from hFcRn recycling tests in transfected MDCK II cells and parental MDCK II cells, encompassing all conditions used to obtain hFcRn recycling scores. The amount internalized during the loading phase was obtained via the remaining 4°C group, as negligible intracellular transport is expected at this temperature. mAb1 reference showed pH-dependent uptake in hFcRn-GFP / hβ2m, which was consistent with the degree of recycling at matched pH values. Furthermore, low nonspecific uptake was measured in parental MDCK II cells. As shown in Figure 11B, hFcRn recycling scores (FREMS) were obtained for eight mAbs and plotted against their human CLind values. Low recycling scores indicated high nonspecific uptake and / or inefficient hFcRn-mediated recycling, with values ​​below 0.25 strongly indicating rapid CLind in vivo. The pH 5.8 loading condition was used for scoring because the amount of internalized mAbs with CLind less than 5 mL / d / kg was very low at pH 7.4. MAb1 served as a reference mAb for a complete mAb panel comparison. *, **, ***FcRn recycling scores were compared with mAb1 using Dunnett's multiple comparison one-way ANOVA, with p<0.05, 0.01, and 0.001. The green, yellow, and red dashed lines represent the 100%, 50%, and 25% recycling scores for mAb1, respectively. The loading period was generated from parental MDCK II cells at 4°C (showing the amount internalized) and showed significantly higher anti-IL-4Rα mAb uptake at pH 7.4 compared to mAb1 (Figure 11C) (p<0.0001, one-way ANOVA with Dunnett's multiple comparison). At pH 5.8, both anti-IL-4Rα mAb (††††, p<0.0001) and mAb9 (†††, p<0.001) showed significantly higher nonspecific uptake compared to mAb1 (one-way ANOVA with Dunnett's multiple comparisons). mAb9 and mAb1 also showed a significant increase in nonspecific uptake at pH 7.4 compared to pH 5.8 (*, **** after multiple t-tests, p<0.05, 0.0001).Nonspecific endocytosis of the mAb panel was measured by incubating CHO-K1 cells with 100 μg / mL of mAb at either pH 5.8 or 7.4 for 60 minutes at 37°C, followed by flow cytometry of fixed and permeabilized cells using anti-human Fc detection (Figure 11D). Only anti-IL-4Rα mAb (p<0.0001) and mAb9 (p<0.01) showed significantly higher nonspecific uptake at pH 7.4 compared to ASAWT (standard one-way ANOVA with Dunnett's multiple comparison test). Furthermore, the degree of internalization of anti-IL-4Rα mAb at pH 7.4 was dramatically higher than that of mAb9. At pH 5.8, all mAbs showed significantly higher nonspecific endocytosis to CHO-K1 cells compared to the pH 7.4 condition, with mAb9 showing the greatest change (multiple unpaired t-tests, p < 0.05). In summary, these results demonstrate that the tested mAbs showed a significantly increased tendency towards nonspecific behavior at pH 5.8 compared to pH 7.4. [Figure 11D]Figures 11A–11D show that low hFcRn recycling scores correspond to high CLind in humans. Figure 11A shows a representative dataset of mAb1 from hFcRn recycling tests in transfected MDCK II cells and parental MDCK II cells, encompassing all conditions used to obtain hFcRn recycling scores. The amount internalized during the loading phase was obtained via the remaining 4°C group, as negligible intracellular transport is expected at this temperature. mAb1 reference showed pH-dependent uptake in hFcRn-GFP / hβ2m, which was consistent with the degree of recycling at matched pH values. Furthermore, low nonspecific uptake was measured in parental MDCK II cells. As shown in Figure 11B, hFcRn recycling scores (FREMS) were obtained for eight mAbs and plotted against their human CLind values. Low recycling scores indicated high nonspecific uptake and / or inefficient hFcRn-mediated recycling, with values ​​below 0.25 strongly indicating rapid CLind in vivo. The pH 5.8 loading condition was used for scoring because the amount of internalized mAbs with CLind less than 5 mL / d / kg was very low at pH 7.4. MAb1 served as a reference mAb for a complete mAb panel comparison. *, **, ***FcRn recycling scores were compared with mAb1 using Dunnett's multiple comparison one-way ANOVA, with p<0.05, 0.01, and 0.001. The green, yellow, and red dashed lines represent the 100%, 50%, and 25% recycling scores for mAb1, respectively. The loading period was generated from parental MDCK II cells at 4°C (showing the amount internalized) and showed significantly higher anti-IL-4Rα mAb uptake at pH 7.4 compared to mAb1 (Figure 11C) (p<0.0001, one-way ANOVA with Dunnett's multiple comparison). At pH 5.8, both anti-IL-4Rα mAb (††††, p<0.0001) and mAb9 (†††, p<0.001) showed significantly higher nonspecific uptake compared to mAb1 (one-way ANOVA with Dunnett's multiple comparisons). mAb9 and mAb1 also showed a significant increase in nonspecific uptake at pH 7.4 compared to pH 5.8 (*, **** after multiple t-tests, p<0.05, 0.0001).Nonspecific endocytosis of the mAb panel was measured by incubating CHO-K1 cells with 100 μg / mL of mAb at either pH 5.8 or 7.4 for 60 minutes at 37°C, followed by flow cytometry of fixed and permeabilized cells using anti-human Fc detection (Figure 11D). Only anti-IL-4Rα mAb (p<0.0001) and mAb9 (p<0.01) showed significantly higher nonspecific uptake at pH 7.4 compared to ASAWT (standard one-way ANOVA with Dunnett's multiple comparison test). Furthermore, the degree of internalization of anti-IL-4Rα mAb at pH 7.4 was dramatically higher than that of mAb9. At pH 5.8, all mAbs showed significantly higher nonspecific endocytosis to CHO-K1 cells compared to the pH 7.4 condition, with mAb9 showing the greatest change (multiple unpaired t-tests, p < 0.05). In summary, these results demonstrate that the tested mAbs showed a significantly increased tendency towards nonspecific behavior at pH 5.8 compared to pH 7.4. [Figure 12A]Figures 12A–12B provide an exemplary overview. Figure 12A is an image illustrating the cellular process of a low-CLind mAb. Small amounts of mAbs enter non-target vascular endothelial cells at pH 7.4 via non-specific endocytosis due to a lack of high nonspecific interactions resulting from the physicochemical traits of the mAbs, such as localized charge patches. Endosomes containing the internalized mAbs are gradually acidified during transport. As shown herein, all mAbs tested showed increased nonspecific interactions at acidic pH compared to pH 7.4. However, these nonspecific behaviors may have only a minimal effect on mAb interactions with hFcRn for low-CLind compounds, resulting in a higher rate of endosomal hFcRn binding. The bound mAbs are then transported to the plasma membrane and dissociate from hFcRn at near-neutral pH. The result is efficient hFcRn recycling, observed as a high hFcRn recycling score in the current tests. Examples of low-CLind mAbs from this study include mAb1 and ASAWT. Figure 12B is an image illustrating how multiple cellular mechanisms can drive high mAb CLind. Some mAbs, such as anti-IL-4Rα mAbs, exhibit a high rate of nonspecific endocytosis due to their harmful physicochemical properties, including strongly charged local patches at neutral pH. This results in relatively large amounts of mAb internalization into non-target cell populations via nonspecific endocytosis. Increased nonspecificity can occur upon endosomal acidification. This pH-dependent shift can be dramatic, as observed in this study for mAb9. These nonspecific aspects can not only impair binding to hFcRn but also potentially lead to other nonspecific interactions with endosomal membrane components. The results are (1) a greater total number of mAbs in endosomes per hFcRn receptor, (2) dysfunctional hFcRn binding resulting from altered charge states, and / or (3) increased nonspecific interactions that act to counteract hFcRn-mAb binding. Furthermore, mAb dissociation from hFcRn can be impaired on the cell surface if its affinity for the receptor is too high at pH 7.4.All of these factors may contribute to a higher rate of intracellular catabolism, and therefore higher Clind, due to a lower proportion of endogenous mAbs undergoing hFcRn-mediated recycling. Experimentally, this was resolved in the studies described herein where a low hFcRn recycling score indicated the occurrence of one or more of these processes. Test mAbs that did not exhibit large negative behavior (e.g., mAb8) were affected by varying degrees of nonspecific behavior, where the amount of hFcRn interaction was negatively offset by the amount of nonspecificity relative to reference mAb1. Image created with BioRender.com. [Figure 12B]Figures 12A–12B provide an exemplary overview. Figure 12A is an image illustrating the cellular process of a low-CLind mAb. Small amounts of mAbs enter non-target vascular endothelial cells at pH 7.4 via non-specific endocytosis due to a lack of high nonspecific interactions resulting from the physicochemical traits of the mAbs, such as localized charge patches. Endosomes containing the internalized mAbs are gradually acidified during transport. As shown herein, all mAbs tested showed increased nonspecific interactions at acidic pH compared to pH 7.4. However, these nonspecific behaviors may have only a minimal effect on mAb interactions with hFcRn for low-CLind compounds, resulting in a higher rate of endosomal hFcRn binding. The bound mAbs are then transported to the plasma membrane and dissociate from hFcRn at near-neutral pH. The result is efficient hFcRn recycling, observed as a high hFcRn recycling score in the current tests. Examples of low-CLind mAbs from this study include mAb1 and ASAWT. Figure 12B is an image illustrating how multiple cellular mechanisms can drive high mAb CLind. Some mAbs, such as anti-IL-4Rα mAbs, exhibit a high rate of nonspecific endocytosis due to their harmful physicochemical properties, including strongly charged local patches at neutral pH. This results in relatively large amounts of mAb internalization into non-target cell populations via nonspecific endocytosis. Increased nonspecificity can occur upon endosomal acidification. This pH-dependent shift can be dramatic, as observed in this study for mAb9. These nonspecific aspects can not only impair binding to hFcRn but also potentially lead to other nonspecific interactions with endosomal membrane components. The results are (1) a greater total number of mAbs in endosomes per hFcRn receptor, (2) dysfunctional hFcRn binding resulting from altered charge states, and / or (3) increased nonspecific interactions that act to counteract hFcRn-mAb binding. Furthermore, mAb dissociation from hFcRn can be impaired on the cell surface if its affinity for the receptor is too high at pH 7.4.All of these factors may contribute to a higher rate of intracellular catabolism, and therefore higher Clind, due to a lower proportion of endogenous mAbs undergoing hFcRn-mediated recycling. Experimentally, this was resolved in the studies described herein where a low hFcRn recycling score indicated the occurrence of one or more of these processes. Test mAbs that did not exhibit large negative behavior (e.g., mAb8) were affected by varying degrees of nonspecific behavior, where the amount of hFcRn interaction was negatively offset by the amount of nonspecificity relative to reference mAb1. Image created with BioRender.com. [Figure 13A] Figure 13A shows the gating scheme for all endocytosis studies with a sequential subpopulation order indicated by underlined numbers (upper left corner of each plot). For parental (GFP-) or hFcRn-GFP / hβ2m(GFP+) MDCK II cells, the median fluorescence intensity was measured within a single viable cell event. Histograms from two separate samples were superimposed for plot 4 to demonstrate differences in GFP expression. At the bottom of Figure 13A are two representative histograms from separate MDCK II cell types for ASAWT-DL650 time-dependent uptake results at pH 5.8. In Figure 13B, the uptake results under pH 5.8 conditions have been removed to allow visualization of results at pH 7.4. [Figure 13B] Figure 13A shows the gating scheme for all endocytosis studies with a sequential subpopulation order indicated by underlined numbers (upper left corner of each plot). For parental (GFP-) or hFcRn-GFP / hβ2m(GFP+) MDCK II cells, the median fluorescence intensity was measured within a single viable cell event. Histograms from two separate samples were superimposed for plot 4 to demonstrate differences in GFP expression. At the bottom of Figure 13A are two representative histograms from separate MDCK II cell types for ASAWT-DL650 time-dependent uptake results at pH 5.8. In Figure 13B, the uptake results under pH 5.8 conditions have been removed to allow visualization of results at pH 7.4. [Figure 14A]Figures 14A and 14B show the recycled (Figure 14A) and residual (Figure 14B) amounts of the mAb panel tested in the hFcRn recycling test in transfected MDCK II cells and parental MDCK II cells, encompassing all conditions used to derive the hFcRn recycling score. The 37°C and 4°C groups are applied to the respective temperatures of the assay plates during the recycling phase of the test. The amount internalized during the loading phase was obtained via the remaining 4°C group, as negligible intracellular transport is expected at this temperature. The nonspecific uptake coefficient (NUC) and FcRn recycling efficiency metric (FREM) scores were calculated using the internalized (uptake) and recycled amounts, as well as the residual concentration, after the pH 5.8 loading phase, respectively. [Figure 14B] Figures 14A and 14B show the recycled (Figure 14A) and residual (Figure 14B) amounts of the mAb panel tested in the hFcRn recycling test in transfected MDCK II cells and parental MDCK II cells, encompassing all conditions used to derive the hFcRn recycling score. The 37°C and 4°C groups are applied to the respective temperatures of the assay plates during the recycling phase of the test. The amount internalized during the loading phase was obtained via the remaining 4°C group, as negligible intracellular transport is expected at this temperature. The nonspecific uptake coefficient (NUC) and FcRn recycling efficiency metric (FREM) scores were calculated using the internalized (uptake) and recycled amounts, as well as the residual concentration, after the pH 5.8 loading phase, respectively. [Figure 15A] Figures 15A and 15B show the measured nonspecific endocytosis in CHO-K1 cells for a proliferating anti-IL-4Rα mAb panel with single-point and double-point mutants at pH 7.4 (Figure 15A) and 5.8 (Figure 15B). Nonspecific endocytosis of all WT and YTE mutants was significantly different compared to the control anti-IL-4Rα wild-type mAb at either pH value (standard one-way ANOVA with Dunnett's multiple comparison test); #: P<0.001 and *: P≦0.0001. Each bar graph represents the mean ± SD, with N=3-4 per group. [Figure 15B] Figures 15A and 15B show the measured nonspecific endocytosis in CHO-K1 cells for a proliferating anti-IL-4Rα mAb panel with single-point and double-point mutants at pH 7.4 (Figure 15A) and 5.8 (Figure 15B). Nonspecific endocytosis of all WT and YTE mutants was significantly different compared to the control anti-IL-4Rα wild-type mAb at either pH value (standard one-way ANOVA with Dunnett's multiple comparison test); #: P<0.001 and *: P≦0.0001. Each bar graph represents the mean ± SD, with N=3-4 per group. [Figure 16A] Figures 16A to 16D demonstrate the concentration-dependent endocytosis assay of hFcRn-mediated endocytosis using ASAWT-hIgG2-DL650 (Figures 16A to 16B) or ASAWT-hIgG2-YTE-DL650 (Figures 16C to 16D) in hFcRn-GFP / hβ2m MDCK II cells at pH 5.8 for 30 minutes. Nonspecific endocytosis measurements in parental MDCK II cells were subtracted from hFcRn-specific internalization data, and the resulting value was expressed as a rate per minute. These data were applied to the Michaelis-Menten equation to obtain estimated concentrations of ASAWT-hIgG2-DL650 (Figure 16B) and ASAWT-hIgG2-YTE-DL650 (Figure 16D) that achieved the maximum half-capacitation rate (Km) and maximum rate (Vmax) in each experimental system. N=3-4 / study, mean ± SD. MeFI, median fluorescence intensity; ASAWT-hIgG2-DL650, anti-streptavidin human immunoglobulin G2 antibody conjugated with DyLight 650; ASAWT-hIgG2-YTE-DL650, anti-streptavidin human immunoglobulin G2 YTE antibody conjugated with DyLight 650. [Figure 16B]Figures 16A to 16D demonstrate the concentration-dependent endocytosis assay of hFcRn-mediated endocytosis using ASAWT-hIgG2-DL650 (Figures 16A to 16B) or ASAWT-hIgG2-YTE-DL650 (Figures 16C to 16D) in hFcRn-GFP / hβ2m MDCK II cells at pH 5.8 for 30 minutes. Nonspecific endocytosis measurements in parental MDCK II cells were subtracted from hFcRn-specific internalization data, and the resulting value was expressed as a rate per minute. These data were applied to the Michaelis-Menten equation to obtain estimated concentrations of ASAWT-hIgG2-DL650 (Figure 16B) and ASAWT-hIgG2-YTE-DL650 (Figure 16D) that achieved the maximum half-capacitation rate (Km) and maximum rate (Vmax) in each experimental system. N=3-4 / study, mean ± SD. MeFI, median fluorescence intensity; ASAWT-hIgG2-DL650, anti-streptavidin human immunoglobulin G2 antibody conjugated with DyLight 650; ASAWT-hIgG2-YTE-DL650, anti-streptavidin human immunoglobulin G2 YTE antibody conjugated with DyLight 650. [Figure 16C]Figures 16A to 16D demonstrate the concentration-dependent endocytosis assay of hFcRn-mediated endocytosis using ASAWT-hIgG2-DL650 (Figures 16A to 16B) or ASAWT-hIgG2-YTE-DL650 (Figures 16C to 16D) in hFcRn-GFP / hβ2m MDCK II cells at pH 5.8 for 30 minutes. Nonspecific endocytosis measurements in parental MDCK II cells were subtracted from hFcRn-specific internalization data, and the resulting value was expressed as a rate per minute. These data were applied to the Michaelis-Menten equation to obtain estimated concentrations of ASAWT-hIgG2-DL650 (Figure 16B) and ASAWT-hIgG2-YTE-DL650 (Figure 16D) that achieved the maximum half-capacitation rate (Km) and maximum rate (Vmax) in each experimental system. N=3-4 / study, mean ± SD. MeFI, median fluorescence intensity; ASAWT-hIgG2-DL650, anti-streptavidin human immunoglobulin G2 antibody conjugated with DyLight 650; ASAWT-hIgG2-YTE-DL650, anti-streptavidin human immunoglobulin G2 YTE antibody conjugated with DyLight 650. [Figure 16D]Figures 16A to 16D demonstrate the concentration-dependent endocytosis assay of hFcRn-mediated endocytosis using ASAWT-hIgG2-DL650 (Figures 16A to 16B) or ASAWT-hIgG2-YTE-DL650 (Figures 16C to 16D) in hFcRn-GFP / hβ2m MDCK II cells at pH 5.8 for 30 minutes. Nonspecific endocytosis measurements in parental MDCK II cells were subtracted from hFcRn-specific internalization data, and the resulting value was expressed as a rate per minute. These data were applied to the Michaelis-Menten equation to obtain estimated concentrations of ASAWT-hIgG2-DL650 (Figure 16B) and ASAWT-hIgG2-YTE-DL650 (Figure 16D) that achieved the maximum half-capacitation rate (Km) and maximum rate (Vmax) in each experimental system. N=3-4 / study, mean ± SD. MeFI, median fluorescence intensity; ASAWT-hIgG2-DL650, anti-streptavidin human immunoglobulin G2 antibody conjugated with DyLight 650; ASAWT-hIgG2-YTE-DL650, anti-streptavidin human immunoglobulin G2 YTE antibody conjugated with DyLight 650. [Figure 17A]Figures 17A–17C demonstrate the cell-based hFcRn recycling assay's ability to successfully and simultaneously reach ranked mAbs with wild-type or manipulated Fc regions. Figure 17A shows the hFcRn efficiency metric (FREM) scores for anti-IL-4Rα WT and YTE charged mutant mAbs. Scores were normalized relative to anti-IL-4Rα mAb-EEES-YTE. The assay was optimized to simultaneously compare both Fc-manipulated YTE and wild-type Fc (i.e., hIgG2) charged mutant mAbs. Half-life extended YTE mAbs showed better FREM scores compared to their WT counterparts. Each bar graph represents mean ± SD, with N=6–8 replicates per group except for anti-IL-4Rα mAb-EEES-YTE (N=16). YTE mutant mAbs were tested in two batches on two different days, and the FREM score for each day was normalized relative to the EEES-YTE mutant mAb for that day as a relative control. Standard one-way ANOVA with Dunnett's multiple comparison test was performed between groups, and significance was compared with that of the control anti-IL-4Rα mAb (#: P ≤ 0.05 and *: P ≤ 0.0001). Figure 17B shows the serum concentration-time profiles of the tested anti-IL-4Rα mAb WT and YTE charged mutant in male homozygous immunodeficient SCID hFcRn Tg32 transgenic mice (N=3 per group). Figure 17C shows a comparison of CLind scores and FREM scores. It was observed that mAbs with relatively low FREM scores showed higher CLind. [Figure 17B]Figures 17A–17C demonstrate the cell-based hFcRn recycling assay's ability to successfully and simultaneously reach ranked mAbs with wild-type or manipulated Fc regions. Figure 17A shows the hFcRn efficiency metric (FREM) scores for anti-IL-4Rα WT and YTE charged mutant mAbs. Scores were normalized relative to anti-IL-4Rα mAb-EEES-YTE. The assay was optimized to simultaneously compare both Fc-manipulated YTE and wild-type Fc (i.e., hIgG2) charged mutant mAbs. Half-life extended YTE mAbs showed better FREM scores compared to their WT counterparts. Each bar graph represents mean ± SD, with N=6–8 replicates per group except for anti-IL-4Rα mAb-EEES-YTE (N=16). YTE mutant mAbs were tested in two batches on two different days, and the FREM score for each day was normalized relative to the EEES-YTE mutant mAb for that day as a relative control. Standard one-way ANOVA with Dunnett's multiple comparison test was performed between groups, and significance was compared with that of the control anti-IL-4Rα mAb (#: P ≤ 0.05 and *: P ≤ 0.0001). Figure 17B shows the serum concentration-time profiles of the tested anti-IL-4Rα mAb WT and YTE charged mutant in male homozygous immunodeficient SCID hFcRn Tg32 transgenic mice (N=3 per group). Figure 17C shows a comparison of CLind scores and FREM scores. It was observed that mAbs with relatively low FREM scores showed higher CLind. [Figure 17C]Figures 17A–17C demonstrate the cell-based hFcRn recycling assay's ability to successfully and simultaneously reach ranked mAbs with wild-type or manipulated Fc regions. Figure 17A shows the hFcRn efficiency metric (FREM) scores for anti-IL-4Rα WT and YTE charged mutant mAbs. Scores were normalized relative to anti-IL-4Rα mAb-EEES-YTE. The assay was optimized to simultaneously compare both Fc-manipulated YTE and wild-type Fc (i.e., hIgG2) charged mutant mAbs. Half-life extended YTE mAbs showed better FREM scores compared to their WT counterparts. Each bar graph represents mean ± SD, with N=6–8 replicates per group except for anti-IL-4Rα mAb-EEES-YTE (N=16). YTE mutant mAbs were tested in two batches on two different days, and the FREM score for each day was normalized relative to the EEES-YTE mutant mAb for that day as a relative control. Standard one-way ANOVA with Dunnett's multiple comparison test was performed between groups, and significance was compared with that of the control anti-IL-4Rα mAb (#: P ≤ 0.05 and *: P ≤ 0.0001). Figure 17B shows the serum concentration-time profiles of the tested anti-IL-4Rα mAb WT and YTE charged mutant in male homozygous immunodeficient SCID hFcRn Tg32 transgenic mice (N=3 per group). Figure 17C shows a comparison of CLind scores and FREM scores. It was observed that mAbs with relatively low FREM scores showed higher CLind. [Figure 18A] Figures 18A–18F show the recycled, uptake, and residual amounts of anti-IL-4RαWT mAb mutants in hFcRn recycling tests in hFcRn-GFP / β2M transfects (Figures 18A–18C) and parental MDCK II cells (Figures 18D–18F) used to induce FREM scores. The mAb loading and recycling stages were tested at 37°C. The nonspecific uptake coefficient (NUC) and FcRn recycling efficiency metric (FREM) scores were calculated using the internalized (uptake), recycled, and residual concentrations after the pH 5.8 loading stage, respectively. [Figure 18B]Figures 18A–18F show the recycled, uptake, and residual amounts of anti-IL-4RαWT mAb mutants in hFcRn recycling tests in hFcRn-GFP / β2M transfects (Figures 18A–18C) and parental MDCK II cells (Figures 18D–18F) used to induce FREM scores. The mAb loading and recycling stages were tested at 37°C. The nonspecific uptake coefficient (NUC) and FcRn recycling efficiency metric (FREM) scores were calculated using the internalized (uptake), recycled, and residual concentrations after the pH 5.8 loading stage, respectively. [Figure 18C] Figures 18A–18F show the recycled, uptake, and residual amounts of anti-IL-4RαWT mAb mutants in hFcRn recycling tests in hFcRn-GFP / β2M transfects (Figures 18A–18C) and parental MDCK II cells (Figures 18D–18F) used to induce FREM scores. The mAb loading and recycling stages were tested at 37°C. The nonspecific uptake coefficient (NUC) and FcRn recycling efficiency metric (FREM) scores were calculated using the internalized (uptake), recycled, and residual concentrations after the pH 5.8 loading stage, respectively. [Figure 18D] Figures 18A–18F show the recycled, uptake, and residual amounts of anti-IL-4RαWT mAb mutants in hFcRn recycling tests in hFcRn-GFP / β2M transfects (Figures 18A–18C) and parental MDCK II cells (Figures 18D–18F) used to induce FREM scores. The mAb loading and recycling stages were tested at 37°C. The nonspecific uptake coefficient (NUC) and FcRn recycling efficiency metric (FREM) scores were calculated using the internalized (uptake), recycled, and residual concentrations after the pH 5.8 loading stage, respectively. [Figure 18E]Figures 18A–18F show the recycled, uptake, and residual amounts of anti-IL-4RαWT mAb mutants in hFcRn recycling tests in hFcRn-GFP / β2M transfects (Figures 18A–18C) and parental MDCK II cells (Figures 18D–18F) used to induce FREM scores. The mAb loading and recycling