IN VITRO TRANSCYTOSIS ASSAY

Abstract

The present disclosure relates to methods and compositions useful for measuring the transcytosis of molecules. In particular, the present disclosure relates to in vitro receptor-mediated transcytosis assays for assessing the clearance rates of therapeutic antibody molecules and Fc-fusion proteins in humans and animals. [Selected Figure] Figure 1

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 719,402, filed Aug. 17, 2018, the contents of which are incorporated herein by reference in their entirety. [Technical field]

[0002] The present disclosure relates to methods and compositions useful for measuring the transcytosis of molecules. In particular, the present disclosure relates to in vitro receptor-mediated transcytosis assays for evaluating the in vivo pharmacokinetic profile, e.g., clearance rate, and / or half-life of molecules that interact with FcRn, e.g., therapeutic antibodies, Fc fusion molecules, and molecules that bind albumin. [Background technology]

[0003] Therapeutic monoclonal antibodies (mAbs) have become a major class of pharmaceutical products worldwide due to their proven efficacy in treating various diseases and their desirable pharmacological properties. One of the favorable pharmacological properties of mAb drugs is their typically long circulating half-life. The extended half-life of biotherapeutic products may allow for less frequent dosing and / or lower doses of drug, as well as lower concentration-dependent cytotoxicity / adverse events, which reduces the cost of care and improves patient compliance.

[0004] Significant progress has been made in understanding the pharmacokinetic (PK) profiles of mAb drugs in animals and humans. While many mAb drugs exhibit similar PK behavior similar to endogenous IgG, substantial heterogeneity in the nonspecific clearance rates of mAb drugs in humans is commonly observed. Thus, evaluation and selection of clinical candidates for desirable PK properties is a critical early step during drug development. A wide range of techniques involving in silico, in vitro and in vivo analyses have been used to evaluate and / or predict the PK behavior of mAbs in humans (Dostalek et al., 2017, MAbs 9(5):756-766). However, the translation of PK data from animals to humans and from in vitro assays to in vivo readouts remains elusive to drug developers (Vugmeyster et al., 2012, World J Biol Chem 3(4):73-92). In addition, several animal models, such as human FcRn transgenic mice (Avery, 2010) and non-human primates (Deng et al., 2011, Drug Metab Dispos 38(4):600-605), have been successfully used to predict human PK of mAbs, but these studies are typically time-consuming, expensive, and involve animal sacrifice.

[0005] There are many complexities in predicting human PK of mAbs from animal studies and in vitro assays. Target-mediated drug disposition (TMDD) is known to affect the dose-dependent PK behavior of mAbs, resulting in nonlinear distribution and elimination. In the absence of TMDD, mAbs are expected to exhibit linear elimination and nonspecific clearance rates reflecting target-independent, molecule-specific drug catabolism. Nonspecific clearance of mAbs is primarily mediated by lysosomal degradation in the reticuloendothelial system, where the neonatal Fc receptor (FcRn)-mediated Savage pathway plays a major role. Structurally, FcRn is a heterodimer composed of a transmembrane heavy chain (FCGRT) homologous to major histocompatibility complex (MHC) class I-like molecules and a soluble light chain, β2-microglobulin (B2M). FcRn binds to the Fc domain of endogenous IgG at acidic pH (<6.5) but only minimally at neutral or basic pH (>7.0). This unique property allows FcRn to protect Fc-containing molecules from degradation by binding in acidic endosomes after uptake of the molecules into the cell and then transporting them back to the cell surface and released into the circulation at physiological pH. In contrast, internalized molecules that are not bound to FcRn are targeted to lysosomes for degradation (see FIG. 6 and Roopenian et al., Nat Rev Immunol. September 2007; 7(9):715-25).

[0006] Although the contribution of FcRn in extending the half-life of Fc-containing proteins is well recognized (Ward, 2015), reports of a lack of correlation between FcRn binding and PK for mAb drugs in humans (Suzuki, 2010; Zheng, 2011; Hotzel, 2012) suggest that binding affinity to FcRn is not the sole determinant for the clearance rate of Fc-containing proteins. Considering the function of FcRn in intracellular trafficking, the kinetics of endosomal sorting and trafficking of Fc-containing molecules / FcRn complexes likely affect the overall efficiency of the FcRn-mediated salvage mechanism and, therefore, the rate of nonspecific clearance of the molecule. In addition, the process of cellular uptake via pinocytosis or endocytosis and degradation in lysosomes may also play a role in determining the rate and extent of mAb degradation (Gurbaxani, 2013). Finally, biochemical features such as charge / pI and glycosylation patterns have been reported to affect mAb pharmacokinetics (Datta-Mannan, 2015; Igawa, 2010; Khawli, 2010). These molecular features may themselves affect mAb structure, altering the "true" interaction with FcRn in vivo, or may contribute to non-specific cell surface binding that alters the mode / rate of internalization and the relative proportions of FcRn-mediated intracellular trafficking pathways, including recycling and transcytosis.

[0007] A variety of in vitro assays have been developed to model IgG Fc-FcRn interactions. An inherent limitation of many of these assays is the absence of nonspecific interactions with cells, plasma proteins, and extracellular matrix (ECM) components, as well as intracellular trafficking parameters related to mAb recycling, transcytosis, and lysosomal degradation. Preferably, an in vitro assay for prediction of mAb clearance would be able to evaluate the combined effects of electrostatic interactions, cellular uptake of Abs, interactions with FcRn, and intracellular trafficking of molecules. However, considering the multiple factors that affect mAb pharmacokinetics in vivo, it is difficult to develop a simple in vitro assay that accurately and consistently predicts the PK behavior of mAb drugs.

[0008] The development of animal models presents challenges as well. Although animal models such as human FcRn transgenic mice (Avery, 2010) and non-human primates (Deng, 2011) have been successfully used to predict human PK of mAbs, these studies are typically time-consuming, expensive, and involve animal sacrifice. Furthermore, the translation of PK behavior from animal studies to humans is not straightforward, in part due to species differences in FcRn binding, TMDD, and induction of anti-drug immune responses (Liu, 2018).

[0009] Thus, there remains a need for methods to predict the in vivo PK properties (e.g., clearance or half-life) of therapeutic molecules that can be performed quickly and at reasonable cost to aid in the evaluation and selection of therapeutic molecules. Summary of the Invention

[0010] The present disclosure relates to methods and compositions useful for measuring the transcytosis of molecules of interest. In particular, the present disclosure relates to in vitro receptor-mediated transcytosis assays for evaluating the in vivo pharmacokinetic profile, e.g., clearance rate, and / or half-life of molecules that interact with FcRn, e.g., therapeutic antibodies, Fc fusion molecules, and molecules that bind albumin.

[0011] In certain embodiments, the present disclosure is directed to a method for determining the pharmacokinetic (PK) parameters of a molecule of interest. For example, such a method can include determining a measure of the transcytosis of a molecule or molecules through a cell or cells under physiological pH conditions. In certain embodiments, the PK parameter is a measure of the in vivo clearance of the molecule. In certain embodiments, the PK parameter is a measure of the in vivo half-life of the molecule.

[0012] In certain embodiments, the measure of transcytosis used in connection with the methods disclosed herein is the rate of transcytosis. In certain embodiments, the measure of transcytosis is a measure of the amount of molecule(s) being transcytosed.

[0013] In certain embodiments, the measure of transcytosis is determined by measuring the transcytosis of a molecule(s) from a solution in a first chamber to a solution in a second chamber, where the first and second chambers are separated by a cell monolayer and the solution in each chamber is at physiological pH. In certain embodiments, the cell or cells are Madin-Darby Canine Kidney (MDCK) cells. In certain embodiments, the cell comprises at least one heterologous gene. In certain embodiments, the at least one heterologous gene is selected from the group consisting of FCGRT gene and B2M gene. In certain embodiments, the cell expresses a heterologous cell surface protein. In certain embodiments, the heterologous cell surface protein comprises neonatal Fc receptor (FcRn). In certain embodiments, measuring the transcytosis of a molecule comprises using enzyme-linked immunosorbent assay (ELISA), liquid scintillation counting (LSC), quantitative PCR, or a fluorescence reader system. In certain embodiments, the methods disclosed herein further comprise determining the in vivo clearance of the molecule based on the number of molecules measured.