stages were tested at 37°C. The nonspecific uptake coefficient (NUC) and FcRn recycling efficiency metric (FREM) scores were calculated using the internalized (uptake), recycled, and residual concentrations after the pH 5.8 loading stage, respectively. [Figure 18F] Figures 18A–18F show the recycled, uptake, and residual amounts of anti-IL-4RαWT mAb mutants in hFcRn recycling tests in hFcRn-GFP / β2M transfects (Figures 18A–18C) and parental MDCK II cells (Figures 18D–18F) used to induce FREM scores. The mAb loading and recycling stages were tested at 37°C. The nonspecific uptake coefficient (NUC) and FcRn recycling efficiency metric (FREM) scores were calculated using the internalized (uptake), recycled, and residual concentrations after the pH 5.8 loading stage, respectively. [Figure 19A] Figures 19A–19F show the recycling, uptake, and residual levels of anti-IL-4RαYTE mAb mutants in hFcRn-GFP / β2M transfected cells (Figures 19A–19C) and parental MDCK II cells (Figures 19D–19F) of EEES-YTE, SSLS-YTE, and WT-YTE mAb mutants used to induce FREM scores in the hFcRn recycling test. All experimental data were collected over one day. Experiments were performed on different days after thawing different cell vials compared to the WT mutant in Figure 18. The mAb loading and recycling stages were tested at 37°C. The nonspecific uptake coefficient (NUC) and FcRn recycling efficiency metric (FREM) scores were calculated using the internalization (uptake), recycling, and residual concentrations after the pH 5.8 loading stage, respectively. [Figure 19B]Figures 19A–19F show the recycling, uptake, and residual levels of anti-IL-4RαYTE mAb mutants in hFcRn-GFP / β2M transfected cells (Figures 19A–19C) and parental MDCK II cells (Figures 19D–19F) of EEES-YTE, SSLS-YTE, and WT-YTE mAb mutants used to induce FREM scores in the hFcRn recycling test. All experimental data were collected over one day. Experiments were performed on different days after thawing different cell vials compared to the WT mutant in Figure 18. The mAb loading and recycling stages were tested at 37°C. The nonspecific uptake coefficient (NUC) and FcRn recycling efficiency metric (FREM) scores were calculated using the internalization (uptake), recycling, and residual concentrations after the pH 5.8 loading stage, respectively. [Figure 19C] Figures 19A–19F show the recycling, uptake, and residual levels of anti-IL-4RαYTE mAb mutants in hFcRn-GFP / β2M transfected cells (Figures 19A–19C) and parental MDCK II cells (Figures 19D–19F) of EEES-YTE, SSLS-YTE, and WT-YTE mAb mutants used to induce FREM scores in the hFcRn recycling test. All experimental data were collected over one day. Experiments were performed on different days after thawing different cell vials compared to the WT mutant in Figure 18. The mAb loading and recycling stages were tested at 37°C. The nonspecific uptake coefficient (NUC) and FcRn recycling efficiency metric (FREM) scores were calculated using the internalization (uptake), recycling, and residual concentrations after the pH 5.8 loading stage, respectively. [Figure 19D]Figures 19A–19F show the recycling, uptake, and residual levels of anti-IL-4RαYTE mAb mutants in hFcRn-GFP / β2M transfected cells (Figures 19A–19C) and parental MDCK II cells (Figures 19D–19F) of EEES-YTE, SSLS-YTE, and WT-YTE mAb mutants used to induce FREM scores in the hFcRn recycling test. All experimental data were collected over one day. Experiments were performed on different days after thawing different cell vials compared to the WT mutant in Figure 18. The mAb loading and recycling stages were tested at 37°C. The nonspecific uptake coefficient (NUC) and FcRn recycling efficiency metric (FREM) scores were calculated using the internalization (uptake), recycling, and residual concentrations after the pH 5.8 loading stage, respectively. [Figure 19E] Figures 19A–19F show the recycling, uptake, and residual levels of anti-IL-4RαYTE mAb mutants in hFcRn-GFP / β2M transfected cells (Figures 19A–19C) and parental MDCK II cells (Figures 19D–19F) of EEES-YTE, SSLS-YTE, and WT-YTE mAb mutants used to induce FREM scores in the hFcRn recycling test. All experimental data were collected over one day. Experiments were performed on different days after thawing different cell vials compared to the WT mutant in Figure 18. The mAb loading and recycling stages were tested at 37°C. The nonspecific uptake coefficient (NUC) and FcRn recycling efficiency metric (FREM) scores were calculated using the internalization (uptake), recycling, and residual concentrations after the pH 5.8 loading stage, respectively. [Figure 19F]Figures 19A–19F show the recycling, uptake, and residual levels of anti-IL-4RαYTE mAb mutants in hFcRn-GFP / β2M transfected cells (Figures 19A–19C) and parental MDCK II cells (Figures 19D–19F) of EEES-YTE, SSLS-YTE, and WT-YTE mAb mutants used to induce FREM scores in the hFcRn recycling test. All experimental data were collected over one day. Experiments were performed on different days after thawing different cell vials compared to the WT mutant in Figure 18. The mAb loading and recycling stages were tested at 37°C. The nonspecific uptake coefficient (NUC) and FcRn recycling efficiency metric (FREM) scores were calculated using the internalization (uptake), recycling, and residual concentrations after the pH 5.8 loading stage, respectively. [Figure 20A] Figures 20A–20F demonstrate the recycling, uptake, and residual levels of anti-IL-4RαYTE mAb mutants in hFcRn-GFP / β2M transfected (A–C) and parental MDCK II cells (D–F) of four YTE mutants in an hFcRn recycling test, along with EEES-YTE as a control mAb used to derive the FREM score. Figure 20 shows experimental data collected on a different day than Figure 19. This experiment was performed together with the WT mutant described in Figure 18 from the same cell flask. The mAb loading and recycling stages were tested at 37°C. The nonspecific uptake coefficient (NUC) and FcRn recycling efficiency metric (FREM) score were calculated using the internalization (uptake) and recycling levels and residual concentrations after the pH 5.8 loading stage, respectively. [Figure 20B]Figures 20A–20F demonstrate the recycling, uptake, and residual levels of anti-IL-4RαYTE mAb mutants in hFcRn-GFP / β2M transfected (A–C) and parental MDCK II cells (D–F) of four YTE mutants in an hFcRn recycling test, along with EEES-YTE as a control mAb used to derive the FREM score. Figure 20 shows experimental data collected on a different day than Figure 19. This experiment was performed together with the WT mutant described in Figure 18 from the same cell flask. The mAb loading and recycling stages were tested at 37°C. The nonspecific uptake coefficient (NUC) and FcRn recycling efficiency metric (FREM) score were calculated using the internalization (uptake) and recycling levels and residual concentrations after the pH 5.8 loading stage, respectively. [Figure 20C] Figures 20A–20F demonstrate the recycling, uptake, and residual levels of anti-IL-4RαYTE mAb mutants in hFcRn-GFP / β2M transfected (A–C) and parental MDCK II cells (D–F) of four YTE mutants in an hFcRn recycling test, along with EEES-YTE as a control mAb used to derive the FREM score. Figure 20 shows experimental data collected on a different day than Figure 19. This experiment was performed together with the WT mutant described in Figure 18 from the same cell flask. The mAb loading and recycling stages were tested at 37°C. The nonspecific uptake coefficient (NUC) and FcRn recycling efficiency metric (FREM) score were calculated using the internalization (uptake) and recycling levels and residual concentrations after the pH 5.8 loading stage, respectively. [Figure 20D]Figures 20A–20F demonstrate the recycling, uptake, and residual levels of anti-IL-4RαYTE mAb mutants in hFcRn-GFP / β2M transfected (A–C) and parental MDCK II cells (D–F) of four YTE mutants in an hFcRn recycling test, along with EEES-YTE as a control mAb used to derive the FREM score. Figure 20 shows experimental data collected on a different day than Figure 19. This experiment was performed together with the WT mutant described in Figure 18 from the same cell flask. The mAb loading and recycling stages were tested at 37°C. The nonspecific uptake coefficient (NUC) and FcRn recycling efficiency metric (FREM) score were calculated using the internalization (uptake) and recycling levels and residual concentrations after the pH 5.8 loading stage, respectively. [Figure 20E] Figures 20A–20F demonstrate the recycling, uptake, and residual levels of anti-IL-4RαYTE mAb mutants in hFcRn-GFP / β2M transfected (A–C) and parental MDCK II cells (D–F) of four YTE mutants in an hFcRn recycling test, along with EEES-YTE as a control mAb used to derive the FREM score. Figure 20 shows experimental data collected on a different day than Figure 19. This experiment was performed together with the WT mutant described in Figure 18 from the same cell flask. The mAb loading and recycling stages were tested at 37°C. The nonspecific uptake coefficient (NUC) and FcRn recycling efficiency metric (FREM) score were calculated using the internalization (uptake) and recycling levels and residual concentrations after the pH 5.8 loading stage, respectively. [Figure 20F]Figures 20A–20F demonstrate the recycling, uptake, and residual levels of anti-IL-4RαYTE mAb mutants in hFcRn-GFP / β2M transfected (A–C) and parental MDCK II cells (D–F) of four YTE mutants in an hFcRn recycling test, along with EEES-YTE as a control mAb used to derive the FREM score. Figure 20 shows experimental data collected on a different day than Figure 19. This experiment was performed together with the WT mutant described in Figure 18 from the same cell flask. The mAb loading and recycling stages were tested at 37°C. The nonspecific uptake coefficient (NUC) and FcRn recycling efficiency metric (FREM) score were calculated using the internalization (uptake) and recycling levels and residual concentrations after the pH 5.8 loading stage, respectively. [Figure 21A]Figure 21A is a graph showing nonspecific endocytosis results from CHO-K1 uptake assays for a panel of fully human and humanized clinical mAbs or their derivatives with known clinical PK data. mAbs were grouped by having one of the following: CLind < 4.5 mL / kg / d in humans after intravenous (IV) dose, CLind > 4.5 mL / kg / d, or an unobtained CLind estimate (e.g., a dose level not high enough to achieve target saturation). This was done because currently disclosed assays cannot provide information on mAb target-mediated kinetics. The 4.5 mL / kg / d value was chosen because it corresponds to a terminal serum half-life of approximately 10 days in an 80 kg human. The graph also shows the generated assay thresholds. The "low-risk" group includes mAbs with a low risk of high CLind and / or low FSQ (due to nonspecific endocytosis). This was defined as the upper limit of the 95% confidence interval for the mean ABC value for mAbs with FSQ greater than 50% and / or CLind less than 4.5 mL / kg / d. The "moderate risk" group included mAbs that may or may not exhibit high CLind and / or low FSQ, and were defined to be two standard deviations above the mean ABC value described above. The "high risk" group highlighted mAbs with apparent nonspecific endocytosis that are likely to be harmful to their placement in humans. All mAbs with CLind estimates from Figure 21A were binned into their respective risk categories as described herein and plotted in Figure 21B. The gradual increase in mean CLind values ​​correlated with an increase in risk classification in the CHO-K1 assay. Furthermore, mAbs that may have had low CLind but still exhibited high nonspecific CHO-K1 endocytosis (i.e., high ABC values) were found to have low FSQ in humans (e.g., mAb 28). Thus, mAbs in the high-risk category did not have good PK in humans. [Figure 21B]Figure 21A is a graph showing nonspecific endocytosis results from CHO-K1 uptake assays for a panel of fully human and humanized clinical mAbs or their derivatives with known clinical PK data. mAbs were grouped by having one of the following: CLind < 4.5 mL / kg / d in humans after intravenous (IV) dose, CLind > 4.5 mL / kg / d, or an unobtained CLind estimate (e.g., a dose level not high enough to achieve target saturation). This was done because currently disclosed assays cannot provide information on mAb target-mediated kinetics. The 4.5 mL / kg / d value was chosen because it corresponds to a terminal serum half-life of approximately 10 days in an 80 kg human. The graph also shows the generated assay thresholds. The "low-risk" group includes mAbs with a low risk of high CLind and / or low FSQ (due to nonspecific endocytosis). This was defined as the upper limit of the 95% confidence interval for the mean ABC value for mAbs with FSQ greater than 50% and / or CLind less than 4.5 mL / kg / d. The "moderate risk" group included mAbs that may or may not exhibit high CLind and / or low FSQ, and were defined to be two standard deviations above the mean ABC value described above. The "high risk" group highlighted mAbs with apparent nonspecific endocytosis that are likely to be harmful to their placement in humans. All mAbs with CLind estimates from Figure 21A were binned into their respective risk categories as described herein and plotted in Figure 21B. The gradual increase in mean CLind values ​​correlated with an increase in risk classification in the CHO-K1 assay. Furthermore, mAbs that may have had low CLind but still exhibited high nonspecific CHO-K1 endocytosis (i.e., high ABC values) were found to have low FSQ in humans (e.g., mAb 28). Thus, mAbs in the high-risk category did not have good PK in humans. [Figure 22A]Figures 22A–22B highlight how nonspecific endocytosis can identify high-risk, multispecific antibodies with increased target-independent clearance. Forty-eight multispecific antibodies exhibiting a wide range of target-independent clearance (CLind) were obtained in Tg32 human FcRn transgenic mice (0.2 to 2000 mL / kg / hour) (Figure 22A). The proteins originated from various molecular formats. Nonspecific endocytosis was measured for the multispecific protein panel by obtaining antibody-binding ability (ABC) in CHO-K1 cells. Compounds were then classified into low, medium, or high-risk categories using ABC, with the same parameters as the human mAb panel in Figure 15 (i.e., low in this embodiment, defined as having the same ABC values ​​as the human mAb panel shown in Figure 21A) (Figure 22B). This approach identified multispecificity in the intermediate and high-risk bins, with a tendency for elevated CLind in Tg32 mice. 94% of high-risk bin proteins and 92% of intermediate-risk bin proteins exhibited CLind levels exceeding 0.5 mL / kg / hour in Tg32 mice. However, 63% of low-risk multispecificity proteins also showed CLind levels exceeding 0.5 mL / kg / hour in Tg32 mice, highlighting alternative clearance mechanisms other than nonspecific endocytosis. This indicates the need for supplementary assays for alternative mechanisms, such as the cell-human FcRn recycling assay defined in current studies. [Figure 22B]Figures 22A–22B highlight how nonspecific endocytosis can identify high-risk, multispecific antibodies with increased target-independent clearance. Forty-eight multispecific antibodies exhibiting a wide range of target-independent clearance (CLind) were obtained in Tg32 human FcRn transgenic mice (0.2 to 2000 mL / kg / hour) (Figure 22A). The proteins originated from various molecular formats. Nonspecific endocytosis was measured for the multispecific protein panel by obtaining antibody-binding ability (ABC) in CHO-K1 cells. Compounds were then classified into low, medium, or high-risk categories using ABC, with the same parameters as the human mAb panel in Figure 15 (i.e., low in this embodiment, defined as having the same ABC values ​​as the human mAb panel shown in Figure 21A) (Figure 22B). This approach identified multispecificity in the intermediate and high-risk bins, with a tendency for elevated CLind in Tg32 mice. 94% of high-risk bin proteins and 92% of intermediate-risk bin proteins exhibited CLind levels exceeding 0.5 mL / kg / hour in Tg32 mice. However, 63% of low-risk multispecificity proteins also showed CLind levels exceeding 0.5 mL / kg / hour in Tg32 mice, highlighting alternative clearance mechanisms other than nonspecific endocytosis. This indicates the need for supplementary assays for alternative mechanisms, such as the cell-human FcRn recycling assay defined in current studies. [Figure 23A]Figures 23A–23F demonstrate how nonspecific endocytosis exhibits increased target-independent clearance across a wide range of protein structures. A panel of Fc fusion proteins containing five different protein structures unrelated to those of human immunoglobulins was constructed. Each construct had two individual proteins fused to a human Fc domain. Single-dose studies (2 mg / kg) in wild-type mice demonstrated a wide range of CLind (0.81–1260 mL / hour / kg), as shown in Figure 23A. All Fc fusion proteins were analyzed using CHO-K1 cell endocytosis. Antibody binding capacity (ABC) was obtained at either pH 7.4 (Figure 23B) or pH 5.8 (Figure 23C) and plotted against their corresponding CLind in wild-type mice. Next, the mean ABC values ​​from CHO-K1 nonspecific endocytosis studies performed at pH 7.4 were placed into low, medium, or high-risk bins using ABC cutoff points derived from the human mAb panel shown in Figure 21 (Figure 23D). 94% of the Fc fusion proteins in the high-risk bin had CLind greater than 3 mL / kg / hour in wild-type mice. Fc fusion proteins considered low or medium risk after the pH 7.4 endocytosis experiments were placed into a new risk threshold based on their ABC values ​​at pH 5.8 (Figure 23E). Three proteins were found to exhibit a wide jump in pH-dependent nonspecific behavior: Fc-fusions 7, 8, and 10. Of these, two of the three had very high CLind values ​​in mice (Figure 23F), supporting high pH-dependent nonspecificity as an undesirable attribute of therapeutic proteins. [Figure 23B]Figures 23A–23F demonstrate how nonspecific endocytosis exhibits increased target-independent clearance across a wide range of protein structures. A panel of Fc fusion proteins containing five different protein structures unrelated to those of human immunoglobulins was constructed. Each construct had two individual proteins fused to a human Fc domain. Single-dose studies (2 mg / kg) in wild-type mice demonstrated a wide range of CLind (0.81–1260 mL / hour / kg), as shown in Figure 23A. All Fc fusion proteins were analyzed using CHO-K1 cell endocytosis. Antibody binding capacity (ABC) was obtained at either pH 7.4 (Figure 23B) or pH 5.8 (Figure 23C) and plotted against their corresponding CLind in wild-type mice. Next, the mean ABC values ​​from CHO-K1 nonspecific endocytosis studies performed at pH 7.4 were placed into low, medium, or high-risk bins using ABC cutoff points derived from the human mAb panel shown in Figure 21 (Figure 23D). 94% of the Fc fusion proteins in the high-risk bin had CLind greater than 3 mL / kg / hour in wild-type mice. Fc fusion proteins considered low or medium risk after the pH 7.4 endocytosis experiments were placed into a new risk threshold based on their ABC values ​​at pH 5.8 (Figure 23E). Three proteins were found to exhibit a wide jump in pH-dependent nonspecific behavior: Fc-fusions 7, 8, and 10. Of these, two of the three had very high CLind values ​​in mice (Figure 23F), supporting high pH-dependent nonspecificity as an undesirable attribute of therapeutic proteins. [Figure 23C]Figures 23A–23F demonstrate how nonspecific endocytosis exhibits increased target-independent clearance across a wide range of protein structures. A panel of Fc fusion proteins containing five different protein structures unrelated to those of human immunoglobulins was constructed. Each construct had two individual proteins fused to a human Fc domain. Single-dose studies (2 mg / kg) in wild-type mice demonstrated a wide range of CLind (0.81–1260 mL / hour / kg), as shown in Figure 23A. All Fc fusion proteins were analyzed using CHO-K1 cell endocytosis. Antibody binding capacity (ABC) was obtained at either pH 7.4 (Figure 23B) or pH 5.8 (Figure 23C) and plotted against their corresponding CLind in wild-type mice. Next, the mean ABC values ​​from CHO-K1 nonspecific endocytosis studies performed at pH 7.4 were placed into low, medium, or high-risk bins using ABC cutoff points derived from the human mAb panel shown in Figure 21 (Figure 23D). 94% of the Fc fusion proteins in the high-risk bin had CLind greater than 3 mL / kg / hour in wild-type mice. Fc fusion proteins considered low or medium risk after the pH 7.4 endocytosis experiments were placed into a new risk threshold based on their ABC values ​​at pH 5.8 (Figure 23E). Three proteins were found to exhibit a wide jump in pH-dependent nonspecific behavior: Fc-fusions 7, 8, and 10. Of these, two of the three had very high CLind values ​​in mice (Figure 23F), supporting high pH-dependent nonspecificity as an undesirable attribute of therapeutic proteins. [Figure 23D]Figures 23A–23F demonstrate how nonspecific endocytosis exhibits increased target-independent clearance across a wide range of protein structures. A panel of Fc fusion proteins containing five different protein structures unrelated to those of human immunoglobulins was constructed. Each construct had two individual proteins fused to a human Fc domain. Single-dose studies (2 mg / kg) in wild-type mice demonstrated a wide range of CLind (0.81–1260 mL / hour / kg), as shown in Figure 23A. All Fc fusion proteins were analyzed using CHO-K1 cell endocytosis. Antibody binding capacity (ABC) was obtained at either pH 7.4 (Figure 23B) or pH 5.8 (Figure 23C) and plotted against their corresponding CLind in wild-type mice. Next, the mean ABC values ​​from CHO-K1 nonspecific endocytosis studies performed at pH 7.4 were placed into low, medium, or high-risk bins using ABC cutoff points derived from the human mAb panel shown in Figure 21 (Figure 23D). 94% of the Fc fusion proteins in the high-risk bin had CLind greater than 3 mL / kg / hour in wild-type mice. Fc fusion proteins considered low or medium risk after the pH 7.4 endocytosis experiments were placed into a new risk threshold based on their ABC values ​​at pH 5.8 (Figure 23E). Three proteins were found to exhibit a wide jump in pH-dependent nonspecific behavior: Fc-fusions 7, 8, and 10. Of these, two of the three had very high CLind values ​​in mice (Figure 23F), supporting high pH-dependent nonspecificity as an undesirable attribute of therapeutic proteins. [Figure 23E]Figures 23A–23F demonstrate how nonspecific endocytosis exhibits increased target-independent clearance across a wide range of protein structures. A panel of Fc fusion proteins containing five different protein structures unrelated to those of human immunoglobulins was constructed. Each construct had two individual proteins fused to a human Fc domain. Single-dose studies (2 mg / kg) in wild-type mice demonstrated a wide range of CLind (0.81–1260 mL / hour / kg), as shown in Figure 23A. All Fc fusion proteins were analyzed using CHO-K1 cell endocytosis. Antibody binding capacity (ABC) was obtained at either pH 7.4 (Figure 23B) or pH 5.8 (Figure 23C) and plotted against their corresponding CLind in wild-type mice. Next, the mean ABC values ​​from CHO-K1 nonspecific endocytosis studies performed at pH 7.4 were placed into low, medium, or high-risk bins using ABC cutoff points derived from the human mAb panel shown in Figure 21 (Figure 23D). 94% of the Fc fusion proteins in the high-risk bin had CLind greater than 3 mL / kg / hour in wild-type mice. Fc fusion proteins considered low or medium risk after the pH 7.4 endocytosis experiments were placed into a new risk threshold based on their ABC values ​​at pH 5.8 (Figure 23E). Three proteins were found to exhibit a wide jump in pH-dependent nonspecific behavior: Fc-fusions 7, 8, and 10. Of these, two of the three had very high CLind values ​​in mice (Figure 23F), supporting high pH-dependent nonspecificity as an undesirable attribute of therapeutic proteins. [Figure 23F]Figures 23A–23F demonstrate how nonspecific endocytosis exhibits increased target-independent clearance across a wide range of protein structures. A panel of Fc fusion proteins containing five different protein structures unrelated to those of human immunoglobulins was constructed. Each construct had two individual proteins fused to a human Fc domain. Single-dose studies (2 mg / kg) in wild-type mice demonstrated a wide range of CLind (0.81–1260 mL / hour / kg), as shown in Figure 23A. All Fc fusion proteins were analyzed using CHO-K1 cell endocytosis. Antibody binding capacity (ABC) was obtained at either pH 7.4 (Figure 23B) or pH 5.8 (Figure 23C) and plotted against their corresponding CLind in wild-type mice. Next, the mean ABC values ​​from CHO-K1 nonspecific endocytosis studies performed at pH 7.4 were placed into low, medium, or high-risk bins using ABC cutoff points derived from the human mAb panel shown in Figure 21 (Figure 23D). 94% of the Fc fusion proteins in the high-risk bin had CLind greater than 3 mL / kg / hour in wild-type mice. Fc fusion proteins considered low or medium risk after the pH 7.4 endocytosis experiments were placed into a new risk threshold based on their ABC values ​​at pH 5.8 (Figure 23E). Three proteins were found to exhibit a wide jump in pH-dependent nonspecific behavior: Fc-fusions 7, 8, and 10. Of these, two of the three had very high CLind values ​​in mice (Figure 23F), supporting high pH-dependent nonspecificity as an undesirable attribute of therapeutic proteins. [Modes for carrying out the invention]

[0018] This disclosure relates to in vitro cell-based methods useful for predicting the in vivo pharmacokinetic properties of candidate therapeutic biomolecules. In particular, the cell-based assays described herein facilitate the prediction or estimation of in vivo non-target-mediated clearance of candidate therapeutic molecules. The cell-based assays described herein may also be used to rank the in vivo non-target-mediated clearance of candidate molecules to assist in the selection and optimization of lead candidates while reducing the number of molecules tested in animal models.

[0019] Please understand that the above summary and the following detailed description are merely illustrative and descriptive, and do not limit the claimed invention.

[0020] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure have meanings generally understood by those skilled in the art. Generally, the nomenclature and techniques used in connection with cell culture, molecular biology, genetics, and protein and nucleic acid chemistry, as well as hybridization, as described herein are well known and commonly used in the art. Unless otherwise stated, the methods and techniques described herein are generally carried out in accordance with prior art methods well known in the art, and as described in the general and more specific references cited and discussed throughout this specification. See Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (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, NY (1990) (incorporated herein by reference).

[0021] In this disclosure, unless otherwise specified, the use of singular terms includes plural terms, and plural terms include singular terms. Where used herein, the singular forms "a," "an," and "the" include both singular and plural referents unless otherwise explicitly indicated by the context.

[0022] In this disclosure, the use of “or” means “and / or” unless otherwise specified. Furthermore, the use of the term “contains” is not limited to other forms such as “contains” and “includes.” Also, terms such as “component” include both components containing one unit and components containing two or more subunits unless otherwise specified.

[0023] As used herein, the terms “comprising,” “comprises,” and “comprised of” are synonymous with “including,” “includes,” “containing,” or “contains,” and are comprehensive or open-ended, not excluding additional unlisted components, compounds, products, elements, or process steps. As used in the context of a composition or method (e.g., “a method essentially consisting of”), the expression “essentially consisting of” means that additional elements, components, or steps may exist, but such additional elements, components, or steps do not alter the properties / activity / functionality of the product, composition, or method. As used in the context of a composition or method, the expression “consisting of” means that the composition or method referred to includes only the elements, steps, or components specifically listed in the particular embodiment or claim.

[0024] In embodiments or claims in which the term "comprising" is used as a transitional clause, such embodiments and claims may also be envisioned by replacing the term "comprising" with the terms "consisting of" or "consisting essentially of".

[0025] The enumeration of numerical ranges by endpoints includes all numbers and fractions contained within each range, as well as the enumerated endpoints.

[0026] As used herein, the term “about” when referring to measurable values ​​such as parameters, quantities, and durations means to include variations of + / -10%, preferably + / -5% or + / -1%, from a specified value, insofar as such variations are appropriate to perform in the disclosed embodiments. It should be understood that the values ​​referred to by the modifier “about” are also specifically, preferably, disclosed.

[0027] A first aspect of this disclosure relates to a method for predicting in vivo non-targeted clearance of a biomolecule. The method includes providing a cell preparation in which the cells of the preparation do not express the target of the biomolecule. The method further includes incubating the cell preparation with a culture medium containing the biomolecule under conditions that mimic physiological conditions in vivo, and determining the amount of the biomolecule taken up by the cells of the preparation after incubation. The method further includes predicting in vivo non-targeted clearance of the biomolecule based on the determination.

[0028] Where used herein, “clearance” or “CL” is defined as the volume of plasma from which a drug has been cleared over a specified period. Therefore, the unit of measurement for drug clearance is volume / hour. Clearance is equal to the rate at which a drug, such as a therapeutic protein or other biomolecule, is removed from plasma (mg / mL) divided by the concentration of that drug in the plasma (mg / mL). For therapeutic biomolecules such as antibodies, antigen-binding antibody fragments, antibody derivatives, multispecific engineered antibodies and proteins, and fusion proteins, clearance is determined by target-dependent and non-target-dependent pathways.

[0029] Target-dependent clearance (CL) resulting from the interaction between a biomolecule and its target depends on the rate at which the therapeutic biomolecule is internalized, the density of the binding target (e.g., antigen), the binding affinity, and the metabolic turnover rate of the binding target. indNonspecific cellular uptake of biomolecules is mediated by adsorbent endocytosis and / or fluid-phase endocytosis. Once inside the cellular endosomal compartment, two competing processes, namely intracellular lysosomal catabolism and neonatal Fc receptor (FcRn)-mediated recycling or transcytosis, determine the clearance of the biomolecule. From a drug development perspective, candidate therapeutic agents exhibiting high levels of nonspecific endocytosis and / or insufficient FcRn capture and recycling will increase non-target-dependent clearance. Furthermore, as described herein, high nonspecific endocytosis has been identified as a key parameter in predicting low subcutaneous bioavailability. Therefore, the cell-based assays described herein are useful quantitative tools for evaluating nonspecific endocytosis and FcRn recycling efficiency to identify pharmacokinetic responsibilities, including increased non-target-dependent clearance and low subcutaneous bioavailability, early in the drug development process.

[0030] In any embodiment, the methods described herein are useful for predicting in vivo non-target-mediated clearance of biomolecules. “Biomolecules” as used herein include, but are not limited to, any therapeutic protein, polypeptide, or nucleic acid molecule. In any embodiment, a biomolecule interacts with or binds to a specific or defined target, such as a cellular target. Such biomolecules in this disclosure are referred to as “binding molecules,” and these include therapeutic proteins, polypeptides, or nucleic acid molecules that contain or are coupled to a domain having binding specificity to a target molecule, i.e., a “binding target.” The binding target or target molecule may be a toxin, drug, protein, nucleic acid molecule, or other biomolecule. In any embodiment, the binding target is a protein expressed by a cell, such as a cell surface protein or cell surface receptor. In any embodiment, the biomolecule has binding specificity to a human target molecule, such as a human cell surface protein or human cell surface receptor.

[0031] 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). The biomolecules of this disclosure may also interact with or bind to neonatal Fc receptors (FcRn). In one embodiment, the biomolecule interacts with or binds to FcRn innately. In one embodiment, the biomolecule is engineered to bind to FcRn. In one embodiment, the biomolecule includes a fragment crystallizable (Fc) region that interacts with FcRn. In one embodiment, the biomolecule includes an Fc region engineered to increase FcRn binding affinity. In one embodiment, the biomolecule includes an Fc moiety that has been manipulated to reduce its FcRn binding affinity or to remove or delete FcRn binding. In one embodiment, the biomolecule includes an albumin domain and its variants that can bind to FcRn (e.g., the C-terminal DIII region of human serum albumin) (see, e.g., Andersen et al., J. Biol. Chem. 289(19):13492-13502 (2014) (incorporated herein by reference)). In one embodiment, the biomolecule includes an albumin-binding protein domain that indirectly binds to FcRn via bound albumin, e.g., albumin-binding domains B2A3 and B1A2B2A3 derived from Streptococcus protein G (see, e.g., Andersen et al., J. Biol. Chem. 286(7):5234-5241 (2011) (in its entirety incorporated herein by reference)).In one embodiment, the biomolecule includes an aphibody molecule, which is a low-affinity protein that directly binds to FcRn in a pH-dependent manner (see Seijsing et al., Applied Biol. Sci. 111(48):17110-17115 (2014) (the whole of which is incorporated herein by reference)).

[0032] According to this aspect and all aspects of this disclosure, a method for predicting in vivo non-target-mediated clearance of a biomolecule as described herein includes the step of providing a cell preparation that does not express the target (e.g., binding target) of the biomolecule. Suitable cell preparations include any mammalian cell preparation, e.g., human cell preparation, primate cell preparation, canine cell preparation, feline cell preparation, porcine cell preparation, rodent cell preparation, or any other suitable mammalian cell preparation. Suitable cells may 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, and tumor, and may be primary cell preparations or immortalized cell preparations, e.g., cell line preparations. Examples of cell line preparations suitable for use in the methods described herein include, but are not limited to, 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 (HUVEC).

[0033] Cell preparations useful in this embodiment of the Disclosure do not express the target molecule of the biomolecule; that is, the cells of the preparation do not express the cell surface protein or receptor to which the biomolecule binds or interacts. The cells of the preparation may not express any protein that is bound to the target or biomolecule in nature. Alternatively, the cells of the preparation may express a variant of the target that is not bound by the biomolecule; for example, a canine cell preparation may express a homolog of the target that is not bound by a human biomolecule, having binding specificity to the human-bound target. In another embodiment, the cells of the preparation are modified so as not to express the target of the biomolecule. For example, the expression of the target can be silenced using siRNA, shRNA, or other inhibitory nucleic acid molecules. Alternatively, the expression of the target can be gene-silenced using gene editing techniques.

[0034] According to this aspect of the Disclosure, if a biomolecule is a molecule that interacts with or binds to an Fc receptor, the cells of the preparation will not express an Fc receptor or protein. For example, if the biomolecule is a human immunoglobulin containing an Fc region, the cells of the preparation will 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 will not naturally express human Fc receptors such as FcαR, FcαRI, FcγRI, FcγRII, FcγRIII, FcRn, FcεRI or FcεRII, or any species equivalent thereof, that bind to the Fc portion of human immunoglobulin. In any embodiment, the cells of the preparation will not express the human FcRn protein and therefore will not express the active human FcRn / hβ2m complex. The cells of the preparation may naturally lack Fc receptor expression or may be engineered not to express such receptors (e.g., by being genetically modified to knock down or silence Fc receptor expression). In any embodiment, a suitable cell preparation includes one that has been manipulated to not express hFcRn or hβ2m, and in the absence of either protein, the cells of the preparation will not be able to express the active FcRn / hβ2m complex.

[0035] 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. Suitable proliferation and culture conditions for various mammalian cell types are well known in the art. The cells of the preparation can be seeded from a suspension onto and / or within a substrate so that they are uniformly distributed at relatively high surface and / or volume densities. The cell suspension is approximately 1 × 10⁻⁶. 4 ~Approx. 5×10 7 Culture medium at a rate of cells / ml, or approximately 2 × 10⁻⁶ 6 cells / ml ~ approx. 2×10 7 Cells / ml, or approximately 5 × 10⁻⁶ 6 It may contain cells / ml. The optimal concentration and absolute number of cells varies depending on the cell type, cell growth rate, substrate material, and various other parameters. The suspension can be formed in any physiologically acceptable medium, preferably one that does not damage the cells or impair their ability to adhere to the substrate. Suitable media include standard cell growth media, such as DMEM containing 10% FBS.

[0036] In carrying out a method to predict in vivo non-target-mediated clearance, the biomolecule of interest is added to the cell culture medium, and the cells are cultured under other normal culture conditions (e.g., 37°C, 5% CO2, and 20% O2) for a period suitable to allow non-target-mediated cell uptake to occur in the presence of the biomolecule, e.g., 30 minutes, 60 minutes, 90 minutes, 120 minutes, 180 minutes, 3 hours, 4 hours, 5 hours, or longer. In any embodiment, the cells are cultured in the medium containing the biomolecule for about 60 to about 90 minutes.

[0037] Cells are cultured in the presence of a medium containing biomolecules under conditions that mimic in vivo physiological conditions, including physiological pH. In any embodiment, physiological pH is a neutral pH, such as the pH of blood. A suitable neutral pH for a medium containing biomolecules is about 7.0 to about 8.0. In any embodiment, the medium containing biomolecules has a pH of about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, or about 8.0.

[0038] In another embodiment, the physiological pH is an acidic pH that mimics the pH typical of endosomal acidification. In any embodiment, the culture medium containing the biomolecules has a pH of about 5.6 to about 6.9. For example, the biomolecules have a pH of about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, or about 6.9.

[0039] After culturing cell preparations in a culture medium containing biomolecules under conditions that mimic physiological conditions in vivo, the amount of biomolecules taken up by the cells of the preparation is determined. In any embodiment, a first cell preparation is cultured in a culture medium containing biomolecules at a neutral pH, and a second cell preparation, otherwise identical to the first cell preparation, is cultured in a culture medium containing biomolecules at an acidic pH. A comparison of the amount of biomolecules taken up by cells exposed to biomolecules under neutral pH conditions and cells exposed to biomolecules under acidic pH conditions demonstrates the pH dependence of non-targeted mediated uptake of biomolecules.

[0040] In one embodiment, determining the amount of biomolecules taken up by cells of a preparation includes permeabilizing cells of the preparation and detecting the amount of biomolecules taken up by cells. To detect biomolecules taken up by cells, biomolecules can be directly or indirectly labeled with a detectable moiety. For example, in one embodiment, biomolecules are directly conjugated to a detectable moiety, such as a fluorescent moiety, before being cultured with cells, and the amount of biomolecules taken up by cells is determined by detecting the amount of the detectable moiety in the permeabilized cells. Alternatively, biomolecules can be directly conjugated or coupled with an enzyme label or a small molecule label (e.g., biotin), and the amount of biomolecules taken up by 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 permeabilized cells with an antibody having binding specificity to the biomolecule (e.g., an IgG or IgM molecule), an antibody fragment (e.g., Fab F(ab'), an Fc fragment, a single-domain antibody, a gamma chain of IgG, an Fc5μ of IgM, or a Mu chain of IgM) or an antibody derivative (e.g., scFv), where the antibody is conjugated to the detectable moiety (i.e., a labeled secondary antibody). Suitable detectable moieties include, but are not limited to, fluorescent molecules, small molecules, enzyme labels, and radioisotopes, as are readily known in the art. After this incubation, the amount of the detectable moiety is detected to determine the amount of biomolecules taken up by the cells.

[0041] Suitable methods for detecting detectable parts include those known in the art, readily available, and described in the examples herein. For example, in any embodiment, the detectable part is a fluorescent part, such as Alexa Fluor dye, fluorescein, Oregon Green dye, rhodamine dye, Texas Red dye, and / or derivatives thereof, which are detected via a fluorophotometer in immunoassays (e.g., ELISA), fluorescence microscopy, or flow cytometry. Other suitable detectable parts include radioisotopes (e.g.,) that can be detected using immunoassays, flow cytometry, spectroscopy, microscopy, confocal microscopy, liquid chromatography with tandem mass spectrometry, or scintigraphy, as are well known in the art. 14 C, 125 I, 32 P, 35 Examples include luminescent proteins, enzyme proteins (e.g., horseradish peroxidase, alkaline phosphatase, glucose oxidase, etc.), and small molecules (e.g., biotin).

[0042] Once a detectable portion is detected, it is compared to one or more reference values ​​to quantify the amount of biomolecules taken up by the cell, thereby providing a value for nonspecific endocytosis. For example, a standard curve or calibration curve representing biomolecular binding capacity can be created, as described herein. The amount of biomolecules detected in the cell is compared to or plotted against the standard curve to quantify the amount of biomolecules taken up by the cell. As described herein, the standard curve can be created using commercially available microspheres coated with an anti-Fc antibody (e.g., an anti-IgG antibody) that binds to the biomolecule of interest. The level of nonspecific endocytosis of the biomolecule determined from the calibration curve is compared to the level of nonspecific endocytosis of one or more internal reference antibodies to predict the nontarget-mediated clearance of the biomolecule. In a preferred embodiment, the amount of biomolecules taken up by the nonspecific endocytosis means, as determined from the calibration curve, is compared to the corresponding values ​​of at least two reference antibodies, one of which has a low non-target clearance (e.g., less than 4.5 mL / kg / d) and the other having a high non-target clearance (e.g., greater than 4.5 mL / kg / d).

[0043] Alternatively, the amount of biomolecules detected in cells is compared with one or more internal reference antibodies included in the assay, and the nonspecific endocytosis and in vivo nontarget-dependent clearance of the reference antibodies, which are detected together with the biomolecules, have been previously quantified and are known. The nontarget-mediated clearance of the biomolecule of interest is predicted by comparing the level of nonspecific endocytosis of the biomolecule being tested with the level of nonspecific endocytosis of one or more internal reference antibodies. In a preferred embodiment, the amount of biomolecules taken up by nonspecific endocytosis is compared with at least two reference antibodies, one of which has a low nontarget clearance (e.g., less than 4.5 mL / kg / d in humans) and the other having a high nontarget clearance (e.g., greater than 4.5 mL / kg / d in humans).

[0044] By utilizing the level of nonspecific endosomal uptake, human subcutaneous bioavailability (F SQ ) can be indicated and predicted, and here, high levels of nonspecific endocytosis are low F SQ (For example, less than 50%), and low levels of nonspecific endocytosis are high F SQ (For example, greater than 50%). In any embodiment, thresholds for "high," "medium," and "low" levels of nonspecific endocytosis can be prepared using multiple reference antibodies as described herein (see, for example, Example 9 and Figure 21). For example, a low nonspecific endocytosis threshold is a CL of less than 4.5 mL / kg / d in humans. ind and / or F exceeding 50% SQ This is identified by defining the mean quantitative value of nonspecific endocytosis for one or more reference mAbs having the following characteristics: The upper limit of the 95% confidence interval for this mean can serve as a reference range for low nonspecific uptake. Moderate nonspecific uptake is defined as a nonspecific endocytosis value corresponding to two standard deviations + low CL. ind This can be established by the mean endocytosis. Tested biomolecules with moderate nonspecific uptake are CL ind Increase and / or F SQ A decrease in [specific factor] is highly likely. Any factor exceeding a moderate uptake threshold is associated with high nonspecific endocytosis and high CL in humans. ind and / or low F SQ It is thought that this may have the potential to be significantly improved.