[0014] In certain embodiments of the present disclosure, the molecule of interest is a molecule that naturally binds to FcRn.In certain embodiments, the molecule is engineered to bind to FcRn.In certain embodiments, the molecule is an Fc-containing molecule.In certain embodiments, the Fc-containing molecule is a receptor-Fc fusion molecule.In certain embodiments, the molecule is an antibody.In certain embodiments, the antibody is a monoclonal antibody.In certain embodiments, the molecule is an albumin-containing molecule.

[0015] In certain embodiments, the present disclosure is directed to a method for measuring transcytosis of a plurality of single molecules or a plurality of different molecules, comprising: a) introducing the plurality of single molecules or the plurality of different molecules into a first of two chambers, where the first chamber is separated from the second chamber by a cell or a plurality of cells, and each of the first and second chambers has a physiological pH value; and b) measuring the number of molecules transcytosed from the first chamber to the second chamber. In certain embodiments, the first chamber comprises a monolayer of cells. In certain embodiments, the cells are Madin-Darby Canine Kidney (MDCK) cells. In certain embodiments, the cells comprise at least one heterologous gene. In certain embodiments, the at least one heterologous gene is selected from the group consisting of FCGRT and B2M genes. In certain embodiments, the cells express a heterologous cell surface protein. In certain embodiments, the heterologous cell surface protein comprises neonatal Fc receptor (FcRn). In certain embodiments, measuring the number of transcytosed molecules comprises using an enzyme-linked immunosorbent assay (ELISA), liquid scintillation counting (LSC), quantitative PCR, or a fluorescence reader system. In certain embodiments, the methods disclosed herein further comprise determining the in vivo clearance of the molecules based on the number of molecules measured.

[0016] In certain embodiments, the present disclosure is directed to a method of determining the in vivo clearance of a molecule, comprising: a) introducing a molecule into a first of two chambers, where the first chamber is separated from the second chamber by a cell or a plurality of cells, each of the first and second chambers having a physiological pH value; b) measuring the number of molecules transcytosed from the first chamber to the second chamber; and c) determining the in vivo clearance of the molecule based on the number of molecules measured. In certain embodiments, the first chamber comprises a monolayer of cells. In certain embodiments, the cells are MDCK cells. In certain embodiments, the cells comprise at least one heterologous gene. In certain embodiments, the at least one heterologous gene is selected from the group consisting of FCGRT and B2M genes. In certain embodiments, the cells express a heterologous cell surface protein. In certain embodiments, the heterologous cell surface protein comprises neonatal Fc receptor (FcRn). In certain embodiments, measuring the number of transcytosed molecules comprises using enzyme-linked immunosorbent assay (ELISA), liquid scintillation counting (LSC), quantitative PCR, or a fluorescence reader system. In certain embodiments, the molecules are molecules that naturally bind to FcRn.

[0017] In certain embodiments, the present disclosure is directed to an assay for measuring transcytosis of a plurality of molecules. For example, but not by way of limitation, the assay can include using a first chamber and a second chamber, each of which has a physiological pH value, where the first chamber is configured to receive a plurality of molecules and transcytosis of the molecules to the second chamber, and the transcytosis of the plurality of molecules is measured. In certain embodiments, the first chamber includes a monolayer of cells. In certain embodiments, the cells are MDCK cells. In certain embodiments, the cells include at least one heterologous gene. In certain embodiments, the at least one heterologous gene is selected from the group consisting of FCGRT and B2M genes. In certain embodiments, the cells express a heterologous cell surface protein. In certain embodiments, the heterologous cell surface protein includes neonatal Fc receptor (FcRn). In certain embodiments, measuring the number of transcytosed molecules comprises using an enzyme-linked immunosorbent assay (ELISA), liquid scintillation counting (LSC), quantitative PCR, or a fluorescence reader system, hi certain embodiments, the assay further comprises determining the in vivo clearance of the molecules based on the number of molecules measured.

[0018] In certain embodiments, the present disclosure is directed to an assay system comprising a first chamber and a second chamber, each of the first and second chambers having a physiological pH, a monolayer of cells positioned within the first chamber such that the cells can mediate transcytosis of a molecule introduced into the first chamber into the second chamber, and a detector for detecting the presence of a molecule in the second chamber. In certain embodiments, the cells are MDCK cells. In certain embodiments, the cells comprise at least one heterologous gene. In certain embodiments, the at least one heterologous gene is selected from the group consisting of FCGRT and B2M genes. In certain embodiments, the cells express a heterologous cell surface protein. In certain embodiments, the heterologous cell surface protein comprises neonatal Fc receptor (FcRn). In certain embodiments, measuring the number of transcytosed molecules comprises the use of enzyme-linked immunosorbent assay (ELISA), liquid scintillation counting (LSC), quantitative PCR, or a fluorescence reader system. [Brief description of the drawings]

[0019] [Figure 1] FIG. 1 depicts a schematic diagram of one particular embodiment of a transcytosis assay of the present disclosure. [Figure 2A-2B] Figures 2A-B depict the expression of human FcRn heavy chain and beta2-microglobulin in the transfected clonal MDCK cell line 305-6. Flow cytometry histograms of cell surface expression of B2M (A) and FCGRT (B) for WT and 305-6 cells.

[0020] [Figure 3A-3E]Figures 3A-E show scatter plots depicting the relationship between clearance in humans (rate in mK / Kg / day) and either transcytosis value (A; concentration in ng / mL), FcRn binding affinity at pH 6.0 (B; Kd in M) or BV ELISA score (C). In addition, clearance rate of mAbs in cynomolgus monkeys (rate in mK / Kg / day) was plotted against either transcytosis value (D) or clearance rate in humans (E). Data were fitted to a linear regression model and both equations and R-squared values ​​are presented. [Figure 4A-4C] 4A to 4C show the relationship between clearance in humans and either transcytosis in MDCK cells (A) or MDCK-hFcRn cells (B), as well as the relationship between binding to MDCK-hFcRn cells and transcytosis in MDCK-hFcRn cells (C). [Figure 5A-5C] 5A-C show the relationship between clearance in humans and either the charge (A), pI (B), or transcytotic output (C) of mAbs. [Figure 6] FIG. 6 depicts a schematic of the potential fates of internalized molecules. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] In practicing the presently disclosed subject matter, many conventional techniques in molecular biology, microbiology, cell biology, biochemistry and immunology are used. These techniques are described in more detail, for example, in Molecular Cloning: a Laboratory Manual, 3rd edition, edited by J.F. Sambrook and D.W. Russell, Cold Spring Harbor Laboratory Press, 2001; Recombinant Antibodies for Immunotherapy, edited by Melvyn Little, Cambridge University Press, 2009; "Oligonucleotide Synthesis" (edited by M.J. Gait, 1984); "Animal Cell Culture" (edited by R.I. Freshney, 1987); "Methods in Enzymology" (Academic Press, Inc.); "Current Protocols in Molecular Biology" (edited by F.M.Ausubel et al., 1987, and regularly updated); "PCR: The Polymerase Chain Reaction" (edited by Mullis et al., 1994); "A Practical Guide to Molecular Cloning" (Perbal Bernard V., 1988); "Phage Display: A Laboratory Manual" (Barbas et al., 2001). The contents of these references, as well as other references containing standard protocols, including manufacturer's instructions, that are widely known and relied upon by those of skill in the art, are incorporated herein by reference as part of this disclosure.

[0022] 1.Definition Unless otherwise defined, all technical terms, designations, and other scientific terms used herein are intended to have the meaning commonly understood by one of ordinary skill in the art to which this disclosure pertains. In some cases, terms having a commonly understood meaning are defined herein for clarity and / or ease of reference, and the inclusion of such definitions herein should not necessarily be construed as representing a substantial departure from what is commonly understood in the art.

[0023] As used herein, the terms "comprise(s) / include(s)," "having / has," "can," "containing," and variations thereof are intended to be open-ended transitional phrases, terms, or words that do not exclude the possibility of additional acts or constructs. The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments that "comprise," "consist," and "consist essentially of" the embodiments or elements presented herein, whether expressly stated or not.

[0024] As used herein, the term "about" or "approximately" refers to an acceptable error range of a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 3 or more standard deviations per one practice in the art in certain embodiments. Alternatively, "about" can mean within a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value in certain embodiments. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude of a value, for example, within 5-fold, or within 2-fold.

[0025] As used herein, the terms "culture medium" and "cell culture medium" refer to a nutrient source used to grow or maintain cells. As will be understood by those skilled in the art, the nutrient source may contain components required by cells for growth and / or survival or may contain components that support cell growth and / or survival. Vitamins, essential or non-essential amino acids (e.g., cysteine ​​and cystine), and trace elements (e.g., copper) are examples of medium components. Any medium provided herein may be supplemented with any one or more of insulin, vegetable hydrolysates, and animal hydrolysates.

[0026] As used herein, the phrase "pharmacokinetic (PK) parameters" includes clearance (CL), half-life (t 1 / 2 ), area under the curve (AUC), volume of distribution (V d ) and maximum / minimum plasma concentration (C max / C 分 PK parameters refer to any of a variety of PK parameters known in the art, including, but not limited to,

[0027] As used herein, the term "clearance" refers to the rate at which a molecule or polypeptide is removed from the bloodstream.

[0028] As used herein, the term "physiological pH" refers to a pH of about 6.5 to about 8.0. In certain embodiments, the physiological pH value is any value between about 6.5 and about 8.0, such as about 6.6, about 6.7, about 6.8, about 6.9, 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, or about 7.9, or any range within the range of about 6.5 to about 8.0.

[0029] "Culturing" a cell means contacting the cell with a cell culture medium under conditions suitable for the survival and / or growth and / or proliferation of the cell.

[0030] As used herein, "molecule" generally refers to a molecule that is the subject of an assay. For example, but not by way of limitation, the molecule may be any molecule that naturally binds to FcRn or that has been engineered to bind to FcRn, such as, but not limited to, albumin-containing molecules as defined below, molecules engineered to bind to FcRn via peptide tags or other amino acid sequences, antibodies, antibody fragments, or polyclonal or monoclonal antibodies. Such polypeptides, proteins, antibodies, antibody fragments, or polyclonal or monoclonal antibodies may include non-naturally occurring aspects, including, but not limited to, non-naturally occurring amino acids, non-amino acid linkers or spacers, and may include conjugates with other compositions, such as small or large molecule therapeutic agents.

[0031] As used herein, "polypeptide" generally refers to peptides and proteins having more than about 10 amino acids. The polypeptide can be homologous to the host cell, or, preferably, exogenous, meaning that it is heterologous, i.e., foreign, to the host cell utilized, e.g., a human protein produced by a Chinese hamster ovary cell, or a yeast polypeptide produced by a mammalian cell. In certain embodiments, mammalian polypeptides (polypeptides originally derived from a mammalian organism) are used, and in certain embodiments, the polypeptides of the invention are secreted directly into the medium.

[0032] The term "protein" is meant to refer to a sequence of amino acids whose chain length is sufficient to produce higher tertiary and / or quaternary structures. This is to distinguish it from "peptides" or other low molecular weight agents that do not have such structures. Typically, proteins herein have a molecular weight of at least about 15-20 kD, and in certain embodiments, at least about 20 kD. Examples of proteins encompassed by the definition herein include all mammalian proteins, particularly therapeutic and diagnostic proteins such as therapeutic and diagnostic antibodies, as well as general proteins that contain one or more disulfide bonds, including multi-chain polypeptides that contain one or more inter- and / or intra-chain disulfide bonds.

[0033] The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, multispecific antibodies, e.g., bispecific antibodies, and antibody fragments, that exhibit the desired antigen-binding activity.

[0034] As used herein, an "antibody fragment," "antigen-binding portion" (or simply "antibody portion") of an antibody, or an "antigen-binding fragment" of an antibody refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds to an antigen and / or epitope. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; bispecific antibodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and antibody fragments formed from multispecific, e.g., bispecific, antibodies.

[0035] The term "monoclonal antibody" or "mAb" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., each antibody in the population is identical and / or binds to the same epitope, excluding possible variant antibodies, including, for example, naturally occurring mutations or mutations that arise during the manufacture of the monoclonal antibody preparation. Such variants are generally present in small amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not constructed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the presently disclosed subject matter can be made by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals that contain all or a portion of the human immunoglobulin loci; such methods and other exemplary methods for making monoclonal antibodies are described herein.

[0036] The term "hybridoma" refers to a hybrid cell line produced by the fusion of an immunogenic immortalized cell line with an antibody-producing cell. The term encompasses the progeny of a heterohybrid myeloma fusion resulting from the fusion of a human cell with a mouse myeloma cell line, followed by a plasma cell commonly known as a trioma cell line. In addition, the term is meant to include any immortalized hybrid cell line that produces antibodies, such as quadromas. See, e.g., Milstein et al., Nature, 537:3053 (1983).

[0037] As used herein, the term "cell" refers to animal cells, mammalian cells, cultured cells, host cells, recombinant cells, and recombinant host cells. Such cells are generally cell lines obtained from or derived from mammalian tissues that are capable of proliferation and viability when placed in medium containing the appropriate nutrients and / or growth factors.

[0038] As used herein, the term "heterologous gene" refers to a gene encoding a protein that is foreign to the host cell being utilized, such as a gene encoding a human protein produced by Chinese hamster ovary cells, or a gene encoding a yeast polypeptide produced in a mammalian cell.

[0039] 2. Overview The present disclosure relates to methods and compositions useful for measuring the transcytosis of a molecule of interest. In particular, the present disclosure relates to an in vitro receptor-mediated transcytosis assay for evaluating PK parameters such as clearance rate and / or half-life of therapeutic antibody molecules. As disclosed in detail herein, the transcytosis of a molecule of interest (e.g., mAb) measured by the method of the present disclosure is highly related to cellular PK properties such as the in vivo clearance of the molecule. Thus, the claimed method for measuring the transcytosis of a molecule of interest can be used to evaluate other PK parameters such as the in vivo clearance and half-life of the molecule. In certain embodiments, the method includes measuring the transcytosis of a plurality of the same molecules of interest from a first chamber to a second chamber, where each of the first and second chambers has a physiological pH value (e.g., pH=about 7.4).

[0040] Thus, for example, in one embodiment, a cell-based assay using MDCK cells stably expressing human FcRn and β2-microglobulin genes is provided herein to measure the transcytosis efficiency of monoclonal antibody (mAb) drugs under conditions relevant to the FcRn-mediated IgG salvage pathway. The transcytosis assay is performed under physiological pH conditions and is designed to evaluate outcomes from the combined effects of non-specific binding of IgG to cells, its uptake via pinocytosis, its pH-dependent interaction with FcRn, and the kinetics of intracellular trafficking and sorting processes. As described in more detail in the Examples, the inventors have evaluated 49 mAbs, including 27 commercially available mAb drugs, and demonstrated a noteworthy correlation between clearance rates in humans and readouts from assays such as those disclosed herein. Expression of FcRn is required to promote transcytosis of mAbs in the assay and directly contributes to the observed correlation. Moreover, assays such as those provided herein were able to correctly rank the clearance rates of charge or glycosylation variants of Fc-containing molecules in preclinical species. The results provided in the Examples support the utility of the assays disclosed herein as cost-effective and animal-sparing screening tools for the evaluation of mAb-based drug candidates during lead selection and optimization, as well as process development for desirable pharmacokinetic properties.

[0041] 3. Transcytosis Assay Transcytosis, which refers to the vesicular transport of macromolecules from one side of a cell to the other, is a strategy used by multicellular organisms to selectively move materials between two environments without changing the native composition of those environments. Transcytosis is a mechanism for transcellular transport in which cells encapsulate extracellular material in invaginations of the cell membrane to form vesicles, then move the vesicles across the cell and eject the material through the opposing cell membrane by the reverse process.

[0042] In a typical FcRn-mediated transcytosis assay, FcRn-expressing MDCK cells are grown to confluency on filter membranes in transwell plates, and a pH gradient is created by filling the inner chamber with an acidic assay buffer (pH<6.0) and the outer chamber with a basic assay buffer (pH>7.4) prior to the assay. The rationale for this assay design is to exploit the pH-dependent binding characteristics of FcRn to facilitate cellular uptake of test molecules via binding to FcRn on the cell surface under acidic pH and release of test molecules under basic pH, both of which improve the robustness and sensitivity of the assay. However, based on the work described herein, the inventors have come to realize that such assays are inherently flawed for predictive evaluation of PK behavior of mAbs. Because transfected cells typically express high levels of FcRn on the cell surface and the test antibodies were incubated with the cells in an acidic environment, it was determined that test antibodies that exhibit high binding affinity to FcRn at acidic pH would readily bind to FcRn and enter the cells via FcRn-mediated endocytosis. The inventors also determined that this is in contrast to what occurs in vivo, where antibodies minimally bind to cell surface FcRn under physiological pH, and that cellular uptake of mAbs is primarily mediated by nonspecific fluid-phase pinocytosis. As a result, the inventors determined that the output of the pH gradient transcytosis assay is heavily influenced by the antibody's FcRn binding affinity at acidic pH, and therefore is unlikely to fully reflect the contribution of other factors that affect PK, such as electrostatic interactions and intracellular trafficking parameters. In addition, it was further determined that the artificial pH conditions are inherently harmful to cells, limiting the duration of the assay, and could potentially create additional assay artifacts.