[0045] Another aspect of the present disclosure relates to a method for predicting in vivo non-target-mediated clearance of neonatal Fc receptor (FcRn) interacting molecules. The method enhances in vivo prediction of non-target-mediated clearance of biomolecules by evaluating both nonspecific endocytosis and FcRn recycling. The method includes providing a first cell preparation in which the cells of the first preparation do not express human FcRn, and providing a second cell preparation in which the cells of the second preparation express a heterodimer of human neonatal Fc receptor (hFcRn) and human β2m (hβ2m). The cells of the first and second cell preparations do not express targets of FcRn interacting molecules. The method further includes subjecting the first and second cell preparations to first and second incubation periods, the first incubation period including incubation of the cell preparations with a medium containing FcRn interacting molecules under acidic conditions, non-acidic conditions, or acidic and non-acidic conditions. The second incubation period comprises incubating the cell preparation with a medium lacking FcRn interacting molecules under non-acidic conditions after the first incubation. The method further comprises determining the amount of FcRn interacting molecules taken up by the cells of the first and second preparations after the first incubation period and / or the second incubation period, and measuring the amount of FcRn interacting molecules in the medium after the second incubation period. The method further comprises quantifying nonspecific endocytosis and FcRn recycling of FcRn interacting molecules based on the determination and measurement steps, i.e., nonspecific endocytosis is quantified based on the amount of FcRn interacting molecules determined to be taken up by the cells, and FcRn recycling of FcRn interacting molecules is quantified based on the amount of FcRn interacting molecules measured in the medium after the second incubation. In vivo nontarget-mediated clearance of FcRn interacting molecules is predicted based on the quantification step.

[0046] The method of this disclosure provides a tool for in vitro assessment of potential responsibility for non-target-mediated clearance of candidate therapeutic molecules. The method can be used to facilitate prediction or estimation of in vivo non-target-mediated clearance of specific candidate therapeutic molecules, or to rank the in vivo non-target-mediated clearance of candidate molecules to assist in lead candidate selection and optimization while reducing the number of molecules tested in animal models.

[0047] In this aspect of the Disclosure, the “FcRn interacting molecule” as referred to herein 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 bonded to β2-microglobulin. FcRn plays a crucial role in maintaining in vivo levels of IgG and albumin by preventing lysosomal degradation of these molecules within cells. Accordingly, exemplary FcRn interacting molecules for the purposes of this disclosure include, but are not limited to, molecules containing an Fc domain (e.g., the Fc domain of IgG), an albumin domain (e.g., full-length albumin or its C-terminal DIII fragment), an albumin domain variant having enhanced FcRn binding affinity (e.g., Andersen et al., J. Biol. Chem. 289(19):13492-13502 (2014) (incorporated herein by reference)), an albumin-binding protein domain derived from Streptococcus protein G or other Gram-positive bacteria (e.g., Andersen et al., J. Biol. Chem. 286(7):5234-5241 (2011) (in its entirety incorporated herein by reference)), or low-affinity proteins (affibodies) that directly bind to FcRn in a pH-dependent manner (Seijsing et al., Applied See Biol.Sci.111(48):17110-17115(2014) (the entire work is incorporated herein by reference).

[0048] In one embodiment, an FcRn interacting molecule comprises a domain or sequence portion that binds to human FcRn. Suitable FcRn interacting molecules for the purposes of this disclosure also include molecules comprising an FcRn-binding domain that has been manipulated to have an enhanced FcRn-binding affinity compared to the binding affinity of an unmanipulated version of the domain (i.e., including substitution, insertion, or deletion of one or more amino acids). FcRn interacting molecules also include molecules comprising an FcRn-binding domain that has been manipulated to have a reduced FcRn-binding affinity compared to the FcRn-binding affinity of an unmanipulated version of the domain. FcRn interacting molecules further include molecules comprising an FcRn-binding domain that has been manipulated to delete or remove FcRn binding. In any embodiment, an FcRn interacting molecule may also interact with or bind to a non-FcRn cell target, such as a cell surface receptor or cell surface ligand.

[0049] Exemplary FcRn-interacting molecules include antibodies (e.g., human immunoglobulins including IgG1, IgG2, IgG3, and IgG4 immunoglobulins), multispecific antibodies (e.g., human bispecific or tripspecific antibody proteins containing an IgG Fc domain or other FcRn-binding domains), antigen-binding fragments of antibodies containing an IgG Fc domain or other FcRn-binding domains, antigen-binding fragments of multispecific antibodies containing an IgG Fc domain or other FcRn-binding domains, and antigen-binding proteins such as antibody derivatives containing an IgG Fc region or other FcRn-binding domains. FcRn-interacting molecules also include therapeutic proteins and candidate therapeutic proteins (e.g., multispecifically engineered proteins, recombinant proteins, Fc fusion proteins, albumin fusion proteins) and nucleic acid molecules containing FcRn-binding domains (e.g., siRNA molecules, antisense oligonucleotides, aptamer molecules, and mRNA molecules).

[0050] According to this aspect of the Disclosure, suitable first and second cell preparations include any of the above-mentioned mammalian cell preparations, for example, human cell preparations, primate cell preparations, canine cell preparations, feline cell preparations, porcine cell preparations, or rodent cell preparations. Suitable cells may 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, and tumor, and may be primary cell preparations or immortalized cell preparations, such as cell lines. Exemplary cell line preparations include, but are not limited to, 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 (HUVEC).

[0051] This aspect of the present disclosure requires the use of first and second cell preparations, which differ in their expression of human neonatal Fc receptor (FcRn). For example, cells of the first cell preparation do not express hFcRn, while cells of the second cell preparation express functional FcRn, i.e., a heterodimer of human FcRn and human β2m (hβ2m).

[0052] The cells of the first cell preparation may be cells that do not naturally express hFcRn, for example, CHO cells are hamster kidney cells that do not express human FcRn. Alternatively, for the purpose of carrying out this method of disclosure, the cells of the preparation may be modified to silence the expression of hFcRn. In any embodiment, cells expressing hFcRn may be made suitable for use as the first cell preparation by silencing the expression of hFcRn and / or hβ2m using siRNA or other inhibitory nucleic acid molecules. Alternatively, cells expressing hFcRn and hβ2m may be genetically modified using gene editing techniques to “knock down” or “knock out” the expression of hFcRn and / or hβ2m.

[0053] The cells of the second cell preparation may spontaneously express the functional hFcRn protein. For example, a suitable second cell preparation may include a human cell preparation expressing hFcRn and hβ2m. Alternatively, the cells of the second preparation may not spontaneously express the functional hFcRn protein and may need to be modified to achieve such expression. In this embodiment, the cells of the second preparation may be modified to express one or more heterologous genes, for example, the FCGRT gene encoding the human neonatal Fc receptor and / or the B2M gene encoding the human β-2-microglobulin protein.

[0054] According to this aspect of the disclosure, the first and second cell preparations do not express targets of FcRn interacting molecules, such as binding targets of FcRn interacting molecules. As described above, suitable cells may not naturally express targets of FcRn interacting proteins. Alternatively, suitable cells may include cells that express target variants (e.g., target species variants) that do not bind to or interact with human FcRn interacting molecules. Furthermore, in another embodiment, suitable cells are cells modified to silence the expression of targets.

[0055] As described above, this method for predicting in vivo non-target-mediated clearance of FcRn interacting molecules includes first and second incubation periods. During the first incubation period, the first and second cell preparations are incubated with a culture medium containing the FcRn interacting molecules. This first incubation period is referred to herein as the “loading phase,” during which non-target-mediated cellular uptake of the FcRn interacting molecules can occur via nonspecific endocytosis, FcRn-mediated uptake, or a combination of both. In one embodiment, this first incubation is carried out under non-acidic, neutral pH conditions. Thus, in one embodiment, a subset of cells from each of the first and second cell preparations is incubated with a culture medium containing the FcRn interacting molecules under neutral pH conditions. Suitable neutral pH conditions include a medium pH of about 7.0 to about 8.0, preferably about 7.2, about 7.3, about 7.4, about 7.5, or about 7.6.

[0056] In some cases, FcRn interacting molecules do not exhibit high levels of nonspecific endocytosis at neutral pH. In other situations, it is desirable to use smaller amounts of FcRn interacting molecules, such as in the early stages of development when material quantities are limited. In these cases, the loading phase is performed under acidic pH conditions to enhance total endocytosis (i.e., nonspecific and FcRn-mediated) to ensure that a sufficient amount of FcRn interacting molecules are loaded into the cells and the FcRn recycling efficiency of the molecules can be evaluated. Thus, in one embodiment, subsets of cells from each of the first and second cell preparations are incubated with a culture medium containing FcRn interacting molecules under acidic pH conditions. As described above, acidic pH conditions include a medium pH of about 5.6 to about 6.9, preferably about 5.6, about 5.7, about 5.8, about 5.9, or about 6.0.

[0057] When the first incubation period is carried out under acidic pH conditions, it is preferable to use FcRn interacting molecules at concentrations that do not saturate the FcRn receptor binding capacity within FcRn-expressing cells. As described herein, FcRn interacting molecules with high FcRn affinity (e.g., antibodies containing YTE substitution (M252Y / S254T / T256E)) can saturate FcRn binding under acidic conditions, leading to inaccurate results. To accurately assess the nonspecific endocytosis and FcRn recycling efficiency of these molecules, the concentration of FcRn interacting molecules added to the culture medium for the initial incubation is optimized to achieve less than 100% FcRn binding (e.g., optimized to achieve approximately 90% FcRn binding occupancy).

[0058] In another embodiment, a subset of cells from each of the first and second cell preparations is incubated with a culture medium containing the 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 a culture medium containing the FcRn interacting molecule under non-acidic neutral pH conditions. Evaluating the cell uptake of novel FcRn interacting molecules under neutral (non-acidic) and acidic conditions provides a means to determine the tendency of molecules to exhibit high levels of nonspecific endocytosis at physiologically relevant pH and simultaneously measure FcRn recycling. As described above, the FcRn interacting molecule may be a molecule containing an FcRn-binding domain that has been manipulated to reduce or eliminate FcRn-binding affinity. These FcRn interacting molecules can be incubated with the first and second cell preparations under both acidic and non-acidic conditions to confirm the reduction or elimination of FcRn-mediated uptake and / or recycling of the manipulated molecule.

[0059] According to a disclosed method for predicting in vivo non-target-mediated clearance of FcRn interacting molecules, first and second cell preparations are subjected to a first incubation period. During this first incubation period, the cell preparations are incubated with a medium containing FcRn interacting molecules at a concentration that does not saturate the FcRn-mediated uptake capacity of the cells in the preparation. As demonstrated herein, FcRn interacting molecules engineered to have enhanced FcRn binding can more readily saturate FcRn binding under acidic conditions. When the FcRn binding capacity per cell is saturated, nonspecific endocytosis and FcRn recycling parameters cannot be properly or accurately assessed. Therefore, in one embodiment, the concentration of FcRn interacting molecules in the medium during the first incubation is such that it achieves less than 100% of the FcRn occupancy per cell of the cells in the second preparation. In one embodiment, the concentration of FcRn interacting molecules in the culture medium is such that the FcRn occupancy rate per cell of the cells in the second preparation is less than 99%. In one embodiment, the concentration of FcRn interacting molecules in the culture medium is such that the FcRn occupancy rate per cell of the cells in the second preparation is more than 25%. In one embodiment, the concentration of FcRn interacting molecules in the culture medium is such that the FcRn occupancy rate per cell of the cells in the second preparation is between 25% and 99%. In one embodiment, the concentration of FcRn interacting molecules in the culture medium is such that the FcRn occupancy rate per cell of the cells in the second preparation is between 25% and 90%. In one embodiment, the concentration of FcRn interacting molecules in the culture medium is such that the FcRn occupancy rate per cell of the cells in the second preparation is between 50% and 99%. In one embodiment, the concentration of FcRn interacting molecules in the culture medium is such that the FcRn occupancy rate per cell of the cells in the second preparation is between 50% and 90%.

[0060] The appropriate concentration of an FcRn interacting molecule can be determined by performing a concentration-dependent FcRn interaction test, which indicates the concentration of the FcRn interacting molecule that achieves a defined level of FcRn occupancy (e.g., 50% FcRn occupancy per cell in cells within a cell preparation). This is preferably determined for each type or format of the FcRn molecule being tested, where the type or format refers to the FcRn interacting portion of the molecule (e.g., the Fc portion). Exemplary types or formats of FcRn interacting molecules include, but are not limited to, wild-type IgG Fc portions, IgG Fc portions engineered to enhance FcRn binding, and IgG Fc portions engineered to reduce or eliminate FcRn binding. When comparing non-target-mediated cell uptake of FcRn interacting molecules having different formats using the assays described herein, it is important to optimize the concentration of each molecular format being tested to equalize the amount of each FcRn interacting molecule being loaded into cells via the FcRn-mediated mode.

[0061] In one embodiment, a suitable concentration-dependent FcRn interaction test includes a concentration-dependent FcRn binding test. Using the binding affinity curve generated in the concentration-dependent FcRn binding test, the equilibrium dissociation constant (K) between the FcRn interacting molecule and FcRn is determined. D ) can be determined. K D K is the concentration of FcRn interacting molecules to which 50% of all FcRn receptors are bound. Therefore, in one embodiment, the appropriate concentration of FcRn interacting molecules in the culture medium for the initial incubation is K D In one embodiment, the appropriate concentration of FcRn interacting molecules in the culture medium for the initial incubation is 1 / 2K. D (In order to achieve an FcRn occupancy of at least 25%). In one embodiment, the appropriate concentration of FcRn interacting molecules in the culture medium for the first incubation is twice the K. D(To achieve an FcRn occupancy rate of approximately 90%). In one embodiment, the appropriate concentration of FcRn interacting molecules in the culture medium for the first incubation is 2 to 10 times K. D This is because (to achieve an FcRn occupancy rate of approximately 90% to 99%).

[0062] In one embodiment, a suitable concentration-dependent FcRn interaction test includes a concentration-dependent endocytosis assay as described herein. The Km of the FcRn interacting molecule can be determined using the cell uptake rate curve generated in the concentration-dependent endocytosis test. As described herein, Km is half the maximum rate or concentration reaching about 50% of the FcRn binding capacity. Thus, in one embodiment, the suitable concentration of the FcRn interacting molecule in the medium for the first incubation is the Km determined from the endocytosis test. In one embodiment, the suitable concentration of the FcRn interacting molecule in the medium for the first incubation is 1 / 2 Km (to achieve at least 25% of the FcRn binding capacity). In one embodiment, the suitable concentration of the FcRn interacting molecule in the medium for the first incubation is twice Km (about 90% of the FcRn binding capacity). In one embodiment, the appropriate concentration of FcRn interacting molecules in the culture medium for the first incubation is 2 to 10 times Km (approximately 90%-99% FcRn binding capacity).

[0063] As described above, the first incubation period in the disclosed method for predicting in vivo non-target-mediated clearance of FcRn interacting molecules is referred to as the “loading phase” in which non-target-mediated cell uptake occurs via nonspecific endocytosis, FcRn-mediated uptake, or a combination thereof. The first and second incubation periods are carried out for a duration sufficient for nonspecific endocytosis and / or FcRn-mediated uptake to occur, e.g., approximately 30 minutes, 60 minutes, 90 minutes, 120 minutes, 180 minutes, 3 hours, 4 hours, or longer at 37°C. The amount of FcRn interacting molecules taken up by cells after the first incubation period is referred herein to as the “total uptake” amount or “uptake concentration.” This amount can be determined in subsets of the first and second cell preparations by permeabilizing the cells and detecting and quantifying the amount of FcRn interacting molecules present in the cells as described above.

[0064] In the remaining first and second cell preparations, the medium containing FcRn interaction molecules is removed, and the cells are washed once or multiple times with the medium or saline to remove any FcRn interaction molecules that have not been taken up by the cells. The cells are then subjected to a second incubation period to evaluate the FcRn recycling of FcRn interaction molecules. This second incubation period is the "recycling phase" and is carried out for a sufficient amount of time for FcRn capture of FcRn interaction molecules in the endosomal compartment, endosomal transport to the cell surface, and release / recycling of FcRn interaction molecules into the medium. This incubation period may be approximately 30 minutes, 60 minutes, 90 minutes, 120 minutes, 180 minutes, 3 hours, 4 hours, 5 hours, 6 hours, or longer. After the second incubation period, the recycling phase medium is collected, recycled, and the amount of FcRn interaction molecules released into the medium is detected and quantified. The amount of detected FcRn interacting molecules is quantified by comparison with the internal reference value or standard curve generated as described above. The "recycled amount" or "recycled concentration" of FcRn interacting molecules is obtained by subtracting any amount of FcRn interacting molecules detected and measured in the medium of the first cell preparation (not expressing FcRn) from the amount of FcRn interacting molecules in the medium of the second cell preparation (expressing FcRn) after the second incubation.

[0065] Finally, the amount of FcRn interacting molecules remaining in the cells of the second cell preparation after the first and second incubations is determined after the second incubation period. This amount of FcRn interacting molecules remaining in the cells is referred to herein as the “remaining amount” or “remaining concentration” of FcRn interacting molecules. The FcRn interacting molecules remaining in the cell preparation are detected and quantified as described above, i.e., by directly or indirectly labeling the FcRn interacting molecules at a detectable portion and detecting the detectable portion by flow cytometry, microscopy, spectroscopy, scintigraphy or other immunoassays.

[0066] In any embodiment, the hFcRn Recycle Efficiency Metric (FREM) score can be calculated by applying the following formula using the recycled concentration, residual concentration, and incorporated concentration of FcRn interacting molecules obtained while carrying out the method of the present disclosure:

number

[0067] In the above formula, R X This is the recycled concentration of the FcRn interacting molecule "X" from the sample at 37°C after loading and the subsequent recycling stage, and RA X This is the residual concentration of the FcRn interacting molecule "X" from the sample at 37°C after loading and subsequent recycling stages (Grevys, et al. iScience 25, 103746 (2022); and Grevys, et al. Nat Commun 9, 621 (2018) (the whole of which is incorporated herein by reference)).

[0068] First and second cell preparations for each FcRn interacting molecule, namely, FcRn ー and FcRn + Using the uptake concentration by expressing cells, the nonspecific uptake coefficient (NUC) is calculated using the following formula:

number

[0069] In the above equation,

number

number

[0070] In one embodiment, when the NUC value is between 0 and 1 (0 < NUC < 1), it is an indicator that the cell uptake process is dominated by FcRn-independent non-specific endocytosis (Grevys, et al. iScience 25, 103746 (2022), which is incorporated herein by reference in its entirety). In these cases, multiply the FREM score by NUC. When NUC > 1, it indicates FcRn-mediated uptake of the molecule ("X") in negligible non-specific endocytosis. For these molecules, do not multiply the FREM score by the NUC value.

[0071] As demonstrated herein, the FREM score is inversely correlated with the target-independent clearance value, and a higher FREM score, i.e., a higher recycling efficiency, predicts low target-independent clearance, and a lower FREM score, i.e., a lower recycling efficiency, predicts high target-independent clearance (see Figure 17C).

[0072] The following examples in which the present invention is described are presented for illustrative purposes and not for limitation.

Examples

[0073] Materials and Methods for Examples 1-3 Cell Culture: Parental Chinese hamster ovary-K1 (CHO-K1) cells were maintained in hamster F-12K medium (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 containing 10% heat-inactivated fetal bovine serum (ATCC; 30th 2003). Cells were cultured without antibiotics in a humidified 5% CO2 incubator at 37°C and subcultured using 0.05% trypsin-EDTA (Thermo, 25300054). Mycoplasma negative testing was performed using a MycoAlert Detection Kit (Lonza). Cells were cryopreserved using complete growth medium containing 5% (v / v) DMSO.

[0074] Monoclonal antibodies (mAbs): Two fully human wild-type mAbs were used for initial characterization: an IgG2 anti-IL-4Rα mAb (AMG 317) and an IgG1 control mAb induced against streptavidin (anti-streptavidin antibody, ASA). Both mAbs were constructed using recombinant DNA technology along with the anti-IL-4Rα mAb mutant lineage and produced in stably transfected CHO cells using a standardized protocol. The preclinical mAb panel included five antibodies produced against G protein-coupled receptors (mAbs B1-B5). Only the anti-IL-4Rα mAb and ASA were wild-type IgG. Other mAbs were produced in stably transfected CHO cells using Amgen's patented stable, effector-free Fc backbone, as previously reported (Liu et al., J. Biol. Chem 292(5):1876-1883(2016) (the whole is incorporated herein by reference)).

[0075] PK Testing and Analysis: PK data were obtained from three separate tests. Female wild-type C57BL / 6J (for ASA and anti-IL-4Rα mAb) and Balb / C (for mAb B1-B5 panel) mice were purchased from Jackson Laboratory (Bar Harbor, MA). The target proteins were administered intravenously via the lateral tail vein as intravenous bolus doses of 1 mg / kg (anti-IL-4Rα mAb), 2 mg / kg (mAb B1-B5 preclinical panel), or 3 mg / kg (ASA). Blood samples were collected at various time points after injection and incubated at ambient temperature for approximately 20 minutes or until complete coagulation occurred, then centrifugation was performed to separate the serum. All serum samples were stored at -70°C (±10°C) until use in analytical assays. Mice were cared for according to the Guide for the Care and Use of Laboratory Animals, 8th Edition, from the internationally accredited facility AAALAC. All mouse protocols were approved by Amgen, Inc. Institutional Animal Care and Use Committee (Thousand Oaks, CA).

[0076] Protein quantification in mouse serum was performed by electrochemiluminescence immunoassay using an MSD Sector600 instrument (Meso Scale Diagnostics, Rockville, Maryland) with anti-human Fc antibodies as both capture and detection reagents. In all assays, serum analyte concentrations were interpolated from standard curves using corresponding analytes prepared in pooled mouse serum with Watson LIMS software (Thermo).

[0077] The mouse PKs of all tested mAbs were described using a two-compartment model with a central compartment (Tang et al., J. Parm Sci. 93(9):2184-204 (2004) (the entire model is incorporated herein by reference)). Parameters included concentration and volume within each compartment, exclusion from the central compartment, and distribution between compartments. Computational modeling was performed using Phoenix (Certara, Princeton, NJ) with mean serum concentration-time profiles, and plotted using GraphPad Prism (Dotmatics, Boston, MA). This method was chosen because linear clearance was assumed for all mAbs used in doses due to the lack of measurable target binding in mice. Specifically, to report the human clearance of anti-IL-4Rα mAbs, previously reported CL ind The value was normalized to 80 kg because this weight was used to model its PK (Kakkar et al., Pharm Res. 28 (10:2530-42 (2011) (the whole is incorporated herein by reference))).