[0043] Therefore, as described in more detail in the Examples, the inventors then developed a different approach to measure transcytosis as a basis for accurately determining measures of PK parameters such as clearance and / or half-life. Thus, for example, in certain embodiments, the present disclosure provides a method for measuring transcytosis of a plurality of single molecules or a plurality of different molecules, comprising: a) introducing the plurality of single molecules or a plurality of different molecules into a first chamber, where the first chamber is separated from a second chamber by a cell or a plurality of cells, each of the first and second chambers having a physiological pH value; and b) measuring the number of molecules transcytosed from the first chamber to the second chamber. In certain embodiments, the physiological pH value is about 6.5 to about 8.0. In certain embodiments, the physiological pH value is any value between about 6.5 and about 8.0, such as about 6.6, about 6.7, about 6.8, about 6.9, 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 or about 7.9, or any range within the range of about 6.5 to about 8.0. In certain embodiments, the pH of each chamber can be different, as long as both chambers have a physiological pH. For example, but not by way of limitation, the first chamber can have a pH of 6.5, while the second chamber can have a pH of 8.0, or vice versa. In certain embodiments, the physiological pH can be about 7.4.

[0044] In certain embodiments, the disclosure provides a method for determining a pharmacokinetic (PK) parameter of a molecule, comprising determining a measure of transcytosis of the molecule or molecules through a cell or a plurality of cells under physiological pH conditions. In certain embodiments, the PK parameter can be a measure of in vivo clearance of the molecule. In certain embodiments, the PK parameter can be a measure of in vivo half-life of the molecule. In certain embodiments, the measure of transcytosis can be a rate of transcytosis. In certain embodiments, the measure of transcytosis can be a measure of the amount of molecule(s) being transcytosed. In certain embodiments, the measure of transcytosis can be determined by measuring the transcytosis of the molecule(s) from a solution in a first chamber to a solution in a second chamber, where the first and second chambers are separated by a cell monolayer and the solution in each chamber is at physiological pH. In certain embodiments, the cell or cells can be Madin-Darby Canine Kidney (MDCK) cells. In certain embodiments, the cell comprises at least one heterologous gene. In certain embodiments, the at least one heterologous gene may be selected from the group consisting of FCGRT gene and B2M gene. In certain embodiments, the cell may express a heterologous cell surface protein. In certain embodiments, the heterologous cell surface protein may comprise neonatal Fc receptor (FcRn). In certain embodiments, measuring the transcytosis of the molecule may comprise using enzyme-linked immunosorbent assay (ELISA), liquid scintillation counting (LSC), quantitative PCR, or a fluorescence reader system. In certain embodiments, the method of the present disclosure may further comprise determining the in vivo clearance of the molecule based on the number of molecules measured. In certain embodiments, the molecule may be a molecule that naturally binds to FcRn or a molecule that has been engineered to bind to FcRn. In certain embodiments, the molecule may be an Fc-containing molecule. In certain embodiments, the molecule may be an antibody.In certain embodiments, the antibody may be a monoclonal antibody. In certain embodiments, the molecule may be an albumin-containing molecule. In certain embodiments, the physiological pH value is about 6.5 to about 8.0. In certain embodiments, the physiological pH value is any value between about 6.5 and about 8.0, such as about 6.6, about 6.7, about 6.8, about 6.9, 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 or about 7.9, or any range within the range of about 6.5 to about 8.0. In certain embodiments, the pH of each chamber may be different, as long as both chambers have a physiological pH. For example, but not by way of limitation, the first chamber may have a pH of 6.5, while the second chamber may have a pH of 8.0, or vice versa. In certain embodiments, the physiological pH may be about 7.4.

[0045] In certain embodiments, the assay comprises seeding a cell(s) in cell growth medium on a 96-well transwell plate with medium in the inner and outer chambers, removing the medium from the inner chamber, adding a test molecule to the inner chamber, incubating for a period of time in a tissue culture incubator, collecting the medium from the outer chamber, and quantifying transcytosed test molecule. In certain embodiments, the cells are at about 1×10 in cell growth medium. 5Cells may be seeded at a density of 100 μg / well. In certain embodiments, the cell growth medium may be DMEM high glucose supplemented with 10% FBS, 100 units penicillin / streptomycin, and 5 μg / mL P-puromycin. In certain embodiments, the medium in the inner chamber may be 100 μL and the medium in the outer chamber may be 200 μL. In certain embodiments, the cells may be used 2 days after plating. In certain embodiments, the test molecule may be added to a final concentration of about 100 μg / mL (0.67 μM) and incubated for 24 hours in a tissue culture incubator at 37° C. In certain embodiments, Lucifer Yellow (Lucifer Yellow CH, dilithium salt; Sigma Aldrich; St. Louis MO) may be prepared in cell growth medium and added until the last 90 minutes of the 24 hour assay incubation. In certain embodiments, the level of passive passage of Lucifer Yellow in the assay may be calculated by dividing the fluorescent signal in the sample from the outer chamber by that of the inner chamber. In certain embodiments, transcytosis results from wells that showed greater than 0.1% passive passage of Lucifer Yellow in the outer chamber may be discarded.

[0046] In certain embodiments, the test molecule can be added to the outer chamber of a transwell assay, and transcytosis can be detected by measuring the amount of test molecule present in the inner chamber after a suitable incubation period. In certain embodiments, the test molecule is transcytosed from the chamber exposed to the apical membrane of the cell to the chamber exposed to the basolateral membrane of the cell.

[0047] In certain embodiments, the present disclosure provides a method for determining or predicting the in vivo clearance of one or more molecules, comprising: a) introducing a molecule into a first chamber of two chambers, where the first chamber is separated from the second chamber by a cell or a plurality of cells, and each of the first and second chambers has a physiological pH value; and b) measuring the number of molecules transcytosed from the first chamber to the second chamber. In certain embodiments, the physiological pH value is about 6.5 to about 8.0. In certain embodiments, the physiological pH value is any value between about 6.5 and about 8.0, such as about 6.6, about 6.7, about 6.8, about 6.9, 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, or about 7.9, or any range within the range of about 6.5 to about 8.0. In certain embodiments, the pH of each chamber can be different, as long as both chambers have physiological pH.For example, but not by way of limitation, the first chamber can have a pH of 6.5, while the second chamber can have a pH of 8.0, or vice versa.In certain embodiments, the physiological pH can be about 7.4.

[0048] In certain embodiments, the present disclosure provides an assay for measuring transcytosis of a plurality of the same molecules or a plurality of different molecules, comprising a first chamber and a second chamber, each of the first and second chambers having a physiological pH value, wherein the first chamber is configured to receive a plurality of the molecules and allow transcytosis of the molecules into the second chamber. In certain embodiments, the physiological pH value is about 6.5 to about 8.0. In certain embodiments, the physiological pH value is any value between about 6.5 and about 8.0, such as about 6.6, about 6.7, about 6.8, about 6.9, 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, or about 7.9, or any range within the range of about 6.5 to about 8.0. In certain embodiments, the pH of each chamber can be different, as long as both chambers have a physiological pH. For example, and not by way of limitation, the first chamber may have a pH of 6.5 while the second chamber may have a pH of 8.0, or vice versa. In certain embodiments, the physiological pH may be about 7.4.

[0049] In certain embodiments, the present disclosure provides an assay for determining or predicting in vivo clearance of one or more molecules, comprising a first chamber and a second chamber, each of the first and second chambers having a physiological pH value, where the first chamber is configured to receive a plurality of the molecules and allow transcytosis of the molecules into the second chamber. In certain embodiments, the physiological pH value is about 7.4.

[0050] In certain embodiments, the first chamber may contain a monolayer of cells. In certain embodiments, the cells used in the assay are selected from the list provided in section 3 below. In certain embodiments, the cells used in the assay are MDCK cells.

[0051] In certain embodiments, the cells used in the methods described in the present disclosure may contain at least one heterologous gene, which may be selected from the group consisting of the FCGRT gene and the B2M gene.