[0078] Cell uptake test: CHO-K1 and Vero cells were seeded in 96-well plates at a density of 150,000 cells per well 24 hours prior to the experiment, or at a density of 75,000 cells per well 48 hours prior to the experiment. On the day of the test, the cells were washed twice with preheated (37°C) or ice-cold (4°C) 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). A third wash of fresh Ringer's solution was added, and the cells were equilibrated at either temperature for 30 minutes. The wells were then aspirated, and the treatment was immediately applied. The test utilized conditions of 37°C (to enable cell surface binding, internalization, and intracellular transport) and 4°C (to enable cell surface binding). For time-dependent studies, CHO-K1 cells were incubated with 100 μg / mL mAb for 15, 30, 60, or 120 minutes. For concentration-dependent studies, CHO-K1 cells were subjected to 1:2 dilutions ranging from 100 to 25 μg / mL for 60 minutes. These conditions were based on preliminary data that showed concentrations were below the detection saturation limit. After incubation, cells were washed four times on ice with ice-cold Ringer's solution and then trypsinized at 37°C for 4 minutes. After detaching the cells, trypsin was inhibited by adding complete ice-cold growth medium in a 1:1 ratio (v / v). Cells were removed from the cell culture plate and placed in a V-bottom 96-well plate (Fisher #249944), and then centrifuged at 300 g for 5 minutes at 4°C. Cells were stained in 100 μL of 1×PBS containing 0.5% (v / v) Zombie UV-fixable viable dye (BioLegend #423108) on ice for 20 minutes. The cells were washed once with 100 μL of FACS buffer (1×PBS, 2% w / v BSA, 1 mM EDTA, 0.1% w / v sodium azide), centrifuged at 300 g for 5 minutes at 4°C, and then fixed / permeabilized with Cyto-Fast fix / perm buffer (Biolegend #426803) in the dark at room temperature for 20 minutes. The cells were then washed twice with 1× Cyto-Fast Perm Wash solution (Biolegend #426803).Next, the sample was incubated with Alexa Fluor 647 (Ab35-AF647) at a fluorescence conjugate concentration of 5 μg / mL (100 μL / well, Cyto-Fast Perm Wash solution) on ice in the dark for 30 minutes (Hall et al., J.Immunol.Methods 393(1-2):70-73 (2013) (the entire sample is incorporated herein by reference)). The sample was then washed three times with Cyto-Fast Perm Wash solution, resuspended in 100 μL of Cyto-Fast Perm Wash solution, and BD Biosciences High Throughput Sampler (catalog no. 338301) equipped with an 18-color, 5-laser configuration (UV-355nm, violet-405nm, blue-488nm, yellow / green-561nm, red-637nm). Analysis was performed using a FACSymphony flow cytometer. Gating was performed as shown in the figure, and 10,000 single cells were analyzed using BD Diva software, Zombie. - (Biological) events were targeted. For Vero cell samples, approximately 5000 single biological events were captured due to the difference in trypsin dissociation rates between Vero and CHO-K1 cell lines, resulting in fewer Vero cells being collected. Data were analyzed using FlowJo software (Becton Dickson, Franklin Lakes, NJ) to obtain the median fluorescence intensity of the indicated cell population.

[0079] Antibody-nonspecific endocytosis (NSE) by fluorescence microscopy: An endocytosis test was performed as described above using either 100 μg / mL of ASA or anti-IL-4Rα mAb at 37°C for 60 minutes, after which the cells were washed four times on ice with ice-cold Ringer's solution. The cells were then fixed with 4% paraformaldehyde in 1× PBS for 15 minutes, followed by two washes with 1× PBS. The samples were then blocked and permeabilized in 1× Cyto-Fast Perm Wash buffer at room temperature for 1 hour. After aspiration, the wells were stained with 0.5 μg / mL Ab35-AF647 in 1× Cyto-Fast Perm Wash buffer at room temperature for 1 hour. Next, the samples were washed three times with 1× Cyto-Fast Perm Wash buffer and then stained with 1 μg / mL Hoechst (Thermo#H1399, diluted with 1× PBS) and 2 μg / mL HCS Cell Mask Blue (Thermo#H32720 diluted with DMSO) in 1× Cyto-Fast Perm Wash buffer for 30 minutes at room temperature. The wells were then washed three times with 1× PBS and imaged using an OperaPhenix high-content screening system (PerkinElmer) with a 40× aqueous objective lens with Z-stack acquisition. Maximum projection was obtained using Columbus software (PerkinElmer).

[0080] Antibody Binding Capacity: Quantum® Simply Cellular® mouse IgG beads were obtained from Bio-Rad (#FCSC815; Lot #15515; Hercules, CA). Initial evaluation utilized final Ab35-AF647 concentrations of 5 or 10 μg / mL, precisely following vendor product information, in all procedures performed on ice or at 4°C. Subsequent tests demonstrated that this concentration saturated each bead population with antibody, so a concentration of 5 μg / mL was used to match cell staining experiments. For each uptake test, freshly stained beads were analyzed using the same flow cytometer with the same instrument parameters for the cell sample of the day. Median fluorescence intensity of the beads was obtained using FlowJo software and then plotted against the known antibody binding capacity per bead population supplied by the vendor via GraphPad Prism. The data were fitted linearly using simple linear regression. The antibody binding capacity for each cell sample was then interpolated from the median cell fluorescence intensity.

[0081] Homology Modeling and Anti-IL-4Rα mAb Mutant Design: Antibody homology models were constructed using the default settings of the Molecular Operating Environment (MOE v2022.02, Chemical Computing Group; Montreal, Canada) and Antibody modeler (Amber10 forcefield, template search by identity, highest-scoring template used in the model, number of models = 1). For each antibody, the Fv region was modeled using the template with the highest identity for the framework and complementarity-determining region (CDR). Following a structural template search of the PDB database based on the similarity and identity (Kabat definition) of the framework and CDR regions, the highest-scoring structural template was used for homology modeling. Then, one homology model was constructed for each antibody. Structural preparation was performed using default settings for charge, protonation, rotomer and steric collision minimization, and energy minimization. Protein properties were calculated for the prepared homology models at pH 7.4 and pH 5.2. The protein properties of focus included Fv charge separation (between heavy and light domains), isoelectric point (pI, structure-based), zeta potential, dipole moment, hydrophobic moment, and mobility. Protein patches exceeding 50 angstroms in area (hydrophobic, positive and negative) were also calculated and visualized. The homology model is represented as a ribbon structure with surface patches colored green for hydrophobicity, blue for positive, and red for negative.

[0082] Comparison of anti-IL-4Rα mAb and ASA homology models with calculated properties highlighted the high positive charge on anti-IL-4Rα mAb Fv(+7) and the resulting higher pI(8.7). Targeted mutation designs were created to reduce charge separation between the charge patch and variable domains. Specifically, residues in the positively charged patch were mutated to negative and neutral residues, and protein attributes were calculated to evaluate the effects on the charged patch (reduction in size) and protein properties (reduction in Fv charge and pI). Triple and double mutants showed the greatest effect on the reduction of Fv charge and pI. Single mutants were also investigated, but were expected to show a small effect on overall protein properties.

[0083] Example 1: ASA and anti-IL-4Rα mAbs exhibit varying degrees of nonspecific uptake and preclinical clearance. Anti-IL-4Rα mAbs are therapeutic proteins that lacked wild-type mouse PK data due to the use of mouse surrogate mAbs in previous preclinical rodent studies. To better understand the mechanism driving high rates of anti-IL-4Rα mAb CL, PK studies were conducted in wild-type animals to compare non-target-mediated elimination of anti-IL-4Rα mAbs with a reference mAb, ASA. Single-dose PK studies in wild-type mice demonstrated that anti-IL-4Rα mAbs had approximately three times higher CL than ASA (Table 1, Figure 1). Previous studies have not reported a significant effect of IgG subclasses on linear PKs of mAbs, so this result was not thought to be due to differences in IgG subclasses between ASA and anti-IL-4Rα mAbs (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 (these are incorporated herein by reference in their entirety)). Rather, observations made in wild-type mice indicated a role in nonspecific clearance based on the absence of the target, which is due to negligible cross-reactivity of anti-IL-4Rα mAbs with mouse IL-4Rα.

[0084] [Table 1]

[0085] Previous analyses of Phase 1 and Phase 2 trials of anti-IL-4Rα mAbs demonstrated that anti-IL-4Rα mAbs exhibited non-dose-proportionality in plasma exposure, showing target-mediated kinetics at the evaluated dose. Clinical outcomes were mathematically described using a two-compartment population PK model with parallel linear (target-independent) and non-linear (target-mediated) CL pathways. From this report, anti-IL-4Rα mAbs were found to have an estimated CL of 10.5 mL / d / kg. ind This was shown (Table 1), which represents the mean CL of 64 clinical mAbs evaluated by Grinshpun et al. indThe value is more than twice the normal value. (4.84 ± 4.72 SD) (Kakkar et al., “Population PK and IgE pharmacodynamic analysis of 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 (these are incorporated herein by reference in their entirety)). This linear CL was also significantly higher when compared to that of dupilumab, a human IgG4 monoclonal antibody that binds to the same target antigen as the anti-IL-4Rα mAb (human IL-4Rα) (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 (the entire article is incorporated herein by reference)). In summary, these findings indicate that target-mediated arrangement was not the sole reason for the high CL of anti-IL-4Rα mAbs in humans.

[0086] Nonspecific endocytosis leads to rapid CL of anti-IL-4Rα mAb indTo determine whether they contribute to the process, the internalization dynamics of ASA and anti-IL-4Rα mAb were analyzed using parental CHO-K1 cells. This strain was selected because it is highly suitable for flow cytometry, easy to proliferate, widely used, and non-human. Of the two mAbs tested, the anti-IL-4Rα mAb showed significant endocytosis with biphasic, time-dependent (Figure 2B) and linear, concentration-dependent (Figure 2C) uptake under the tested conditions. ASA was barely distinguishable from the untreated control, with both populations showing strong overlap (Figure 2A, lower left panel). Furthermore, very low signals were observed for both mAbs at 4°C, indicating negligible cell surface binding compared to the degree of internalization at 37°C. Confocal microscopy performed after separate 60-minute uptake studies at 37°C using 100 μg / mL of ASA or anti-IL-4Rα mAb confirmed extensive internalization of anti-IL-4Rα mAb compared to ASA and untreated controls (Figure 2D). These combined observations supported extensive nonspecific endocytosis of anti-IL-4Rα mAb compared to ASA in CHO-K1 cells. Furthermore, nonspecific uptake of ASA was essentially undetectable compared to untreated controls. This result highlights the detectable responsibility that has been proposed as a contributing factor to the rapid elimination of anti-IL-4Rα mAb in vivo.

[0087] Example 2: Development of a quantitative flow cytometry method enabling day-to-day comparisons One limitation of the initial endocytosis assay replication was the inability to compare the degree of uptake across experimental days. This is because the readout is the median fluorescence intensity, a variable influenced by various factors, including different voltage settings on the cytometer and the use of separate instruments over several days. To standardize the assay to enable day-to-day comparisons and reliable database creation of molecular attributes, we optimized a quantitative flow cytometry method using commercially available anti-mouse IgG microspheres. Each population of these beads binds to a predetermined amount of antibody, which can be used to calculate the antibody-binding capacity (ABC) of a cell population (i.e., the amount of antibody associated with a single viable cell event). Each bead standard was saturated under experimental conditions. When stained with 5 or 10 μg / mL anti-human Fc mouse mAb directly conjugated with Alexa Fluor 647, the histograms of each bead population overlapped (Figure 3A). Both concentrations yielded standard curves with a similar degree of linearity (r 2 (=0.999).

[0088] A concentration of 5 μg / mL was selected because it was the same concentration used to stain CHO-K1 cells after uptake. Assay reproducibility was assessed by performing endocytosis tests separately over 3 days using anti-IL-4Rα mAb or ASA, with two different cytometers and two different scientists (n=20 total samples). The antibodies were incubated with cells at 100 μg / mL for 60 minutes at 37°C based on initial kinetic characterization (Figure 2). The 60-minute incubation time was chosen to allow positive detection of any future mAbs exhibiting intermediate nonspecific uptake that is not as rapid as or as similar as anti-IL-4Rα mAb. Under these conditions, mean ABCs were measured for anti-IL-4Rα mAb at 40,839 (SD 6614, CV 16.2%) and for ASA at 921 (SD 315, CV 34.2%) (Figure 3B). To demonstrate significant signal overlap indicating weak internalization of ASA in CHO-K1 cells, population histograms were included for representative ASA and untreated samples (Figure 3C). A schematic of the assay is shown in Figure 3D.

[0089] Example 3: High nonspecific adsorption endocytosis is driven by charge-dependent interactions and is conserved across species and cell types. One possible mechanism that leads to a high rate of anti-IL-4Rα mAb uptake is charge. Differences in protein charge have been shown to affect mAb PKs, with extreme values ​​of positive and negative net charge typically associated with the strongest influence (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 has been previously shown that charge effects in more specific structural regions, such as CDRs, can influence nonspecific mAb endocytosis even without a measurable shift in isoelectric point (pI) (Datta-Mannan et al., MAbs 7(3):483-93 (2015)). To provide a better understanding of the relationship between these variables and NSE, structural modeling was performed to investigate the charge distribution of anti-IL-4Rα mAbs and ASAs (Figures 4A and 4B). The pIs of anti-IL-4Rα mAbs and ASAs were calculated as 8.75 and 7.24, respectively, from their amino acid sequences. Antibody modeling identified several charge patches on anti-IL-4Rα mAbs, including two positive patches in the heavy chain CDR. Anti-IL-4Rα mAbs had a higher calculated charge of Fv(+7) compared to ASA Fv(+2).

[0090] Therefore, the extra positive charge patch on the anti-IL-4Rα mAb, along with its higher Fv charge, leads to high nonspecific uptake and CL. indThis may have contributed to the increase in nonspecific adsorption endocytosis. To confirm this, point mutations were performed on the anti-IL-4Rα mAb within its CDR and light chain framework to mitigate its positive charge attribute. The mutations were located at amino acid positions that exhibited positive charge clusters and / or were important for driving the protein's net charge (Table 2). ABC values ​​obtained by endocytosis assays in CHO-K1 cells demonstrated that each mutation resulted in a substantial reduction in nonspecific adsorption endocytosis (Figure 4C). These findings support the positive charge exposed on the surface of the anti-IL-4Rα mAb as a key factor causing the increased rate of nonspecific adsorption endocytosis.

[0091] [Table 2]

[0092] Nonspecific endocytosis was evaluated using a separate panel of preclinical mAbs (mAb B1-B5) targeting G protein-coupled cell surface receptors. While the mAbs were expected to be unable to bind to similar pI values ​​and mouse targets, they showed dramatically different CLs in wild-type mice. indThese cells possessed the following characteristics (Figure 5, Tables 3 and 4). Basic local alignment search tool (BLAST) analysis identified that the mAbs differed at amino acids 27 (light chain), 54, and 56 (both on the heavy chain) within the CDR. The amino acid variation would result in the expected charge difference between mAb B1, which has the lowest positive charge, and mAb B5, which has the highest. The results of CHO-K1 endocytosis were consistent with this trend, with mAb B1 showing the lowest ABC and mAb B5 showing the highest (Figure 6A). Repeated experiments in green monkey kidney epithelial cells (Vero cells, savanna monkey (Cercopithecus aethiops)), which are unrelated to CHO-K1 in terms of species (Chinese hamster (Cricetulus griseus)) and tissue origin (kidney vs. ovary), were in direct agreement with the results using CHO-K1, confirming that internalization is nonspecific (Figures 6B, 6C). Furthermore, uptake tests conducted at 4°C supported the absence of target expression in either cell line and highlighted the magnitude of nonspecific adsorbent endocytosis compared to cell surface binding. Subsequently, in vivo-in vitro correlation was performed at 37°C using ABC readings from either CHO-K1 or Vero cells, and CL ind、マウス A strong relationship was demonstrated (Figure 6D). Furthermore, these results confirm that the exposed positive charge on the surface of therapeutic proteins leads to a higher rate of nonspecific adsorption endocytosis, revealing that the nonspecific adsorption internalization pattern is clearly shared across different cell types, and that CL can be performed using the currently disclosed methods. ind Infer the ability to inform about this.

[0093] [Table 3]

[0094] [Table 4]

[0095] Discussion of Examples 1-3 The tests described herein demonstrated the sensitivity of the cell-based assays of this disclosure for identifying different rates of nonspecific endocytosis of mammalian cells by different mAbs.

[0096] The kinetic characterization of ASA and anti-IL-4Rα mAbs supports nonspecific uptake of mAbs in cell-based experimental platforms lacking targets for several reasons. First, there was a linear relationship between the concentration of anti-IL-4Rα mAb and the degree of its endocytosis. Second, a significant discrepancy was observed between the cell-associated amounts of anti-IL-4Rα mAb at separate temperatures. 37°C enabled cell binding, endocytosis, and transport, while 4°C enabled only surface binding. Therefore, the low signal at 4°C indicated internalization of anti-IL-4Rα mAb at 37°C, supporting the conclusion that no target exists within CHO-K1 cells. Notably, the biphasic, time-dependent uptake of anti-IL-4Rα mAb was observed. This same observation has been made previously by others and is thought to be due to rapid and constitutive analyte exocytosis after initial nonspecific 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.USA 83(1986)9488–9492 (the entire article is incorporated herein by reference).Therefore, the results obtained when combined with anti-IL-4Rα mAbs are consistent with previous reports describing the nonspecific dynamics of various solutes in several different cell types.

[0097] Apart from nonspecific uptake, recycling facilitated by FcRn 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 (these are incorporated herein by reference in their entirety). FcRn is important for the long half-life of Fc-containing therapeutics due to its pH-dependent association with the Fc region. Following endocytosis, FcRn binds to its ligand during endosomal acidification, then mediates ligand transport to the plasma membrane, where dissociation from FcRn occurs due to the low affinity of FcRn-Fc interactions 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 (the entire article is incorporated herein by reference). FcRn is a key aspect of mAb PK, and preclinical mAb CL variability may be caused by differences in mouse FcRn binding, but the efforts here focused on determining the relationship between nonspecific uptake and CL. However, as described herein (Examples 4-8), combined evaluation of nonspecific and FcRn interactions for mAbs may provide further insight into potential PK shortcomings.

[0098] The cell-based assays described herein are useful in vitro tools for identifying high-risk compounds in preclinical polymer development. The current CHO-K1 format has a moderate throughput, allowing individual users to run approximately 50 samples per day. This can be increased by the removal of 4°C groups, which is partially used here to help identify the presence of target binding. This group can be replaced with publicly available transcriptome / genomic-level information to assess target expression.

[0099] Because many variables can influence mAb PK, cell-based measurements of nonspecific uptake can be used in conjunction with accompanying approaches to support preclinical candidate selection and risk elimination strategies. Current techniques provide per-cell quantitative outputs that can be incorporated into computational models to support deeper, more biologically relevant evaluations of preclinical compounds. For example, readings from current assays can directly provide analyte-specific single-cell internalization rates for PK modeling efforts, rather than inferring this parameter using biophysical techniques or from general measures of nonspecific endocytosis. Furthermore, other cells can be used instead of CHO cells to tailor internalization kinetics measurements to the tissue being modeled, providing a more representative level of site-specific information.

[0100] Materials and methods of Examples 4-8 Antibody and corresponding CL value ASA WT and ASA AAA Based on previous reports, wild-type Fc region or H310A, I253A, H435A (ASA AAAThis was a fully human IgG1 mAb produced in response to streptavidin containing the mutation (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 USA 105,9337-9342 (these are incorporated herein by reference in their entirety). Clinical mAbs included commercially available antibody products, including anti-CD20 antibody (mAb5), anti-PD-1 antibody (mAb3), and anti-CD38 antibody (mAb2), as well as test analogs of several clinical antibodies, including anti-PCSK9 antibody (mAb4), anti-IL-15 antibody (mAb6), IL-4R antibody (AMG-317), IL-12 / IL-23 antibody (mAb1 and mAb9), EGFR / HER3 antibody (mAb7), IL-2Rα antibody (mAb8), and phosphatidylserine antibody (mAb10). The mAb analogs were acidified on the Fc scaffold, which lacks stable effector function, in stably transformed CHO cells, as previously described. (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 (the entire article is incorporated herein by reference).

[0101] To investigate the effects of Fv mutations and half-life extension (HLE) Fc-manipulated YTE mutations, a series of fully human wild-type hIgG2 anti-IL-4RαWT and M252Y / S254T / T256E(YTE) charged mutant mAbs were prepared in addition to the mAbs listed in Table 2. Furthermore, two hIgG2 control mAbs and ASA were also prepared. WT -hIgG2-DL650 and ASA WT-hIgG2-YTE was also prepared 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.

[0102] Many human CL values ​​for clinical mAbs, internal data, FDA monographs, or CL for various mAbs. ind The estimations were obtained from one of the previous reports (Grinshpun et al., Identifying biophysical assays and in silico properties that enrich for slow clearance in clinical-stage therapeutic antibodies, MAbs 13(2021)1932230 (the entire report is incorporated herein by reference)). A body weight of 80 kg was used for normalization. Publicly available clinical data are limited for some antibodies. For these cases, CL ind The reported volume of distribution (V) obtained under linear pk conditions using the following formula (Schoenwald, RD (2002) Basic Principles. in Pharmacokinetics in Drug Discovery and Development (Schoenwald, RD ed.), 1 Ed. pp 3-33 (the entire formula is incorporated herein by reference)) is obtained using the following formula. D ) and terminal half-life (t 1 / 2β Estimated by the value:

number

[0103] Cell culture parent MDCK II cells (Sigma#MTOX1300) were maintained in 5% thermally inactivated FBS EMEM (ATCC#30-2003) and subcultured using 0.25% trypsin-EDTA. Maintaining subconfluence was found to be essential for proper monolayer formation. Optimal subculture conditions were found to maintain a cell density of less than 90% by subculturing at 75 cm every 2-3 days. 2 A density of 2.5–3e6 cells per well was determined. Parental MDCK II cells were seeded at 100,000 cells per well in a flat-bottomed 96-well plate (Corning #353072). Transfected MDCK II cells (see below) were seeded at 120,000 cells / well in the same plate. Monolayers of both cells were achieved in 2 days. CHO-K1 cells were seeded and grown as described with reference to Examples 1–3 herein. All cells were cultured in a 5% CO2-95% O2 humidified incubator at 37°C. Negative mycoplasma was confirmed using the Lonza MycoAlert detection kit (Lonza, Walkersville, MD #LT07-318). Growth medium was used, consisting of cryopreservation medium completely supplemented with 5% DMSO (v / v).

[0104] Plasmid and transfection. The hFcRn construct was developed on a pcDNA3.1 vector incorporating 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 the blastosidine resistance gene.

[0105] In a sterile 1.5 mL Eppendorf tube A, two plasmids with a total of 4 μg of DNA in a 1:1 molar ratio were diluted in 250 μl of Opti-MEM I Reduced Serum Medium, without serum, while gently mixing. Lipofectamine 2000 (10 μL) was diluted in 250 μL of Opti-MEM I Medium and incubated at room temperature for 5 minutes. After 5 minutes of incubation, the diluted DNA was combined with diluted Lipofectamine 2000 (total volume = 500 μl), gently mixed, and incubated at room temperature for 20 minutes. 500 microliters of the complex were dropped into each well containing cells, while gently mixing by shaking the plate back and forth. After incubating the cells in a humidified CO2 incubator at 37°C for 48 hours, transgene expression was tested. hFcRn-GFP / hβ2m-MDCK II was consistently maintained in selective media (1000 μg / mL G418, 7.5 μg / mL blastosidine) because transgene loss was observed when cells were cultured in normal growth medium.