[0052] In certain embodiments, the cells used in the methods described in this disclosure may express a cell surface protein. In certain embodiments, the cell surface protein comprises an Fc receptor (FcR). In certain embodiments, the cell surface protein comprises a neonatal Fc receptor (FcRn).

[0053] In certain embodiments, the molecule can be labeled or unlabeled. Non-limiting examples of labeled molecules include: 3 H-labeled, fluorescently labeled molecules, or radioisotopes, such as I-125 and P-32.

[0054] In certain embodiments, the methods of the present disclosure include measuring the number of transcytosed molecules. In certain non-limiting embodiments, measuring the number of transcytosed molecules can be performed by enzyme-linked immunosorbent assay (ELISA), liquid scintillation counting (LSC), quantitative PCR, a fluorescence reader system, a confocal microscope, or a live cell imaging system.

[0055] In certain embodiments, the present disclosure provides an FcRn-dependent cell-based assay to measure the transcytosis of mAbs, such as IgG mAbs, through MDCK cells expressing human FcRn and B2M under conditions similar to the FcRn-mediated IgG salvage pathway. In certain embodiments, the output of this assay involves not only Fc-FcRn interactions in physiological conditions, but also non-specific binding, cellular uptake, sorting and intracellular trafficking processes that belong to the in vivo PK behavior of mAbs.

[0056] In certain embodiments, the present disclosure provides methods for correlating transcytosis measured in the assays described in this disclosure with reported clearance rates in humans of molecules, such as polypeptides, with diverse structural, functional and pharmacological properties.

[0057] In certain embodiments, expression of FcRn may be required to facilitate transcytosis of mAbs in the assay and contribute to the observed correlation between transcytosis and clearance. In certain embodiments, the assays described in this disclosure can correctly rank-order the clearance rates of charge or glycosylation variants of Fc-containing molecules in preclinical species. In certain embodiments, the assays described in this disclosure can be used as a time-efficient, cost-effective and animal-saving tool for the evaluation of mAb-based drug candidates during lead selection and optimization, as well as process development for desirable pharmacokinetic properties.

[0058] In certain embodiments, the present disclosure provides cell-based assays that measure the transcytosis efficiency of mAb drugs under physiologically relevant conditions. In certain embodiments, MDCK cells stably expressing human FcRn and β2-microglobulin genes are used in conjunction with the assays described herein.

[0059] In certain embodiments, the present disclosure provides that the assay is performed in normal cell culture medium supplemented with fetal bovine serum, which provides bovine albumin, which is known to bind human FcRn at physiological pH (Chaudhury C. et al., 2003, J. Exp. Med. 197:315-322).

[0060] In certain embodiments, the disclosure provides assays in which molecules are taken up by cells via non-specific pinocytosis, interact with human FcRn in the presence of albumin, and traverse the cells under conditions relevant to FcRn-mediated mechanisms of action.

[0061] In certain embodiments, the disclosure provides assays to determine or predict clearance of mAbs in humans due to the ability of the assay to assess the combined effects of non-specific binding to cells, uptake via pinocytosis, pH-dependent interactions with FcRn, and the kinetics of intracellular trafficking and sorting processes.

[0062] In certain embodiments, the present disclosure provides an assay that is used to correlate the obtained transcytosis output with the clearance rate of mAbs in humans. In certain embodiments, the output of the assay of the present disclosure can be the concentration (ng / mL) of transcytosed molecules in the medium of the outer chamber, and the reportable value of the assay can be the average concentration of at least duplicate wells from the same plate. In certain embodiments, the output of the assay can be a measure of the rate of transcytosis, or a measure of the relative transcytosis of the transcytosed molecule compared to a control molecule.

[0063] In certain embodiments, the selection of test molecules for the assays of the present disclosure may be based on the availability of clinical grade materials and human clearance data from reliable sources, such as, but not limited to, prescribing information, publications based on population PK models, or in-house clinical trials from which linear PK data were generated. In certain embodiments, the use of clinical grade materials can ensure that the quality of the test materials can be consistent with that used in the clinical studies from which the human PK data were generated.

[0064] In certain embodiments, the assays described in this disclosure may be used as tools for in vitro assessment of potential liability in non-specific clearance of drug candidates to support lead selection and optimization, with the aim of prioritizing candidates and reducing the number of molecules tested in animal models.

[0065] In certain embodiments, the assays described in this disclosure may be used to support the investigation of mAbs exhibiting undesirable or atypical PK behavior, or the development of novel mAb drugs with improved transcytosis properties for specialized applications such as crossing the blood-brain barrier for enhanced brain exposure or enhanced disposition in the tumor microenvironment for improved tumor targeting.

[0066] In certain embodiments, the assays described in this disclosure may be used for the development of improved mechanism-based PK models to support the design of optimal doses and dosing schemes in clinical studies.

[0067] In certain embodiments, the assays described in this disclosure can be used to demonstrate correlation between in vitro readouts and in vivo K data for test molecules.

[0068] In certain embodiments, the test molecule may be an antibody. In certain embodiments, the antibody may be a monoclonal or polyclonal antibody. In certain embodiments, the antibody may have different structural compositions (chimeric, humanized and fully human). In certain embodiments, the antibody may consist of various heavy and light chain sequences (IgG1, IgG2 and IgG4 heavy chains, kappa and lambda light chains). In certain embodiments, the antibody may recognize different types of targets (membrane bound and soluble). In certain embodiments, the antibody may exert different therapeutic mechanisms of action (agonistic, antagonistic and cytotoxic). In certain embodiments, the antibody may be and is given to the patient via different routes (e.g., intravenous, intramuscular and subcutaneous).

[0069] In certain embodiments, the test molecules may carry engineered mutations that may alter their effector functions (such as, but not limited to, atezolizumab, durvalumab), enable bispecific half-antibody association (such as, but not limited to, emicizumab), or stabilize IgG4 Fab arms (such as, but not limited to, nivolumab, pembrolizumab).

[0070] In certain embodiments, the test molecule may be a mAb that exhibits extreme FcRn binding affinity. In certain embodiments, the observed correlation between transcytosis readings and clearance in humans may apply to a wide range of mAbs that carry typical Fc regions. In certain embodiments, the test molecule may be an antibody that is engineered to have altered FcRn binding activity. In certain embodiments, the test molecule may be an albumin-containing molecule, or any molecule that is engineered to bind to FcRn via a peptide tag or recombinant protein. In certain embodiments, the test molecule may be a molecule that naturally binds to FcRn. In certain embodiments, the test molecule may be a molecule that is engineered to bind to FcRn.

[0071] In certain embodiments, the assays described in this disclosure may be used to indicate whether expression of human FcRn is required for efficient transcytosis in a transcytosis assay.

[0072] In certain embodiments, the assays of the present disclosure may be used to detect and / or indicate that interaction with FcRn contributes to an observed correlation between transcytosis readout and clearance of a test molecule.

[0073] In certain embodiments, the assay of the present disclosure may be used to analyze the binding of test molecules to receptors. In certain embodiments, the receptor may be FcRn receptor. In certain embodiments, the test molecule may be mAb.

[0074] In certain embodiments, the assays of the present invention may be used to assess the contribution of multiple factors, including but not limited to, the kinetics of internalization, sorting and intracellular trafficking, and exocytosis to the overall efficiency of the recovery process of a test molecule.

[0075] In certain embodiments, the assays of the present disclosure may be used to identify and / or detect multiple parameters involved in the FcRn-mediated salvage pathway for determining or predicting the PK of a mAb.

[0076] In certain embodiments, the assays of the present disclosure can be used to detect correlations between in vitro transcytosis and in vivo clearance of molecules. In certain embodiments, the assays of the present disclosure can be used to detect correlations between in vitro transcytosis and in vivo clearance of molecules, as compared to BV ELISA or Fv charge / pI.

[0077] In certain embodiments, the assays of the present disclosure may be used to assess and / or investigate the role of glycosylation in the distribution and catabolism of mAbs in vivo, hi certain embodiments, the molecules may be glycoform variants.

[0078] In certain embodiments, the assays of the present disclosure may be used to assess the role of sialic acid addition to the molecule in intracellular trafficking parameters, hi certain embodiments, the assays of the present disclosure may be used to analyze the involvement of additional mechanisms in the clearance of high mannose and highly sialylated glycoform variants of the molecule in vivo.

[0079] In certain embodiments, the assay of the present disclosure may be used as a tool for the study of FcRn-mediated transcytosis as a clearance mechanism for Abs belonging to their PK.