[0106] Immunostaining by flow cytometry. Immunostaining was performed using the fluorescent conjugate antibodies shown in Table 5. Parental cells and FcRn-GFP / β2m-MDCK II cells grown to confluence were resuspended in 1× PBS containing Zombie UV-fixable viability dye (BioLegend #423108) under the manufacturer's recommended conditions and incubated at room temperature for 20 minutes. The cells were washed once in FACS buffer (1× PBS, 2% w / v BSA, 1 mM EDTA, 0.1% w / v sodium azide) and then fixed and permeabilized in the dark at room temperature for 20 minutes using Cyto-Fast fix / perm buffer (Biolegend #426803) or (1) kept on ice for cell surface staining. Next, all cells were transferred to a V-bottom 96-well plate (Fisher #249944) and stained on ice in the dark for 30 minutes using the antibody amounts (100 μL volume) shown in Table 5. Either FACS buffer (unfixed cells) or 1× Cyto-Fast Perm Wash solution (fixed cells) was used for staining. The cells were then washed three times with the respective buffers and analyzed using an 18-color, 5-laser configuration (UV-355 nm, Violet-405 nm, Blue-488 nm, Yellow / Green-561 nm, Red-637 nm) BD FACSymphony flow cytometer and a BD Biosciences High Throughput Sampler (catalog number 338301). Where necessary, spectral compensation was achieved using single-stained UltraComp eBeads (Thermo#01-2222-42), ArC® amine-reactive compensation beads (Thermo#A10346), and / or AcGFP Flow Cytometer Calibration Beads (Takara#632594), according to vendor instructions.

[0107] [Table 5]

[0108] Fluorescence-activated cell sorting (FACS). Clone #8 FcRn-GFP / β2m-MDCK II cells were selected for proliferation based on preliminary evaluation due to high transgene expression. Different GFP + and GFP - The population (corresponding to hFcRn expression) was first observed. Therefore, GFP + FACS was performed to enrich the population. Cloned #8 cells were subcultured, resuspended in azide-free FACS buffer at 7e6 cells / mL, filtered through a 70μm filter in a 12mm × 75mm polystyrene tube, and GFP within the single-cell clone #8 population was analyzed using a BD FACS Melody Cell Sorter (equipped with violet-405nm, blue-488nm, and red-640nm lasers). + Cells were selected. However, subsequent evaluation showed insufficient hβ2m expression (in multiple populations). GFP + Clones #8 FcRn-GFP / β2m-MDCK II cells were grown and their cell surface staining was performed using anti-hFcRn-AF647 and anti-hβ2m-PerCP / Cy5.5 (Table 6) with a BDFACSAria Fusion cell separator (18 colors, 5 laser configuration (UV-355nm, violet-405nm, blue-488nm, yellow / green-561nm, red-640nm)) to obtain the highest 10% GFP. + hFcRn + hβ2m + Single cells were re-selected. Even after FACS and further selection, approximately 2-3% of the total FcRn-GFP / β2m-MDCK II cell population consistently showed GFP. - Therefore, internalization analysis using fluorescently conjugated proteins was performed using GFP. + It was done with cells.

[0109] [Table 6]

[0110] Fluorescent conjugation. Human serum albumin (Sigma#A3782) and ASA WTThe protein (final concentration of 2 mg / mL per reaction; 1 mL volume) was conjugated in 50 mM borate buffer (Thermo#28384) at pH 8.5 with DyLight 650-NHS (Thermo#62265) in a 10:1 protein:dye ratio, according to the manufacturer's protocol. The protein was dialyzed overnight at 4°C in a 7 kDa molecular weight cutoff cassette (Thermo#66372) in 3 L (1 L increments, a total of 3 changes) of 1 × PBS. The final protein concentration was obtained spectrophotometrically as described by the manufacturer.

[0111] ASA internalization study using flow cytometry. Parental cells and FcRn-GFP / β2m-MDCK II cells were seeded in 96-well plates and grown into a confluent monolayer as described above. On the day of the study, the 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 minutes. For time-dependent studies, the cells were aspirated and immediately treated with 10 or 100 μg / mL of DyLight 650 conjugated ASA. WT (ASA WT -Incubated for 2 hours with DL650 (Ringer's solution at pH 5.8, 7.4, or 8.0). ASA WT - For concentration-dependent studies of DL650 and DyLight 650 conjugated human serum albumin (HSA-DL650), cells were subjected to excess unlabeled protein (50 mg / mL; ASA). WTRegardless of the presence or absence of human serum-derived IgG used in -DL650 and Sigma#I4506, incubation was carried out at pH 5.8 for 20 minutes to confirm receptor-mediated endocytosis. After the treatment was completed, the cells were placed on ice and washed 4 times with ice-cold pH 7.4 Ringer's solution (200 μL / well / wash), and then incubated at 37 °C for 8 - 10 minutes with 100 μL / well of 0.25% EDTA-trypsin. Once the cells were rounded up, the wells were first stirred by gentle but rapid pipetting, and then 100 μL of MDCK II growth medium (without selection reagent) per well was added to inhibit trypsin. The cells were transferred to a V-bottom 96-well plate and centrifuged at 300 g for 5 minutes at 4 °C, and then stained with 0.5% v / v UV Zombie dye in 1×PBS for 20 minutes on ice. The cells were washed once with 100 μL FACS buffer / well and centrifuged at 300 g for 5 minutes at 4 °C, and then fixed in the dark at room temperature using Cyto-Fast fix / perm buffer (50 μL / well). The fixed cells were washed twice with 1×Cyto-Fast Perm Wash solution (150 μL / well), resuspended in 100 μL FACS buffer, and then analyzed on a BD FACSymphony flow cytometer. 10,000 targeted single cells, live, GFP + (For FcRn-GFP / β2m-MDCK II only) The median fluorescence intensity of the events was obtained by BD Diva software. The data files were analyzed using FlowJo (BD). The concentration-dependent receptor-specific uptake data (i.e., the total signal minus the median fluorescence intensity per concentration of the average (+) non-labeled condition and untreated control) was divided by the total incubation time, and then fitted to the Michaelis-Menten equation using GraphPad Prism (Dotmatics, Boston, MA): [Number] <000,0902> V max is the maximum velocity achieved within the experimental system, and [S] is the concentration of ASA WT -DL650 or HSA-DL650, and Km is the concentration that achieves half of the maximum speed.

[0112] ASA, except that the cells were incubated at pH 5.8 for 30 minutes in both the parental cells and the hFcRn-GFP / β2m-MDCK II cells. WT -hIgG2-DL650 and ASA WT -hIgG2-YTE-DL650 concentration-dependent tests were performed in the same manner as described above. After analyzing the data files using FlowJo (BD), the concentration-dependent receptor-specific uptake data (i.e., the total signal of hFcRn-GFP / β2m-MDCK II cells minus the average signal of parental MDCK II cells per concentration and the median fluorescence intensity of untreated parental control cells) was divided by the total incubation time and then fitted using the Michaelis-Menten equation as described in the previous paragraph, where V max is the maximum speed achieved within the experimental system, [S] is the concentration of ASA WT -hIgG2-DL650 or ASA WT -hIgG2-YTE-DL650, and K m is the concentration that achieves half of the maximum speed.

[0113] FcRn-ASA WT -Lysosome imaging and co-localization analysis. Human FcRn-GFP / β2m-MDCK II cells were seeded into a PerkinElmer CellCarrier Ultra 96-well plate (catalog number 6055302) as described above. The day before the imaging test, confluent cell monolayers were incubated with 10 kDa Texas Red-conjugated dextran (Thermo#D1828) at 0.25 mg / mL in selective medium for 6 hours. The dextran concentration was based on a pilot titration test one week earlier. After incubation, the cells were washed with HBSS ++The samples were washed three times in the medium and then incubated overnight in selective medium. This approach allows internalized dextran to accumulate within the lysosomes and thus label the lysosomes. The following day, time-dependent ASA was performed in Ringer's solution at pH 5.8 or 7.4 as described in "ASA Internalization Studies with Flow Cytometry". WT -DL650 uptake tests (10 and 100 μg / mL) were performed. After incubation was complete, FcRn-GFP / β2m-MDCK II cells were washed four times with ice-cold pH 7.4 Ringer's solution and then fixed with 4% v / v paraformaldehyde in 1×PBS at room temperature for 15 minutes. After fixation, the cells were washed twice with 1×PBS and then stained with 1 μg / mL Hoechst (Thermo#H1399) diluted in 1×PBS at room temperature for 30 minutes. The cells were washed twice with 1×PBS and then stored at 4°C in 200 μL / well of 1×PBS. The following day, images were acquired using an OperaPhenix high-content screening system (PerkinElmer) with a 63x aqueous objective lens with Z-stack acquisition. ASA was scanned using Columbus software (PerkinElmer). WT - The mean fluorescence intensity (MFI) measurements of the DL650 channel were analyzed. Analytical 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, Hoechst channels were used for nuclear and cytoplasmic segmentation. Pearson correlation coefficients were used to analyze different channel pairs (ASA). WT Colocalization in the cytoplasmic region between lysosomes loaded with -DL650, hFcRn-GFP, and Texas Red-dextran was measured. The Costes correction method was used to help distinguish labeled regions from the background. MFI and colocalization metrics were plotted using GraphPad software.

[0114] Immunofluorescence and confocal microscopy. Parental cells and hFcRn-GFP / β2m-MDCK II cells were seeded and grown to a confluent monolayer in PerkinElmer CellCarrier Ultra as described above. Cells were washed twice with 1×PBS, fixed in 4% PFA (1×PBS), and blocked / permeabilized in 1× Cyto-Fast Perm Wash solution for 1 hour at room temperature. All subsequent volumes were 100 μL / well. Anti-FcRn antibody (Sigma#HPA012122, 0.2 mg / mL stock concentration) was added in 1× Cyto-Fast Perm Wash solution at a 1:5000 dilution and incubated at room temperature for 1 hour. Cells were washed twice with 1× Cyto-Fast Perm Wash solution and then incubated with goat anti-rabbit secondary antibody conjugated with Alexa Fluor 594 (Thermo#A11037, 2 mg / mL stock concentration, 1:1000 dilution in 1× Cyto-Fast Perm Wash solution) for 1 hour at room temperature. Cells were washed twice with 1× Cyto-Fast Perm Wash solution and then stained with 1 μg / mL Hoechst + 2 μg / mL CellMask Blue (Thermo#H32720) for 30 minutes at room temperature, both of which were diluted with 1× Cyto-Fast Perm Wash solution. Cells were washed twice with 1× PBS and imaged using an OperaPhenix high-content screening system with a 40× water objective lens and Z-stack acquisition.

[0115] Human FcRn mAb recycling tests for Examples 5 and 6, and Figures 10 and 11. Parental and FcRn-GFP / β2m MDCK II cells were seeded in 200 μL of individual growth medium in 96-well plates at 1e5 cells and 1.2e5 cells / well, respectively, and cultured for 48 hours. The cell medium was aspirated, and the cells were washed twice with preheated (37°C) Ringer's solution, pH 7.4. The cells were then equilibrated for 30 minutes in Ringer's solution supplemented with MEM non-essential amino acids 1X (catalog no. 25-025-CI, Corning®) and 1 mM sodium pyruvate solution (Corning #25-000-CI), pH 7.4. ++Subsequently, the wells were aspirated and the cells were incubated with the mAb solution prepared at 666.67 nM (100 μg / mL) in pH 5.8 and pH 7.4 Ringer's solution ++ at 37 °C for 2 hours. This is called the loading stage. After incubation, the cells were washed 4 times at 20 °C with 200 μL / well of room temperature Ringer's solution, pH 7.4. Then, serum-free EMEM growth medium (150 μL / well) was added to the cells and the plate was incubated at either 37 °C or 4 °C for 4 hours. This is designated as the recycling stage. Since there was no active recycling at 4 °C, the remaining data at 4 °C showed the uptake process after a 2-hour loading stage at 37 °C using 100 μg / mL of mAb. Next, the supernatant (recycling sample) was collected into a 96-deep well plate (Axygen, #P-DW-500-C) and stored at -80 °C until analysis. Thereafter, the cells were further washed 4 times with 4 °C Ringer's solution on ice. Then, the cells were lysed with 150 μL / well of ice-cold MOPS lysis buffer pH 7.4 (20 mM MOPS in 0.1% (v / v) Triton X-100) containing an EDTA-free protease inhibitor. To prepare the lysis buffer, one EDTA-free mini cOmplete tablet containing a protease inhibitor cocktail (Roche #04693159001) was added to each 10 mL of MOPS lysis buffer and vortexed. The plate was incubated on ice at 4 °C for 30 minutes while shaking at 600 rpm, and then pipetted up and down 30 times to uniformly remove the cells from the bottom of the plate using a multi-channel pipettor. The lysate was collected into a 96-deep well plate and stored at -80 °C until further analysis.

[0116] Human FcRn recycling test with anti-IL-4RαWT and YTE mutant mAb for Example 8 and Figure 17: This human FcRn recycling test was performed at 37°C to load with anti-IL-4RαWT and YTE mutant mAb, as active recycling was not observed at 4°C in previous recycling tests. Parental and FcRn-GFP / β2m MDCK II cells were seeded at 75,000 and 85,000 cells / well, respectively, in 200 μL of individual growth medium in 96-well plates and cultured for 72 hours. The cell medium was aspirated, and the cells were washed twice with pre-warmed (37°C) Ringer's solution, pH 7.4. The cells were then equilibrated for 30 minutes in Ringer's solution, pH 7.4, supplemented with 1×GlutaMAX® supplement (catalog no. 35050-061, Gibco®) and 1 mM sodium pyruvate solution (Corning #25-000-CI). Ringer's liquid + / + This is called [a specific term]. This is due to the absence of non-essential amino acids that have not been found to be necessary for cell viability, as described above in Ringer's solution. ++ This is different. Subsequently, the wells are aspirated, and the cells are mixed with either an anti-IL-4RαWT mutant mAb solution prepared at 666.67 nM (100 μg / mL) or an anti-IL-4RαYTE mutant mAb solution prepared at 166.67 nM (25 μg / mL), and Ringer's solution at pH 5.8. + / +The cells were incubated at 37°C for 2 hours. This incubation period is called the uptake phase, and it is the net result of both uptake and recycling that occur during this loading phase. After incubation, the cells were washed four times on ice with 200 μL / well ice-cold Ringer's solution pH 7.4. The cells were then lysed with 200 μL / well ice-cold lysis buffer pH 8.0 (10 mM TrisHCl pH 8.0, 1% IGEPAL CA-630, 0.5% sodium deoxycholate, 0.1% SDS, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA) (referred to herein as ULS lysis buffer) containing an EDTA-free protease inhibitor. To prepare the final ULS lysis buffer, one EDTA-free mini cOmplete tablet containing a protease inhibitor cocktail (Roche #04693159001) was added to each 10 mL of ULS lysis buffer and vortexed. The plates were incubated on ice at 4°C for 30 minutes with shaking at 600 rpm, and then the cells were uniformly removed from the bottom of the plates by pipetting 50 times up and down using a Tecan Freedom EVO 200 liquid handler controlled by Freedom EVOware® version 2.8 software. The lysates were collected in 96 deep-well plates and stored at -80°C until further analysis. These samples constituted the incorporation fraction for FcRn scoring. To obtain recycled samples, the exact incorporation procedure described above was followed, except that after the loading step, the cells were washed four times at 20°C with 200 μL / well of room temperature Ringer's solution pH 7.4. Then, serum-free EMEM growth medium (200 μL / well) was added to the cells, and the plates were incubated at 37°C for 4 hours. This was referred to as the recycling step. Next, the supernatant (recycled sample) was collected in a 96-deep-well plate (Axygen, #P-DW-500-C) and stored at -80°C until analysis. The cells were then washed four more times on ice with Ringer's solution at 4°C. Subsequently, 200 μL / well of ice-cold ULS pH 8.0 lysis buffer was added, and the cells were lysed in the same manner as above. The lysates were collected in a 96-deep-well plate and stored at -80°C until further analysis. These lysates are shown as residual samples.

[0117] Analysis of recycled assay samples using MesoScale Discovery Immunoassay. Electrochemiluminescence (ECL) immunoassays were performed using a MesoScale Discovery (MSD) Sector S600 instrument (MesoScale Diagnostics, Rockville, MD, USA), similar to the previously developed protocol (91). First, plates stored at -80°C were thawed on ice. The thawed lysed samples were centrifuged at 4°C and 1,000g for 10 minutes to remove cell debris, and the supernatant was transferred to a new 96-well plate. For the MSD experiment, a 96-well MSD Gold® Streptavidin SECTOR® assay plate (MesoScale diagnostic, #L15SA-1) was coated with 50 μL of 2 μg / mL biotinylated proprietary anti-human Fc antibody (clone 35; Ab35) in blocking buffer (Tris-buffered saline [TBS], Blocker® BLOTTO in Thermo #37530) for 1 hour with shaking at approximately 600 rpm at room temperature. The plate was then washed four times in a plate washer (Biotek 405 Select microplate washer) with 1× KPL washing buffer (LGC seracare). Standards, quality control samples (100, 10, and 1 ng / mL), and diluted recycled samples (1:1 in 1×PBS) were prepared by the following medium matching experiments: serum-free EMEM medium, MOPS lysis buffer pH 7.4 (above) for recycled samples, or ULS lysis buffer pH 8.0 (above) containing an EDTA-free complete protease inhibitor for residual samples. The lysis buffer mixtures used to prepare standards and quality control samples for residual sample analysis were produced using matched amounts of lysate based on the average protein content of the residual samples on the plate. Protein content was measured by either Bradford (Examples 5 and 6, Bryniarski et al., Am.J. Physiol Renal Physiol. 315(5):F1191-F1207 (2018) (the whole thereof is incorporated herein by reference) or the manufacturer's protocol (Example 8) post-assay BCA.To prepare the matrix for the residual sample standard, equal volumes (approximately 1e6 cells) of hFcRn-GFP / hβ2M MDCK II and parental MDCK II cells were grown in a confluent monolayer on a 100 mm round culture dish (Corning #353003) and lysed in either 1 mL of MOPS lysis buffer pH 7.4 or ULS lysis buffer pH 8.0 containing an EDTA-free complete protease inhibitor. The mixture was then centrifuged at 1,000 g for 10 minutes to remove cell debris. The supernatant was collected, and an appropriate amount of lysate from this stock was added to the standard and quality control sample lysis buffer mixture (v / v) to match the average protein content of the residual sample on the plate. Standard, quality control, and recycled assay samples (50 μL / well) were added to the plate and incubated at room temperature with shaking for 2 hours, followed by four further washes. Subsequently, the plate was incubated at room temperature for 1 hour with 50 μL of 2 μg / mL sulfo-labeled Ab35 (ruthenium) in BLOTTO blocking buffer as the detection reagent. Next, the plate was washed four times, and 150 μL of 2X MSD read buffer (diluted with DI water from 4× MSD read buffer T containing surfactant, cat#R92TC-1, and MesoScale Diagnostics) was added to each well, after which it was read using an MSD instrument. Analyte concentrations were interpolated from sigmoid 4-parameter least-squares fitted antibody-specific standard curves in GraphPad Prism on the same plate and validated using quality control samples.

[0118] Human FcRn Recycling Efficiency Metric (FREM) score. Using the recycled, residual, and uptake (4°C recycling stage) concentrations derived from the initial pH 5.8 loading stage of the mAb (Examples 5 and 6), the hFcRn Recycling Efficiency Metric (FREM) score was calculated using the following formula:

number

[0119] The nonspecific uptake coefficient (NUC) for each mAb was calculated using the following formula, based on the uptake concentrations in FcRn-GFP / hβ2m MDCK II cells and parental MDCK II cells:

number

number

number

[0120] When the NUC value was 0 to 1 (0 < NUC < 1), it was shown that the cell uptake process was dominated by FcRn-independent non-specific endocytosis. In these cases, NUC was multiplied by the FREM score. When NUC > 1, it indicated FcRn-mediated uptake of mAb "X" in negligible non-specific endocytosis. For these mAbs, the NUC value was not multiplied by the FREM score. Using GraphPad Prism software, with n = 4 (mAb panel) and / or the Rout method (when n = 8; ASA hIgG1 and anti-IL-4Rα WT and YTE mutant mAbs), and an alpha value of 0.05, outliers in the assay were identified by using the Grubbs' method.

[0121] CHO-K1 non-specific uptake test. The non-specific endocytosis test was performed at pH 7.4 or 5.8 as described above in Examples 1 to 3. The antibody binding capacity (ABC) value is a quantitative technique that facilitates inter-day comparison between mAbs through the creation of a standard curve using IgG-binding microspheres.

[0122] PK test and analysis of ASA and anti-IL-4Rα variant mAbs. Mice were housed in groups at an internationally accredited facility, AALAC. Mice were bred at an internationally accredited facility, AAALAC. Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals, 8 thCare was provided according to the Edition. All study protocols were reviewed and approved by the Amgen Institutional Animal Care and Use Committee. 6-8 week old male homozygous Tg32 (strain number 014565), homozygous Tg276 (strain number 004919), and immunodeficient homozygous hFcRn SCID Tg32 (strain number 018441) mice were purchased from Jackson Laboratory (Bar Harbor, MA). ASA WT (1 mg / kg), ASA YTE (1 mg / kg) and ASA AAA (3 mg / kg), anti-IL-4RαWT and YTE mutant mAbs (1 mg / kg each) were administered as intravenous boluses via the lateral tail vein at the above doses. Blood samples were collected at various time points after injection and incubated at ambient temperature for approximately 20 minutes or until complete coagulation occurred, then centrifuged to separate the serum. All serum samples were stored at -70°C (±10°C) until used in analytical assays.

[0123] Quantitative analysis of proteins in mouse serum was performed by electrochemiluminescence immunoassay on an MSD Sector 600 instrument, using Ab35 anti-human Fc antibody as both the capture and detection reagents, as in previous 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 (the entire work is incorporated herein by reference)). In all assays, analyte serum concentrations were interpolated from standard curves using corresponding analytes prepared in pooled mouse serum using WatsonLIMS software.

[0124] Linear CL was predicted for the ASA variant due to lack of target expression in mice. Serum concentration data were fit to a two-compartment disposition model with central and peripheral compartments (Tang et al., (2004) Pharmacokinetic aspects of biotechnology products. J Pharm Sci 93, 2184-2204 (incorporated herein by reference in its entirety)). Parameterization included primary elimination (clearance, CL) from the central compartment, intercompartmental distribution clearance (CL D ), and volumes of the central (V1) and peripheral (V2) compartments. Modeling was performed using the Ubiquity package workflow within R (v4.2.2).