[0080] In certain embodiments, the assays of the present disclosure may be used as tools for studying and elucidating the molecular mechanisms governing the distribution of FcRn-complexed IgG.

[0081] In certain embodiments, the assays of the present disclosure may be used to provide an output that reflects the target-independent, non-specific clearance mechanism of mAbs with typical FcRn binding affinities in humans.

[0082] In certain embodiments, the assays of the present disclosure may be used to analyze a diverse group of Fc-containing molecules and may respond to factors known to affect PK.

[0083] In certain embodiments, the assays of the present embodiments may be used to analyze factors involved in transcytosis assays to understand how these factors correlate with in vivo clearance and to define the quantitative nature of this correlation.

[0084] In certain embodiments, the assays of the present disclosure may be used to investigate the distribution of pinocytosed mAbs among recycling, transcytosis and degradation pathways in various cell types / tissue compartments, as well as the molecular mechanisms governing such distribution within cells involved in IgG clearance.

[0085] In certain embodiments, the present disclosure is directed to a transcytosis assay system. For example, but not by way of limitation, such a system may comprise a first chamber and a second chamber, each of the first and second chambers having a physiological pH, a monolayer of cells positioned in the first chamber such that the cells can mediate transcytosis of a molecule introduced into the first chamber into the second chamber, and a detector for detecting the presence of a molecule in the second chamber. In certain embodiments, the assay system disclosed herein employs a monolayer of MDCK cells. In certain embodiments, the assay system disclosed herein employs cells comprising at least one heterologous gene. In certain embodiments, the assay system disclosed herein employs cells comprising at least one heterologous gene selected from the group consisting of FCGRT and B2M genes. In certain embodiments, the assay system disclosed herein employs cells expressing a heterologous cell surface protein. In certain embodiments, the assay systems disclosed herein use cells expressing a heterologous cell surface protein, where the heterologous cell surface protein comprises neonatal Fc receptor (FcRn). In certain embodiments, the assay systems disclosed herein can measure the number of transcytosed molecules through the use of enzyme-linked immunosorbent assay (ELISA), liquid scintillation counting (LSC), quantitative PCR, or a fluorescence reader system.

[0086] 4.Cells Cells suitable for use in the assays of the present disclosure include prokaryotic or eukaryotic cells as described herein.

[0087] In certain embodiments, vertebrate cells may also be used. Non-limiting examples of useful mammalian cell lines include monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney lines (e.g., 293 or 293 cells described in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells described in Mather, Biol. Reprod. 23:243-251 (1980); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary carcinoma (MMT 060562); TRI cells, e.g., Mather et al., Annals NYAcad. Sci. 383:44-68 (1982); MRC5 cells; and FS4 cells. Further non-limiting examples of useful mammalian cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)). In certain embodiments, the methods of the disclosure can use hybridoma cells. For example, but not by way of limitation, hybrid cell lines can be of any species, including human and mouse. In certain embodiments, the methods of the disclosure can use primary and established endothelial and / or epithelial cells.For example, but not by way of limitation, the endothelial and / or epithelial cells can be caco-2, T-84, HMEC-1, MHEC 2.6, HUVEC, and induced pluripotent stem cell (iPSC) derived cells (Lidington, E. A., DL Moyes et al. (1999). "A comparison of primary endothelial cells and endothelial cell lines for studies of immune interactions." Transpl Immunol 7(4):239-246; Yamaura, Y., BD Chapron et al. (2016) "Functional Comparison of Human Colonic Carcinoma Cell Lines and Primary Small Intestinal Epithelial Cells for Investigations of Intestinal Drug Permeability and First-Pass Metabolism." Drug Metab Dispos 44(3):329-335).

[0088] In certain embodiments, cells for use in the disclosed methods may contain a nucleic acid encoding a molecule, such as a receptor. In certain embodiments, the nucleic acid may be present in one or more vectors, such as expression vectors. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Certain vectors can autonomously replicate in the cell into which they are introduced (e.g., bacterial vectors with a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the cell upon introduction into the cell, and are thereby replicated along with the host genome. Furthermore, certain vectors, expression vectors, can induce the expression of genes to which they are operably linked. In general, expression vectors utilized in recombinant DNA techniques are often in the form of plasmids (vectors). Further non-limiting examples of expression vectors for use in the present disclosure include viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses) that serve equivalent functional roles.

[0089] In certain embodiments, a nucleic acid encoding a molecule, e.g., a receptor, can be introduced into a cell. In certain embodiments, introduction of the nucleic acid into a cell can be performed by transfection, electroporation, microinjection, infection with a nucleic acid sequence-containing viral or bacteriophage vector, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, etc. In certain embodiments, the cell is eukaryotic, e.g., an MDCK cell.

[0090] 5. Transwell Assay Suitable transwell assays for use in the disclosed methods include, but are not limited to, Boyden chamber assays, cell migration assays, cell invasion assays, microfluidic migration devices, in vitro scratch assays, and extracellular matrix (ECM) protein assays. The disclosed methods can be performed using a wide variety of assay platforms, including "universal" transwell platforms (e.g., 12-well, 24-well, or 96-well multiwell arrays). Non-limiting examples of assay platforms include MILLICELL® cell culture inserts and insert plates, and CORNING® TRANSWELL® polycarbonate membrane cell culture inserts.

[0091] Embodiments of the present disclosure Non-limiting embodiments of the present disclosure include the following:

[0092] 1. A method for determining a pharmacokinetic (PK) parameter of a molecule, comprising determining a measure of transcytosis of said molecule or a plurality of said molecules through a cell or a plurality of cells under physiological pH conditions.

[0093] 2. The method of embodiment 1, wherein said PK parameter is a measure of the in vivo clearance of said molecule.

[0094] 3. The method of embodiment 1, wherein said PK parameter is a measure of the in vivo half-life of said molecule.

[0095] 4. The method of any one of embodiments 1 to 3, wherein the measure of transcytosis is the rate of transcytosis.

[0096] 5. The method of any one of embodiments 1 to 3, wherein the measure of transcytosis is a measure of the amount of molecule(s) that is transcytosed.

[0097] 6. The method of embodiment 1, wherein the measure of transcytosis is determined by measuring transcytosis of a molecule(s) from a solution in a first chamber to a solution in a second chamber, wherein the first and second chambers are separated by a cell monolayer and the solutions in each chamber are at physiological pH.

[0098] 7. The method of embodiment 1, wherein the cell or cells are Madin-Darby canine kidney (MDCK) cells.

[0099] 8. The method of embodiment 1 or 7, wherein the cell comprises at least one heterologous gene.

[0100] 9. The method of embodiment 8, wherein the at least one heterologous gene is selected from the group consisting of the FCGRT gene and the B2M gene.

[0101] 10. The method of any one of embodiments 2 to 9, wherein the cells express a heterologous cell surface protein.

[0102] 11. The method of embodiment 10, wherein the heterologous cell surface protein comprises neonatal Fc receptor (FcRn).

[0103] 12. The method according to any one of the preceding embodiments, wherein measuring the transcytosis of the molecule comprises the use of an enzyme-linked immunosorbent assay (ELISA), liquid scintillation counting (LSC), quantitative PCR or a fluorescence reader system.

[0104] 13. The method of any one of embodiments 1-12, further comprising determining the in vivo clearance of the molecule based on the number of the molecules measured.

[0105] 14. The method according to any one of embodiments 1 to 13, wherein the molecule is a molecule that naturally binds to FcRn.

[0106] 15. The method of any one of embodiments 1 to 13, wherein the molecule is engineered to bind to FcRn.

[0107] 16. The method of any one of embodiments 1 to 15, wherein the molecule is an Fc-containing molecule.

[0108] 17. The embodiment of embodiment 16, wherein the Fc-containing molecule is a receptor-Fc fusion molecule.

[0109] 18. The method of any one of embodiments 16, wherein the molecule is an antibody.

[0110] 19. The method of embodiment 18, wherein the antibody is a monoclonal antibody.

[0111] 20. The method of any one of the preceding embodiments, wherein the molecule is an albumin-containing molecule.

[0112] 21. The method of any one of the preceding embodiments, wherein the physiological pH value is about 7.4.

[0113] 22. A method for measuring transcellular transport of a plurality of single molecules or a plurality of different molecules, comprising: a) introducing the plurality of single molecules or the plurality of different molecules into a first of two chambers, wherein the first chamber is separated from a second chamber by a cell or a plurality of cells, and each of the first and second chambers has a physiological pH value; b) measuring the number of molecules transcytosed from the first chamber to the second chamber.