[0125] Example 4: Generation and functional characterization of MDCK II cells with stable co-expression of hFcRn-GFP and hβ2m MDCK II cells were stably transfected with constructs of hβ2m and hFcRn-GFP. Clones co-expressing high levels of 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 (referred to as hFcRn-GFP / hβ2m-MDCK II cells) by immunofluorescence with confocal microscopy (Figure 7A) and flow cytometry (Figures 7B, 7C).

[0126] Since FcRn exhibits pH-dependent affinity for its ligand, internalization assays were performed at various pH values to evaluate the function of the co-transfected hFcRn-GFP / hβ2m complex. Parental and hFcRn-GFP / hβ2m-MDCK II cells were incubated with DyLight 650 (ASA WTCells were incubated with 10 or 100 μg / mL of wild-type hIgG1 anti-streptavidin antibody (-DL650) fluorescently conjugated at pH 5.8, 7.4, or 8.0 for increasing periods. Analysis was performed by flow cytometry (Figure 13A). Only hFcRn-GFP / hβ2m-MDCK II cells showed concentration and time-dependent uptake in a pH-dependent manner, thus acidic conditions were favorable for a wide range of ASA cells. WT - This resulted in DL650 endocytosis (Figures 8A and 13B). This behavior is consistent with the enhanced affinity of FcRn to its ligand within acidified endosomes. Furthermore, ASA in parental MDCK II cells at any pH. WT - No difference in DL650 uptake was observed, thus indicating an undetectable effect on endocytosis at various pH levels (Figure 8A). Therefore, under the conditions tested, these results support the hFcRn-dependent ASA in co-transfected MDCK II cells. WT - We demonstrate that DL650 endocytosis decreased with increasing culture medium pH. Furthermore, nonspecific uptake was observed as the endocytosis mechanism of IgG in parental MDCK II cells.

[0127] To provide further evidence of FcRn-mediated endocytosis, ASA WT Concentration-dependent uptake studies were performed at pH 5.8 using -DL650 or fluorescently labeled human serum albumin (HSA-DL650). Both compounds were competitively inhibited by excess unlabeled protein (Figures 8B and 8C). The Michaelis-Menten equation was used as a function to fit specific uptake data, and the concentration that achieved half of the maximum uptake in the experimental system was determined as ASA. WT For -DL650, the estimated concentration was 50 μg / mL [95% confidence interval], and for HSA-DL650, it was estimated to be 44 μg / mL. These are approximately 333 μM ASA WT-DL650 and 660 μM HSA-DL650 are equivalent. Furthermore, nonspecific internalization was observed to be linear with concentration, which is consistent with the nonspecific internalization kinetics (Figures 8B and 8C, bottom plot). In summary, these data support (1) the function of hFcRn-GFP / hβ2m in co-transfected MDCK II cells, and (2) the function of albumin and ASA in the absence of the target receptor. WT This demonstrated nonspecific endocytosis.

[0128] The next objective was to evaluate the cellular localization and transport behavior of hFcRn-GFP. To this end, concentration-dependent, pH-dependent, and time-dependent uptake studies were performed in hFcRn-GFP / hβ2m-MDCK II cells using high-concentration confocal microscopy. 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 incubation for 15, 30, 60, or 120 minutes. Lysosomes were labeled by preloading cells with 10 kDa Texas Red-conjugated dextran the day before (Figure 9A). Intracellular ASA WT - The DL650 imaging results were consistent with the results reported in Figure 8, which quantified the time, concentration, and pH dependencies (Figure 9B). hFcRn-GFP, ASA WT Colocalization analysis by Costes' method for image signal thresholding of DL650 and Texas Red Dextran (i.e., lysosomes) shows that hFcRn-GFP and ASA WT -This yielded a Pearson correlation coefficient score showing apparent co-localization only for DL650 (Figure 9C). Minimal lysosome co-localization was observed for hFcRn-GFP and ASA. WT These findings are consistent with previous assessments of GFP-tagged FcRn behavior and provide further support for appropriate hFcRn-GFP and hβ2m function in stably transfected MDCK II cells.

[0129] Example 5: Development of a quantitative FcRn recycling assay using hFcRn-GFP / hβ2m-MDCK II cells The hFcRn-GFP / hβ2m-MDCK II recycling assay described herein is an ASA WT Developed in standard 96-well plates optimized by DL650 dynamic analysis (Figure 10A). After a 2-hour loading period at pH 5.8 or 7.4, cells were washed and then incubated in serum-free cell medium at 37°C or 4°C for 4 hours. The 4°C group served as a control, providing a means to estimate the amount of mAb internalized during the loading period, as intracellular processing stops on ice. Furthermore, this control group also allowed for the demonstration of active recycling compared to a parallel 37°C group (i.e., a 4-hour recycling period at 37°C). In summary, the workflow enabled the quantification of both specific (hFcRn-GFP / hβ2m-MDCK II) and nonspecific (parent MDCK II) amounts of test mAb internalized and recycled under various assay conditions.

[0130] The first test to evaluate assay performance is ASA WT and excised hFcRn affinity mutants (H310A, I253A, H435A; ASA AAA This was achieved using [method / tool ​​name]. FcRn recycling tests in parental and hFcRn-GFP / hβ2m-MDCK II cells demonstrated the following: (1) higher ASA at pH 5.8 compared to pH 7.4 WT Intake / Recycling, (2) Compared with parent MDCK II data, ASA under all pH conditions AAA There was no difference in uptake / recycling, (3) higher recycling under 37°C conditions for 4 hours compared to 4°C (when detectable uptake was observed), and (4) nonspecific uptake of ASA hIgG1 into parental MDCK II cells was negligible (Figure 10B). Notably, ASA after pH 7.4 loading WT The extremely low recycling signal was not a result of inefficient hFcRn-mediated recycling, but rather due to the minimal amount of mAb entering the cell during the loading phase.

[0131] To characterize the difference in total CL of ASA mAbs, rodent PK studies were performed (Figure 10C). For in vivo studies, hFcRn transgenic mice (Tg32) were selected to preserve the species-dependent interaction between ASA hIgG1 and FcRn and for previous reports on the fidelity of this preclinical model for human mAb PK translation. AAA ASA WT Compared to Tg32 mice, Tg32 mice showed higher systemic CL (Table 6). To confirm these results, matching tests were performed in homozygous Tg276 hFcRn mice. Tg276 has a promoter distinct from that in Tg32 and exhibits mutations in hFcRn tissue expression, but can provide an alternative humanized mouse model to depict changes in hIgG arrangement. Again, ASA AAA ASA WT It showed a much faster CL (Table 6).

[0132] One of the goals of this project is mAb CL indThe goal was to generate an in vitro assay that could rank a series of compounds to inform about [the subject]. Previous reports outlined metrics that facilitate the calculation of hFcRn-mediated recycling efficiency in cultured endothelial cells. Since the initial approach alone could not describe the cellular arrangement of mAbs with a wide range of nonspecific features, the scoring system in this report has recently been expanded to also account for nonspecific features (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 (the whole is incorporated herein by reference)). These published studies utilized pH 7.4 loading conditions to replicate physiological conditions within the vascular system. However, based on the internalization results, ASA WT To improve signal sensitivity, a similar scoring method was applied here to the MDCK II cell study using a pH 5.8 loading condition (Figures 8A, 8B, and 10B). The human FcRn recycling efficiency metric (FREM) score in this study was calculated as described above by multiplying the recycled fraction by the nonspecific uptake coefficient (NUC) (Grevys, et al. (2022) Antibody variable sequences have a pronounced effect on cellular transport and plasma half-life. iScience 25, 103746 (the whole is incorporated herein by reference)). The NUC was incorporated only if the analyzed mAb showed elevated nonspecific endocytosis in hFcRn-GFP / hβ2m-MDCK II cells compared to uptake in parental MDCK II. ASA AAAThe FREM score is ASA WT Compared to ASA, it can be ignored. AAA This highlights the lack of hFcRn interaction in the mutants (Figure 10E). These results showed a trend in CL values ​​obtained from both hFcRn transgenic mouse models (Figures 10C and 10D).

[0133] In summary, to validate the cell lineage, we defined hFcRn-specific and nonspecific transport of two hIgG1 mAbs with varying hFcRn affinities using both parental and transfected MDCK II cells. ASA WT ASA AAA A scoring system was applied that allows for easy differentiation. Furthermore, the calculated hFcRn recycling score was consistent with the CL value measured in a single-dose PK test in hFcRn transgenic mice.

[0134] Example 6: Human FcRn recycling score from MDCK II cells can identify mAbs with rapid human CL. Next, the MDCK II cell line has high non-target-mediated CL (i.e., CL) indThe ability to detect mAbs with ) was investigated. A total of 10 mAbs with human CL values ​​ranging from 2.45 to 32.2 mL / d / kg were selected (Table 7). All mAbs were assayed in parental cells and hFcRn-GFP / hβ2m-MDCK II cells under the same conditions as the ASA hIgG1 series (Figure 14). The clinical trial analog mAb, mAb1, was selected as a reference for all subsequent comparisons because it behaved well in the MDCK II cell test (appropriate hFcRn interaction, low nonspecific uptake) and is an FDA-approved drug with low CL (Figure 11A, Table 7). As described above for ASA mAbs, the NUC metric indicated that it was incorporated only when the NUC value was below 1, resulting in excessive nonspecific uptake compared to what is internalized by hFcRn. FREM scores with a mean of less than 25% for mAb1 correlated with human CL values ​​greater than 4.8 mL / kg / d, thus highlighting the potential of this approach to identify mAbs with PK properties (Figure 11B).

[0135] [Table 7]

[0136] Example 7: High mAb CL ind A clear mechanism Observations from the hFcRn recycling test showed an association between nonspecific interactions (by NUC value) and the final hFcRn recycling score (Table 7), indicating a strong role for nonspecific behavior in hFcRn recycling efficiency. Examination of parental MDCK II uptake results revealed that anti-IL-4Rα mAb and mAb9 were unique outliers in terms of nonspecific characteristics. Compared with the mAb1 analogue, anti-IL-4Rα mAb exhibited significantly higher endocytosis into parental MDCK II cells at pH 7.4 (Figure 11C, ####p<0.0001). This is consistent with Examples 1-3, which demonstrate widespread nonspecific cellular uptake of anti-IL-4Rα mAb at this pH value, which may have contributed to its rapid CL in wild-type mice and humans. A large charge patch was identified within the anti-IL-4Rα mAb Fv region, which is likely a determinant of this observation. Therefore, in the case of anti-IL-4Rα mAbs, widespread nonspecific endocytosis from extracellular fluid (e.g., blood) may displace large amounts of mAbs into non-target cell populations, which would increase the probability of intracellular catabolism.

[0137] mAb9 is an anti-interleukin-12 / 23 antibody with unusual nonspecific and hFcRn interactions that result in its high CL rate, as reported in the literature. In contrast to anti-IL-4R α mAbs, mAb9 showed elevated, but not statistically significant, parental MDCK II uptake at pH 7.4 compared to mAb1 (which is also an anti-IL-12 / 23 antibody) (Figure 11C). However, a significant increase in nonspecificity for mAb9 was measured at pH 5.8 (†††, p<0.001). Recently, it has been shown that the high positively charged surface area within the Fv region of mAb9 distinguishes its anti-IL12 / 23 variable domain from mAb1, and that it is also pH sensitive, with increased separation under acidic conditions. This is supported by current findings that the previously reported Fv domain charge difference between mAb1 and mAb9 likely promotes the elevated nonspecific uptake at pH 7.4, which is larger and significantly different at pH 5.8. Therefore, mAb9 has a pH-dependent charge patch that can not only affect its association / dissociation from hFcRn and initial nonspecific endocytosis rate at pH 7.4, but also acidify it to impair mAb9 dynamics with hFcRn and reduce its recycling efficiency, thus leading to increased nonspecific interactions within the endosome.

[0138] Furthermore, a slight increase in pH-dependent nonspecific uptake of mAb1 in parental MDCK II cells was observed (Figure 11C, *p<0.05). To better characterize these results across the entire mAb series, a follow-up study (described above) was conducted to directly measure mAb nonspecific endocytosis in CHO-K1 cells (Figure 11D). This method involves incubation of parental CHO-K1 cells with the mAb of interest at 37°C for 60 minutes. Analysis of cell-associated mAbs (i.e., intracellular and surface) is achieved by anti-human Fc immunostaining and flow cytometry. To allow for comparisons between days, standard curves are created daily using quantitative microspheres bound to a fixed amount of detection antibody (antibody binding capacity, ABC). The use of the CHO-K1 assay provides a means to reproduce the potential for altered nonspecific interactions within endosomal compartments by performing incubation at acidic pH. Higher cell uptake rates using this method would suggest increased nonspecific interactions at the tested pH values.

[0139] In CHO-K1 cells, ASA WT Increased nonspecific endocytosis was measured for mAb9 (p<0.01) and anti-IL-4Rα mAb (p<0.0001) at pH 7.4 compared to the above (Figure 11D). At pH 5.8, all mAbs showed increased endocytosis compared to the pH 7.4 condition, indicating an increase in nonspecific interactions with decreasing pH. Similar to the observations in parental MDCK II cells (Figure 11C), the nonspecificity of mAb9 increased to an amount comparable to that of anti-IL-4Rα mAb only at pH 5.8. These results are relevant to CL ind This provides evidence for two different mechanisms of elevation. The first is high general nonspecificity within endosomes, likely due to marked nonspecific uptake from extracellular fluid at pH 7.4 and mitigation of mAb-FcRn interactions (e.g., anti-IL-4Rα mAb). The second (e.g., mAb9) is low CL due to increased nonspecific extracellular interactions. indCompared to mAbs, moderate nonspecific endocytosis occurs at pH 7.4. Following endocytosis, these nonspecific behaviors increase during endosomal transport, potentially leading to either unintended association with the endosomal membrane and / or impaired endosomal interaction with FcRn, both of which can result in increased intracellular catabolism. Higher CLs do not exhibit extreme degrees of these aspects. ind For the tested mAbs, the rate of extracellular nonspecific endocytosis and / or the degree of acid nonspecificity are likely to exceed the mAb's ability to efficiently interact with FcRn within endosomes, resulting in reduced FcRn recycling efficiency (Figure 12).

[0140] Example 8: The cell hFcRn recycling score can successfully and simultaneously rank WT and Fc-operated mAbs in order of decreasing mouse CLind. CL ind One approach to reduce the half-life of Fc-interacting molecules (e.g., Fc biologics) and extend their serum half-life is to enhance the affinity of the Fc region to FcRn. Doing so requires selectively increasing Fc-FcRn binding at acidic pH values ​​while still maintaining the lowest 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 FcRn recycling assay described herein provides an important in vitro tool for evaluating the potential success of Fc manipulation strategies. However, assay conditions must be specifically optimized to properly evaluate therapeutic proteins with altered FcRn interactions. Otherwise, it may lead to assay failures that correlate with in vivo behavior. For example, previous studies using cell-based FcRn transcytosis assays have failed to clearly explain the CL of antibodies with enhanced FcRn binding.ind We were unable to successfully communicate this (Chung et al., MAbs 11(5):942-955(2019)).

[0141] CL of Fc-interacting molecules with enhanced FcRn affinity ind To demonstrate the feasibility of the cell hFcRn recycling assay described herein for accurate information, we expanded the physicochemical diversity of the anti-IL-4Rα mAb panel detailed in Example 3. In particular, we generated additional one-point and two-point mutants with higher positive charge attributes within the variable (Fv) domain compared to the quadruple mutant of Example 3 (Table 8). NSE measurements were performed in parental CHO-K1 cells at both pH 7.4 and 5.8, which showed a much higher spread of nonspecific behavior than observed in the original panel (Figures 15A-15B). Subsequently, the M252Y / S254T / T256E(YTE) mutation (EU: European Union number) was used to demonstrate clinically proven half-life extension and CL ind As a model for increased FcRn affinity accompanied by a decrease, it was incorporated into each anti-IL-4Rα mAb mutant (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 addition of YTE generally maintained the overall nonspecific trend between mAb and YTE, but compared to the parental WT Fc mAb, it resulted in an increase in NSE at pH 7.4 and a decrease in nonspecificity at pH 5.8 (Figures 15A-15B).

[0142] Enhanced Fc-FcRn interaction strategies, such as YTE mutations, result in higher internalization in FcRn-expressing cells across pH values ​​from acidic to near-neutral, with a net degree driven by relative affinity changes across these conditions. This increases the amount of mAb-YTE entering hFcRn-GFP / hβ2m MDCK II cells at any given concentration compared to non-YTE mAbs. This can saturate the FcRn capacity per cell, leading to misassessment, thereby falsely suggesting that YTE mutants behave worse than their matched WT parents after cell hFcRn recycling tests. To offset this affinity effect and equalize intracellular loading conditions, ASA of DyLight 650 conjugated control mAbs is used. WT -hIgG2 and ASA WT Concentration-dependent endocytosis tests were performed using -hIgG2-YTE at pH 5.8 (Figures 16A-16D). Uptake tests and K m and V max The value was estimated as described above. ASA WT -hIgG2-YTE-DL650's K m (15[10,21]μg / mL) is ASA WT -hIgG2-DL650's K m It was observed that the level was approximately 3.5 times lower than (41[26,64]μg / mL). Based on this data, in a subsequent FcRn recycling test using hFcRn-GFP / hβ2m MDCK II cells, approximately 2×K of WT (100μg / mL) and YTE (25μg / mL) were used for acidic uptake incubation to ensure that similar amounts of WT and YTE mAbs were reliably loaded into the cells in an FcRn-dependent manner. m The concentration was used.

[0143] Cell-based hFcRn recycling assays were performed using 6 WT / YTE pairs of anti-IL-4Rα mAbs. FREM scores were calculated by analyzing uptake, recycling, and residual samples, and were based on either / both FcRn recycling efficiency (RE) and nonspecific uptake coefficient (NUC) scores, as outlined above (Figure 17A). The assay was optimized to simultaneously compare both Fc-operated YTE and WT mAbs by normalizing all results to anti-IL-4RαEEES-YTE mAbs.

[0144] [Table 8]

[0145] As exemplified by the WT anti-IL-4Rα-mAb, the higher nonspecificity and / or lower FcRn recycling efficiency of the mAb resulted in lower FREM scores. A single point mutation at position R20 did not significantly improve the FREM score of the 20S-WT variant (0.006±0.001) compared to the WT (0.004±0.001). However, the R33S mutation in heavy chain (HC)CDR1 improved the FREM score (0.155±0.048) compared to the R20S mutation. Dual point mutations in both HC CDR1 and CDR3 dramatically improved the FREM score of anti-IL-4Rα mAb-33S, 110L (0.504±0.085) compared to a single point mutation in the mAb Fv region. The anti-IL-4Rα mAb-SSLS mAb showed a slightly lower FREM score (0.400 ± 0.095) compared to the double-point variant. The most exposed charge-mediated WT mAb variant, anti-IL-4Rα mAb-EEES, showed a higher FREM score (0.584 ± 0.127) compared to all other WT mAbs. The trend of relative FREM score improvement was conserved between the WT variant mAb series and the YTE variant mAb series. When tested with WT mAbs, the half-life extended YTE mAbs showed better FREM scores compared to their WT counterparts, with the exception of the SSLS-YTE variant which showed minimal improvement. In one-to-one comparisons, anti-IL-4Rα mAb-EEES-YTE showed the highest FcRn recycling efficiency metric score (1.000 ± 0.196).

[0146] [Table 9]

[0147] PK testing was performed in male homozygous immunodeficient SCID hFcRn Tg32 transgenic mice. Due to the lack of mature T cells or B cells, this mouse model facilitated the ability to obtain PK over long sampling time courses without interference from anti-drug antibodies. Serum concentration-time profiles of mAbs are shown in Figure 17B. From the PK results, it was clear that mAbs with higher NSE and lower FcRn recycling efficiency had inferior PK overall (e.g., anti-IL-4Rα mAbs). Next, both WT and YTE mAbs were ranked simultaneously using the FREM score. We performed CL for all mAbs. ind We observed a very strong correlation between the score and the FREM score, with lower FREM scores being associated with higher CL scores. ind It was related to this (Figure 17C).

[0148] Discussion of Examples 4-8 This study provides a quantitative and mechanistic examination of endocytosis and intracellular handling of a panel of mAbs to describe differences in hFcRn-mediated recycling efficiency. The experimental outline provided a means to simultaneously evaluate the hFcRn-dependent endocytosis, recycling, and nonspecific endocytosis properties of mAbs assessed within a cell assay. The degree of nonspecific interaction strongly determined the recycling results, which were distinguished by the mass balance method performed in this study. These findings suggest that mAb CL ind When testing, it is important to emphasize the need to account for nonspecific endocytosis rate, total mAb internalization, and hFcRn recycling.

[0149] Wild-type mice have low CL ind It possesses and exhibits negligible nonspecific endocytosis in mammalian cells, so ASA can be used as a model compound. WT We used ASA at pH 7.4 in parental MDCK II cells to measure mAb endogenous dynamics in both parental and hFcRn-GFP / hβ2m MDCK II cells. This study compared ASA at pH 7.4 in parental MDCK II cells with receptor-mediated conditions at pH 5.8 in transfected MDCK II cells. WT-Demonstrated very low time-dependent uptake of DL650. Concentration-dependent endocytosis in hFcRn-GFP / hβ2m MDCK II cells at pH 5.8 under competitive conditions was observed in extracellular ASA WT - Directly proportional to DL650 concentration, and in the absence of receptor-mediated pharmacokinetics, ASA WT This was consistent with nonspecific endocytosis.

[0150] These measurements indicate low CL ind We confirmed that the mAb containing should show minimal nonspecific uptake at pH 7.4, which was important for testing hFcRn recycling efficiency. This leads to a negligible amount of recycling when 100 μg / mL is used. The data herein show that pH 5.8 promotes FcRn-mediated endocytosis in hFcRn-GFP / hβ2m MDCK II cells, and substantially more ASA when compared to that internalized via nonspecific endocytosis alone. WT -DL650 was introduced into the cells. The concentration required to achieve detectable recycling after the pH 7.4 loading phase was ASA WT Alternatively, the use of the material for other mAbs exhibiting low nonspecific uptake would be unreasonable. Therefore, these results, by indicating that acid loading is necessary to achieve detectable concentrations for mAbs with limited nonspecific endocytosis, led to subsequent recycling tests.

[0151] ASA WT - The DL650 kinetic study also demonstrated the contribution of hFcRn to initial mAb internalization at pH 7.4. ASA to parental MDCK II cells WT -DL650 uptake was lower in transfected cells throughout each time point tested at pH 7.4, and at pH 8.0 compared to pH 7.4, ASA in hFcRn-GFP / hβ2m MDCK II cells. WT - The DL650 resulted in a reduction in endocytosis.

[0152] Cell surface FcRn could contribute to IgG endocytosis outside of acidic environments, but this was not the focus of this project. The data herein indicate that nonspecific endocytosis is the primary IgG endocytosis mechanism at near-neutral pH where neither FcRn-containing targets nor IgG receptors are present. ASA WT Results using DL650 showed that FcRn can promote IgG internalization to some extent at pH 7.4, but results from mAb panel recycling studies suggested minimal FcRn involvement for internalization at this pH value. The extent of FcRn's contribution at pH 7.4 in vivo remains unclear. Future studies evaluating this issue should consider utilizing other physiologically relevant cell lines.

[0153] The success of using acidic incubation was confirmed using a clinically relevant mAb panel, showing that very small amounts of multiple mAbs were internalized in hFcRn-GFP / hβ2m MDCK II cells at pH 7.4, resulting in essentially undetectable concentrations of recycled material after incubation at 100 μg / mL. It is important to note that, as demonstrated above, cell-based FcRn recycling tests at near-neutral pH are more physiological and can be performed. However, ASA WT - Based on the internalization kinetics of DL650 (Figures 8 and 13), to achieve similar internalization levels of the same mAb at pH 7.4, and therefore assay resolution, low nonspecific endocytosis is required. indFor mAbs with low efficacy, at least 30 times higher concentrations would have been required at pH 5.8. This observation and explanation are based on the aforementioned studies ((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 (the entire assay is incorporated herein by reference)).

[0154] The specificity of the MDCK II hFcRn recycling assay was established by testing for ASA hIgG1. AAA The results showed no discernible difference between parental cells and hFcRn-GFP / hβ2m MDCK II cells under any conditions, supporting the deletion of hFcRn binding affinity in the mutant amino acid. Furthermore, by including several experimental groups for the MDCK II cell test, mAb CL ind The mechanisms driving the differences were fully described. This framework facilitated mAb internalization, intracellular transport, hFcRn-mediated recycling, residual accumulation, and capture of nonspecific interactions. In vitro hFcRn recycling scores were modified from previous reports to provide a means of pooling cell datasets into cumulative metrics to describe hFcRn recycling efficiency. Mean FREM scores greater than 0.5 compared to mAb1 analogs indicated mAbs with attributes associated with low elimination rates in humans, including low nonspecific uptake and the ability to effectively undergo hFcRn-mediated recycling. hFcRn recycling scores less than 0.25 strongly indicated mAbs with CL greater than 5 mL / d / kg. FREM scores between 0.25 and 0.5 indicated CL ind This suggested a potential debt that could lead to an increase.

[0155] When tested with the in vitro cell-based recycling assay described herein, the FREM score of the YTE molecule was found to be relatively higher than that of the WT variant. In general, the target-independent PK profiles of these engineered mAb variants were well described by the in vitro cell-based assay discussed in this example. Considering the involvement of both NSE and FcRn via the FREM score, the conclusions about these engineered proteins were reinforced, indicating increased PK predictive capacity. Furthermore, the use of acidic conditions required optimization of the experimental workflow to accommodate the evaluation of Fc-engineered mAbs with increased FcRn affinity. This would have been necessary for the intrinsic increase in FcRn affinity over a certain range of pH values, even if physiologically relevant endocytosis conditions were used (i.e., approximately pH 7.4) (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)).

[0156] The result was mAb CL indWhile supporting the importance of nonspecific and hFcRn interactions in determining these processes, this also provides further insight into the diversity of mechanisms governing these cellular processes (summarized in Figures 12A-12B). Here, a significant effect of pH on nonspecific interactions for all mAbs was demonstrated, as indicated by the higher rate of nonspecific endocytosis in CHO-K1 cells at pH 5.8 compared to pH 7.4. This observation suggests that endogenized mAbs may exhibit nonspecific interactions to a different degree and morphology than those shown on the cell surface at pH around 7.4. The isoelectric points (pI) of all mAbs tested were greater than 7.0, which can result in an increase in positive charge at acidic pH (Table 7). This may have contributed to the universal increase in nonspecific endocytosis, due to increased interaction with the negatively charged plasma membrane. However, pI alone cannot adequately explain the large difference in nonspecific endocytosis between anti-IL-4Rα mAbs or mAb9 compared to other mAbs.

[0157] Regarding cellular outcomes, the observed pH-dependent characteristics led to harmful nonspecific association with the endosomal membrane, as well as reduced hFcRn recycling efficiency and higher CL. ind This can lead to both harmful nonspecific association with hFcRn itself, resulting in a high rate (Figure 12). What can be concluded now is the importance of incorporating appropriate experimental controls when evaluating mAb cell placement and hFcRn interactions. The hFcRn recycling score was obtained at pH 5.8, and the NUC metric was used to assess the degree of nonspecific uptake related to mAb interactions with hFcRn. Where measuring nonspecific interactions alone provided partial insight (e.g., CHO-K1 endocytosis), NUC provided a means to simultaneously define both mAb-hFcRn interactions and nonspecific interactions within the context of the same experimental setting. This is crucial for in vitro performance and critical engagement. ind It was important in the ability to outline the relationship between them.

[0158] Single-parameter in vitro evaluation to predict the in vivo behavior of mAbs is mAb CL indOften, there is no direct correlation. This may be because it is not possible to fully reproduce the biological dynamics important for mAb placement, or because it focuses on only a single aspect. A key advantage of using the cell hFcRn recycling assay described herein is the integration of multiple processes important for mAb placement. The results reported herein are for mAb CL ind This is consistent with the capabilities of IgG-FcRn demonstrated by other cell-based evaluations, further highlighting the multifaceted relationship between cellular uptake and FcRn-mediated recycling. Cell-based systems provide reads within a biological framework, which have been directly shown to be advantageous for testing FcRn-IgG and FcRn-Fc interactions compared to standard biophysical approaches. Subsequently, physiologically based PK models will play a crucial role for expanding in vitro outcomes to patients by helping to describe the net impact of in vitro behavior on mAb placement. As the biopharmaceutical industry moves towards more complex structures for its biological pipelines, combination methodologies that leverage the known drug-like attributes of mAbs will be critically needed to develop low-CL Fc-HLEs.

[0159] Example 9: High nonspecific endocytosis predicts low subcutaneous bioavailability in humans. Subcutaneous administration remains the preferred injection route for therapeutic proteins because it facilitates drug delivery at a more convenient physical location for the patient. However, drugs administered subcutaneously must first be absorbed into the bloodstream from the injection site. (Subcutaneous bioavailability, F) SQ) depends on several factors including temperature, solubility, lymphatic 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 (the whole is incorporated herein by reference). The complexity of these factors and the insufficient mechanistic understanding of their underlying mechanisms are relevant to human F SQ This resulted in a lack of ability to use in vitro technology to inform about this.

[0160] As demonstrated herein, nonspecific endocytosis and FcRn recycling are CL for intravenously administered monoclonal antibodies (mAbs). ind These are important determinants. Defining the cellular mechanisms governing these processes and establishing quantitative experimental workflows are crucial for rapid in vitro clinical trials (CL). ind This led to an improved ability to identify therapeutic mAbs possessing these characteristics. These same factors, particularly nonspecific endocytosis, were hypothesized to be important determinants of FSQ. This is because a high rate of nonspecific endocytosis at the injection site increases therapeutic protein catabolism, resulting in less drug available for absorption into the circulation. Furthermore, mechanisms driving high nonspecific endocytosis are also thought to include nonspecific interactions with the subcutaneous extracellular matrix, such as F SQ This can lead to other in vivo properties that are harmful to human F. Therefore, the use of cell-based assays to quantify nonspecific endocytosis is not recommended. SQ It was expected to be beneficial in this regard.

[0161] A panel of therapeutic mAbs with clinical pharmacokinetic (PK) data was obtained, of which 17 showed target-independent clearance (CL). ind They had estimates for ) mAbs were binned into three groups: (1) CL < 4.5 mL / kg / d after intravenous (IV) dose in humans ind (2) CL exceeding 4.5 mL / kg / d ind , or (3)CLind No estimate was obtained. CL of 4.5 mL / kg / d ind This corresponds to an approximate final half-life of 10 days in a human individual weighing 80 kg.

[0162] In Examples 1-3, as described above, antibodies were analyzed using the CHO-K1 assay to obtain cell antibody binding capacity (ABC), and a series of behaviors were observed as shown in Figure 21A. Assay thresholds were then generated to better interpret the data. The "low-risk" group exhibited high CL. ind and / or low F SQ Contains mAbs with a low risk (due to nonspecific endocytosis). This is used in a formula with a F content exceeding 50%. SQ and / or CL less than 4.5 mL / kg / d ind The upper limit of the 95% confidence interval for the mean ABC of all mAbs with the same CL was defined as the upper limit of the CL. The "intermediate risk" group had high CL. ind and / or low F SQ The study included mAbs that may or may not exhibit cytosis and were set to be 2 standard deviations above the mean ABC directly mentioned above (i.e., mean + 2 SD). The "high-risk" group highlights mAbs with apparent nonspecific endocytosis that are likely to be harmful to their configuration in humans.

[0163] Next, the mAbs were grouped according to their performance in the CHO-K1 assay (i.e., low-risk, medium-risk, or high-risk), and their respective CLs were selected. ind The values ​​were plotted. Figure 21B shows that a higher percentage of nonspecific endocytosis corresponds to an increased risk of higher CL after IV administration. ind In addition, F in humans SQ This demonstrates that it was an indicator of the decrease. Notably, all mAbs characterized as high risk were those with a human dose of more than 4.5 mL / kg / d and less than 50% F SQ CL in either or both ind This was shown.

[0164] Example 10: Nonspecific intracellular endocytosis can identify therapeutic proteins with high target-independent clearance across a wide range of structural modes. Multispecifically engineered proteins (MS-ePRs) are protein-based therapeutics that can simultaneously target two or more different targets. These compounds offer novel mechanisms for treating a variety of diseases that have not been previously achievable with monoclonal antibodies (mAbs). These encompass a wide range of molecular formats that can be tailored to the intended therapeutic use, including engineered structures to achieve optimal efficacy (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 Pt A):95-106(2015); Dickopf et al., Comput. Struct. Biotechnol. J. 18:1221-1227(2020)). MS-ePRs may also contain an Fc region or utilize an immunoglobulin G scaffold (mAb-ePR) to reduce clearance (CL) and decrease administration 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 Pt A):95-106(2015)). The Fc portion facilitates interaction with FcRn, which recycles endogenous immunoglobulin G, albumin, and Fc / albumin fusion proteins via intracellular pH-dependent binding, thereby increasing their serum half-life (Challa et al. al.,Curr.Top.Microbiol.Immunol.382:249-72(2014)).

[0165] The plasticity of MS-ePRs can not only broaden therapeutic prospects but also lead to development obstacles. Ideal structural configurations for efficacy may also be difficult to synthesize. Certain manipulated designs may be prone to encountering immunogenicity or stability issues in vivo. Furthermore, the configuration of mAb-ePR drugs is far more complex than that of mAbs (Deshaies R., Nature 580(7803):329-338(2020); Husain and Ellerman, BioDrugs 32(5):441-464(2018)). Target-mediated pharmacokinetics play a crucial role in the overall pharmacokinetic (PK) profile of mAbs. Target involvement can lead to target-mediated drug placement (TMDD), where the mAb CL becomes strongly intertwined with the biology of its target, resulting in dose-dependent changes in mAb placement (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)). Since at least two distinct targets exist, the degree of TMDD inherently grows with mAb-ePRs, which can be further complicated by changes in the site of their expression and / or binding affinity to mAb-ePRs (Deshaies R., Nature 580(7803):329-338(2020)).

[0166] The target-independent mechanisms of mAb CL are also important for mAb-ePR, including FcRn-mediated recycling to non-target cell types and nonspecific adsorption endocytosis as described above. Complex structures that may arise from protein engineering may lead to poor interaction with FcRn or rapid target-independent CL (CL) of mAb-ePR. indThis can lead to a higher proportion of nonspecific adsorption uptake by unintended cell types (Datta-Mannan, Antibodies (Basel) 11(1):2 (2021); Datta-Mannan et al., Biochemistry 58(28):3116-3132 (2019)). However, direct cytological measurement of mAb-ePR nonspecific endocytosis is particularly important for identifying broad structural features and preclinical clinical clusters. ind Numerous proteins that possess values ​​have not been reported.

[0167] To address this limitation, we formed two different panels of proteins and quantified their nonspecific endocytosis rates by obtaining ABC values ​​at pH 7.4 and / or pH 5.8 in CHO-K1 cells. The first protein series contained 48 mAb-ePRs generated on Amgen's patented stable, effector-less Fc scaffold (Liu et al., J. Biol. Chem. 295(5):1876-1883 (2017)). The proteins exhibited different targeting valencies, structural configurations of their binding sites, and broad CLs in Tg32 human FcRn transgenic mice after a single intravenous administration. ind The molecules had various molecular formats, including (Figure 22A). ABC values ​​were calculated as shown in Figure 3. mAb-ePRs were placed into low, medium, or high-risk bins based on their ABCs by using the same numerical thresholds and approaches outlined above and shown in Figure 21. Consistent with observations for the clinical mAb panel, 94% of mAb-ePRs placed in the high-risk bin (i.e., exhibiting high nonspecific endocytosis quantified via their ABCs) and 92% of mAb-ePRs in the medium-risk group exhibited CLs exceeding 0.5 mL / kg / hour in Tg32 mice. ind This showed that the high-risk group had the highest CL. ind We were able to successfully identify mAb-ePRs possessing this characteristic (Figure 22B).

[0168] The second group of proteins consisted of five distinct protein structures bivalently fused to a human Fc domain, comprising a total of 28 different Fc fusion proteins that did not affect human IgG or typical mAb-ePRs. The primary objective of this panel was to use nonspecific endocytosis to determine the CL of any protein, not just those based on IgG or the corresponding scaffold. ind The objective was to determine whether it was possible to describe the Fc fusion series in C57BL / 6 wild-type mice after a single intravenous administration of 2 mg / kg. ind The values ​​are shown (Figure 23A). CHO-K1 ABC values ​​were obtained at both pH 5.8 and pH 7.4, and their CL values ​​were obtained. ind The values ​​were plotted against (Figures 23B and 23C). The pH 7.4 value directly measures nonspecific endocytosis by mammalian cells. The pH 5.8 condition mimics the endosomal environment and quantifies the pH-dependent shift of positive charge and nonspecificity expected to result from endosomal acidification during intracellular transport as described above. Using the same ABC cutoff values ​​for the above mAb and mAb-ePR protein panel, proteins were placed into low, medium, and high-risk groups based on the degree of nonspecific endocytosis (calculated using the ABC method). 94% of the proteins in the high-risk bin had CLs exceeding 3 mL / kg / hour. ind It had the highest CL, similar to mAb-ePR. ind The protein containing [the specified element] was successfully captured.

[0169] High-risk CL ind The usefulness of using a pH 5.8 surrogate scale for endosomal nonspecificity in protein identification was evaluated. All proteins showing high nonspecific endocytosis at both pH 5.8 and pH 7.4 showed high CL. ind (Figures 23B and 23C) were shown. Next, the inventors filtered the proteins by examining the pH 5.8 ABC values ​​of only Fc-fusion proteins that had low-risk or medium-risk ABC at pH 7.4 (Figure 23E). Of these, three proteins showed a significant increase in nonspecific behavior at lower pH levels, and two showed very high CL in wild-type mice.ind It had (Figure 23F). This was achieved using conditions of pH 5.8, CL ind This means we can identify more treatment patterns that carry a high risk of increased risk.

[0170] Results from mAb-ePR proteins and Fc-fusion proteins showed high nonspecific endocytosis and high CL. ind It has been demonstrated that it provides in vitro metrics strongly related to CL. The data presented herein show that the described cell-based endocytosis assays provide an in vitro metric strongly related to CL in rodents. ind Further demonstrating the ability to identify structurally unrelated proteins possessing [specific characteristics]. Furthermore, the described approach offers a unique and novel capability for quantifying pH-dependent changes in the nonspecific behavior of biologics. However, evaluating nonspecific endocytosis alone is insufficient. ind This is insufficient to successfully identify all compounds in which CL levels were elevated in rodents. ind This was observed in low and intermediate-risk groups, including several constructs that showed elevated levels. As detailed above, CL for mAbs, mAb-ePRs, and any other biological modalities. ind Further factors drive this. One key factor for Fc-containing proteins is FcRn interaction. Therefore, combining nonspecific endocytosis, nonspecific / pH-dependent shifts of charge, and cell-based readout for engagement with FcRn significantly increases the ability to identify high-risk biologics that interact with FcRn, leading to protein engineering efforts to improve the PK attributes of preclinical candidates.

[0171] Each reference cited herein is incorporated herein in its entirety by reference for all the purposes it teaches.

[0172] The present invention is not limited in scope by a single description of individual embodiments of the invention, nor by any specific embodiments described herein that are intended to be functionally equivalent methods and components of the invention. In fact, various modifications of the invention, in addition to those shown and described herein, will be apparent to those skilled in the art from the above and the accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

Claims

1. A method for predicting in vivo non-target-mediated clearance of biomolecules, To provide a cell preparation in which the cells of the preparation do not express the target of the biomolecule; Incubating the cell preparation with a culture medium containing the biomolecules under conditions that mimic in vivo physiological conditions; Determining the amount of biomolecules taken up by the cells of the preparation after the incubation; and Predicting the in vivo non-target-mediated clearance of the biomolecule based on the above determination. Methods that include...

2. The method according to claim 1, wherein the culture medium during incubation has a pH of about 7.0 to about 8.

0.

3. The method according to claim 1, wherein the culture medium during incubation has a pH of about 5.6 to about 7.

0.

4. The method according to claim 1, wherein the incubation is performed at 37°C for approximately 60 minutes.

5. The aforementioned decision is: Permeabilizing the cells of the preparation after incubation; To detect the amount of biomolecules taken up by the cells of the preparation; and Based on the above detection, the amount of biomolecules taken up by the cells of the preparation is quantified. The method according to claim 1, comprising predicting the in vivo non-target-mediated clearance of the biomolecule, based on the quantification thereof.

6. The method according to claim 5, further comprising incubating the permeated cells with a detectable portion bound to the biomolecule after the permeation treatment, wherein the amount of the detectable portion bound to the biomolecule is detectable.

7. The above quantitative determination is The method according to claim 5, comprising comparing the detected amount of the biomolecule taken up by the cells with one or more quantitative reference values.

8. The method according to claim 7, wherein the one or more quantitative reference values ​​include a calibration curve for biomolecular binding capacity.

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

10. The method according to claim 5, wherein the detection is performed using flow cytometry, immunoassay, or microscopy.

11. The method according to 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 according to claim 1, wherein the biomolecule is a multispecific antibody.

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

14. The method according to claim 1, wherein the cells of the preparation do not express Fc receptors.

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

16. The method according to claim 1, wherein the cell preparation is a cell preparation 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 for predicting in vivo non-target-mediated clearance of FcRn interacting molecules, To provide a first cell preparation wherein the cells of the first preparation do not express hFcRn; To provide a second cell preparation in which the cells of the second preparation express a heterodimer of human neonatal Fc receptor (hFcRn) and human β2m (hβ2m), and the cells of the first and second preparations do not express the binding target of the FcRn interacting molecule; The first and second cell preparations are subjected to first and second incubation periods, wherein the first incubation period comprises incubating the cell preparations with a medium containing the FcRn interaction molecule under acidic and / or non-acidic conditions, and the second incubation period comprises incubating the cell preparations after the first incubation with a medium lacking the FcRn interaction molecule under non-acidic conditions; Determining the amount of the FcRn interacting molecule taken up by the cells of the first and second preparations after the first incubation period and / or the second incubation period; Measuring the amount of the FcRn interaction molecule in the culture medium collected at the end of the second incubation period; Based on the aforementioned determination and measurement, respectively, to quantify the nonspecific endocytosis and FcRn recycling of the FcRn interacting molecule; and Based on the above quantification, predict the in vivo non-target-mediated clearance of the FcRn interaction molecule. Methods that include...

18. The method according to claim 17, wherein the first incubation comprises incubating the first and second cell preparations with a culture medium containing the FcRn interacting molecule under acidic and non-acidic conditions.

19. The method according to claim 17, wherein the first incubation comprises incubating the first and second cell preparations in a culture medium containing the FcRn interacting molecule under acidic or non-acidic conditions.

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

0.

21. The method according to any one of claims 17 to 19, wherein the acidic conditions include a medium pH of 5.6 to 6.

9.

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

23. The method according to claim 17, wherein the first incubation period comprises incubating the first and second cell preparations with a medium containing FcRn interacting molecules at a concentration that does not saturate the FcRn binding occupancy of the cells of the second cell preparation.

24. The method according to claim 23, wherein the concentration of the FcRn interacting molecule in the culture medium achieves an FcRn binding occupancy rate of 25-90% of the cells in the second cell preparation.

25. The method according to claim 17, wherein the second incubation period comprises incubating the first and second cell preparations with a medium that does not contain the FcRn interacting molecule at 37°C for about 3 to about 5 hours.

26. The above decision is Permeabilizing the cells of the first and second cell preparations after the first incubation period and / or the second incubation period; To detect the amount of FcRn interaction molecules taken up by cells in each preparation; and Based on the detection, the amount of FcRn interaction molecules taken up by cells of each preparation is quantified, and the in vivo non-target-mediated clearance of the FcRn interaction molecules is predicted, which is based on the quantification. The method according to claim 17, including the method described in claim 17.

27. The method according to claim 26, further comprising, after the permeabilization treatment, bringing the permeabilized cells into contact with a detectable portion bound to the FcRn interacting molecule, wherein the amount of the detectable portion bound to the FcRn interacting molecule is detectable.

28. The above quantitative determination is The method according to claim 26, comprising comparing the detected amount of FcRn interaction molecules taken up by the cells with one or more quantitative reference values.

29. The method according to claim 28, wherein the one or more quantitative reference values ​​include a calibration curve for FcRn interaction molecule binding capacity.

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

31. The method according to claim 26, wherein the detection is performed using flow cytometry, immunoassay, or microscopy.

32. The method according to claim 17, wherein the FcRn interacting molecule includes a fragment crystallizable (Fc) region.

33. The method according to claim 32, wherein the Fc region of the FcRn interacting molecule is manipulated to enhance binding to FcRn compared to the corresponding unmanipulated Fc region.

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

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

36. The method according to claim 17, wherein the first and second cell preparations are 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.

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