[0114] 23. The method of embodiment 22, wherein the first chamber comprises a monolayer of cells.

[0115] 24. The method of embodiment 22 or 23, wherein the cells are Madin-Darby canine kidney (MDCK) cells.

[0116] 25. The method according to any one of embodiments 22 to 24, wherein the cell contains at least one heterologous gene.

[0117] 26. The method of embodiment 25, wherein the at least one heterologous gene is selected from the group consisting of the FCGRT gene and the B2M gene.

[0118] 27. The method according to any one of embodiments 22 to 26, wherein the cells express a heterologous cell surface protein.

[0119] 28. The method of embodiment 27, wherein the heterologous cell surface protein comprises neonatal Fc receptor (FcRn).

[0120] 29. The method of any one of embodiments 22 to 28, wherein measuring the number of transcytosed molecules comprises the use of an enzyme-linked immunosorbent assay (ELISA), liquid scintillation counting (LSC), quantitative PCR or a fluorescence reader system.

[0121] 30. The method of any one of embodiments 22-29, further comprising a step of determining the in vivo clearance of the molecule based on the number of the molecules measured.

[0122] 31. The method according to any one of embodiments 22 to 30, wherein the molecule is a molecule that naturally binds to FcRn.

[0123] 32. The method of any one of embodiments 22-31, wherein the molecule is engineered to bind to FcRn.

[0124] 33. The method of any one of embodiments 22-32, wherein the molecule is an Fc-containing molecule.

[0125] 34. The method of embodiment 33, wherein the Fc-containing molecule is a receptor-Fc fusion molecule.

[0126] 35. The method of any one of claims 22 to 34, wherein the molecule is an antibody.

[0127] 36. The method of claim 35, wherein the antibody is a monoclonal antibody.

[0128] 37. The method of any one of embodiments 22-36, wherein the molecule is an albumin-containing molecule.

[0129] 38. The method of any one of embodiments 22 to 37, wherein the physiological pH value is about 7.4.

[0130] 39. A method for determining the in vivo clearance of a molecule, comprising: a) introducing the molecule into a first of two chambers, wherein the first chamber is separated from a second chamber by a cell or a plurality of cells, and each of the first and second chambers has a physiological pH value; b) measuring the number of molecules transcytosed from the first chamber to the second chamber; and c) determining the in vivo clearance of the molecule based on the number of the molecules measured.

[0131] 40. The method of embodiment 39, wherein the first chamber comprises a monolayer of cells.

[0132] 41. The method of embodiment 39 or 40, wherein the cell is an MDCK cell.

[0133] 42. The method of any one of embodiments 39 to 41, wherein the cells contain at least one heterologous gene.

[0134] 43. The method of embodiment 42, wherein the at least one heterologous gene is selected from the group consisting of the FCGRT gene and the B2M gene.

[0135] 44. The method of any one of embodiments 39 to 43, wherein the cells express a heterologous cell surface protein.

[0136] 45. The method of embodiment 44, wherein the heterologous cell surface protein comprises neonatal Fc receptor (FcRn).

[0137] 46. ​​The method of any one of embodiments 39 to 45, wherein measuring the number of transcytosed molecules comprises the use of an enzyme-linked immunosorbent assay (ELISA), liquid scintillation counting (LSC), quantitative PCR or a fluorescence reader system.

[0138] 47. The method according to any one of embodiments 39 to 46, wherein the molecule is a molecule that naturally binds to FcRn.

[0139] 48. The method of any one of embodiments 39-46, wherein the molecule is engineered to bind to FcRn.

[0140] 49. The method of any one of embodiments 39 to 48, wherein the molecule is an Fc-containing molecule.

[0141] 50. The embodiment of embodiment 49, wherein the Fc-containing molecule is a receptor-Fc fusion molecule.

[0142] 51. The method of any one of claims 39 to 50, wherein the molecule is an antibody.

[0143] 52. The method of claim 51, wherein the antibody is a monoclonal antibody.

[0144] 53. The method of any one of embodiments 39-52, wherein the molecule is an albumin-containing molecule.

[0145] 54. The method of any one of embodiments 39 to 53, wherein the physiological pH value is about 7.4.

[0146] 55. An assay for measuring transcytosis of a plurality of molecules, comprising a first chamber and a second chamber, each of the first and second chambers having a physiological pH value, wherein the first chamber is configured to receive a plurality of molecules and transcytosis of the molecules to the second chamber, and wherein the transcytosis of the plurality of molecules is measured.

[0147] 56. The assay of embodiment 55, wherein the first chamber comprises a monolayer of cells.

[0148] 57. The assay of embodiment 55 or 56, wherein the cells are MDCK cells.

[0149] 58. The assay according to any one of embodiments 55 to 57, wherein the cells contain at least one heterologous gene.

[0150] 59. The assay of embodiment 58, wherein the at least one heterologous gene is selected from the group consisting of the FCGRT gene and the B2M gene.

[0151] 60. The assay of any one of embodiments 55-59, wherein the cells express a heterologous cell surface protein.

[0152] 61. The assay of embodiment 60, wherein the heterologous cell surface protein comprises neonatal Fc receptor (FcRn).

[0153] 62. The assay of any one of embodiments 55 to 61, wherein measuring the number of transcytosed molecules comprises the use of an enzyme-linked immunosorbent assay (ELISA), liquid scintillation counting (LSC), quantitative PCR or a fluorescence reader system.

[0154] 63. The assay of any one of embodiments 55-62, further comprising a step of determining the in vivo clearance of the molecule based on the number of the molecules measured.

[0155] 64. The method according to any one of embodiments 55 to 63, wherein the molecule is a molecule that naturally binds to FcRn.

[0156] 65. The method of any one of embodiments 55-62, wherein the molecule is engineered to bind to FcRn.

[0157] 66. The method of any one of embodiments 55-65, wherein the molecule is an Fc-containing molecule.

[0158] 67. The method of embodiment 66, wherein the Fc-containing molecule is a receptor-Fc fusion molecule.

[0159] 68. The method of any one of claims 55 to 67, wherein the molecule is an antibody.

[0160] 69. The method of embodiment 68, wherein the antibody is a monoclonal antibody.

[0161] 70. The method of any one of embodiments 55-69, wherein the molecule is an albumin-containing molecule.

[0162] 71. The method of any one of embodiments 55 to 70, wherein the physiological pH value is about 7.4.

[0163] 72. An assay system comprising: a) a first chamber and a second chamber, wherein the first and second chambers have a physiological pH; b) a monolayer of cells positioned within the first chamber such that the cells can mediate transcytosis of a molecule introduced into the first chamber into the second chamber; and c) a detector for detecting the presence of a molecule in the second chamber.

[0164] 73. The assay system of embodiment 72, wherein the cells are MDCK cells.

[0165] 74. The assay system of embodiment 72 or 73, wherein the cells contain at least one heterologous gene.

[0166] 75. The assay system of embodiment 74, wherein the at least one heterologous gene is selected from the group consisting of the FCGRT gene and the B2M gene.

[0167] 76. The assay system of any one of embodiments 72 to 75, wherein the cells express a heterologous cell surface protein.

[0168] 77. The assay system of embodiment 76, wherein the heterologous cell surface protein comprises neonatal Fc receptor (FcRn).

[0169] 78. The assay system of any one of embodiments 72 to 77, wherein measuring the number of transcytosed molecules comprises the use of an enzyme-linked immunosorbent assay (ELISA), liquid scintillation counting (LSC), quantitative PCR or a fluorescence reader system. EXAMPLES

[0170] The following examples are merely illustrative of the presently disclosed subject matter and should not be construed as limiting in any way.

[0171] (method) Test molecule The test molecules included 53 mAbs with human clearance data, 5 Fc fusion proteins, and an additional 9 mAbs (4 charge variants, 3 glycosylation variants, and 5 FcRn binding variants). Thirty mAbs were commercially available therapeutic antibodies, 19 of which (ofatumumab, vedolizumab, adalimumab, natalizumab, nivolumab, pembrolizumab, avelumab, cetuximab, palivizumab, infliximab, olaratumab, panitumumab, reslizumab, basiliximab, ixekizumab, zulvalumab, evolocumab, alirocumab, golimumab) were purchased from manufacturers. The rest were all produced in engineered Chinese Hamster Ovary (CHO) cells at Genentech (South San Francisco, CA).

[0172] Generation and characterization of MDCK cells expressing human FcRn and beta2-cloglobulin Stable cell lines were developed in a similar manner as previously described (Claypool, 2002). Madin-Darby canine kidney II (MDCK II) cells (European Collection of Authenticated Cell Cultures (ECACC), Salisbury, UK) were grown in Dulbecco's modified minimal essential medium (DMEM) containing 10% fetal bovine serum (Clontech, Mountain View, CA), 100 units / mL penicillin, 100 μg / mL streptomycin, and 0.292 mg / mL L-glutamine (Thermo Fisher Scientific, Waltham, MA) at 37°C in a humidified incubator with 5% CO2. Cells were transfected by electroporation (Lonza Nucleofactor Kit L) with a modified pRK plasmid (Eaton DL et al., 1986, Biochemistry 25(26):8343-8347), which contains cDNA for human FcRn (FCGRT (UniProtKB-P55899, FCGRTN_HUMAN) and β2m (UniProtKB-P61769, B2MG_HUMAN)) under the control of the cytomegalovirus (CMV) promoter, separated by the P2A sequence (Kim, JH et al., 2011, PLoS One 6(4):e18556). After 3 days, cells were selected with 5 μg / mL puromycin and expanded for 2 weeks.

[0173] Cells were then sorted for clonality and FCGRT expression by fluorescence-activated cell sorting (FACS) using an anti-FCGRT antibody (ADM31, Aldevron, Fargo, ND) and a secondary anti-mouse PE-conjugated antibody (A10543, Thermo Fisher Scientific, Waltham, MA). All clones were maintained in a constant selection agent (5 μg / mL puromycin). Final clones were selected based on cell surface expression of both FCGRT and β2m assessed by flow cytometry using a FITC-anti-human β2m FITC-conjugated antibody (2M2, BioLegend, San Diego, CA) using the methods described below.

[0174] MDCK-II cells were grown to confluence and detached using a non-enzymatic cell dissociation reagent (Sigma). 6 Cells / ml were first stained with anti-FCGRT antibody (1:100 dilution) in PBS-0.05M EDTA 2% BSA for 1 h on ice. After washing, cells were incubated with anti-mouse PE-conjugated secondary antibody (1:200 dilution) and anti-β2M FITC-conjugated antibody (1:50 dilution) for 1 h on ice in the dark. Samples were washed and resuspended in PBS-0.05M EDTA 2% BSA for flow cytometry analysis on a BD FACSCanto II (BD Biosciences, San Jose, CA).

[0175] Transcytosis assay Cells were plated at 1 × 10 in cell growth medium DMEM high glucose supplemented with 10% FBS, 100 units penicillin / streptomycin and 5 μg / mL puromycin on 96-well transwell plates (Corning Costar, Acton, MA, USA). 5Cells were seeded at a density of 100 and 200 μL of medium in the inner and outer chambers, respectively. Cells were used for experiments 2 days after plating. Medium in the inner chamber was removed and test molecules were added to a final concentration of 100 μg / mL (0.67 μM) and incubated for 24 h in a tissue culture incubator at 37°C. Lucifer Yellow (Lucifer Yellow CH, dilithium salt; Sigma Aldrich; St. Louis MO) prepared in cell growth medium was added until the last 90 min of the 24 h assay incubation. Medium from the outer chamber was collected and the amount of transcytosed molecules was quantified (Figure 1). The level of passive passage of Lucifer Yellow during the assay was calculated by dividing the fluorescent signal in samples from the outer chamber by that of the inner chamber. Transcytosis results from wells that showed greater than 0.1% passive passage of Lucifer Yellow into the outer chamber were discarded.

[0176] The output of the assay is the concentration (ng / mL) of transcytosed molecules in the medium of the outer chamber, and the reportable value of the assay is the average concentration of duplicate wells from the same plate. In certain embodiments, the output of the assay can be a measure of the rate of transcytosis, or a measure of the relative transcytosis of transcytosed molecules.

[0177] Quantification of transcytosis A 96-well microtiter plate was coated with 100 μL of goat anti-human IgG-F(ab)' (Jackson ImmunoResearch Laboratories, West Grove, PA) at 1 μg / mL in sodium carbonate (pH 9.6) overnight at 2-8°C. After washing with PBS / 0.05% polysorbate-20, the plate was incubated with 200 μL of blocking buffer (PBS / 0.5%, BSA / 0.1%, casein / 0.05%, P20 / 0.05%, Proclin 300) for 2 h at room temperature with gentle agitation. After washing, 100 μL of serially diluted assay standards, controls or samples in assay diluent (PBS / 0.5%, BSA / 0.05%, P20 / 0.05%, Proclin 300) was added to the plate (Figure 1). After another hour of incubation at room temperature with gentle agitation, the plate was washed again and then incubated with 100 μL of goat anti-human IgG-Fc conjugated to HRP (Jackson ImmunoResearch Laboratories, West Grove, PA) at a 1:10000 dilution in assay diluent. The plate was incubated for 1 hour with gentle agitation, washed, and 100 μL of freshly mixed 3,3',5,5'-tetramethylbenzidine (TMB) solution (Kirkegaard & Perry Laboratories, Gaithersburg, MD) was added. Color was allowed to develop for 15 minutes without agitation, and the reaction was stopped by the addition of 100 μL of 1M H3PO4. Absorbance was read at a wavelength of 450 nm with a 650 nm reference on a Spectramax I3 plate reader (Molecular Devices Corporation, CA), and data was processed using Softmax Pro software provided by the manufacturer. The concentration of test antibody in the samples was interpolated from a four parameter fit of the standard curve on the same plate.

[0178] For Fc fusion proteins, test molecules were captured by protein-specific antibody binding and detected by anti-human IgG-F(ab)' conjugated to HRP following the procedure described above.

[0179] BV ELISA A 1% baculovirus particle suspension was prepared in coating buffer (0.05 sodium carbonate pH 9.6) and 25 μL was added per well in a 384-well plate (Nunc Immunoplate Maxisorp Surface, Cat. No. 464718) and incubated overnight at 4° C. The wells were blocked with 50 μL assay buffer (PBS containing 5% BSA and 10 PPM Proclin) for 1 hour at room temperature with gentle shaking. After washing the wells three times with 100 μL wash buffer (PBS), 25 μL samples prepared in assay buffer were loaded in duplicate and incubated for 1 hour at room temperature with gentle shaking. The plate was washed six times with 100 μl of washing buffer, and 25 μl of anti-goat human FcRn fragment specifically conjugated to horseradish peroxidase (Jackson ImmunoResearch) at 10 ng / mL in assay buffer was added to each well and incubated at room temperature for 1 hour with gentle shaking. The well was washed six times with 100 μl of washing buffer, and 25 μL of TMB (Moss, Inc., Cat. No. TMBE-1000) substrate was added per well for 15 minutes at room temperature with gentle shaking, and the reaction was stopped by adding 25 μL of 1M phosphoric acid per well, and the absorbance was measured at 450 nm using a plate reader.

[0180] BLI-based assessment of mAb-FcRn interactions Octet RED (PALL / ForteBio) was used for all FcRn binding in vitro assays in 96-well solid black plates (Greiner Bio-One, 655900) at 30°C. First, FcRn was immobilized on a nickel-nitrilotriacetic acid coated biosensor (ForteBio, 18-0029) at an optimized concentration for 180 seconds. After a baseline step, the mAb-FcRn binding kinetics was determined when the FcRn immobilized biosensor was exposed to mAb samples in PBS adjusted to pH 6.0 with HCl for 30 seconds. Prior to analysis, all antibodies were dialyzed into PBS pH 6.0, diluted to 100 mg / mL in PBS pH 6.0, and used in a volume of 200 mL. Each assay was performed in quintuplicate for a specific mAb. Data analysis was performed using software version 7.0 (PALL, ForteBio).

[0181] pI determination by imaging capillary isoelectric focusing Imaging capillary isoelectric focusing (icIEF) was performed on an iCE280 analyzer (ProteinSimple, Toronto, ON) as previously described (Li et al., 2014). The anolyte and catholyte were 80 mM phosphoric acid and 100 mM NaOH, respectively, prepared with 0.1% methylcellulose. mAb samples were prepared in water containing carboxypeptidase and incubated at 37°C for 20 min to remove C-terminal lysine residues. An ampholyte mixture consisting of 3.1% (v / v) Pharmalyte (GE Life Sciences), 2.5 M urea, 0.1% (v / v) methylcellulose, and pI 5.5 and 9.77 markers (Protein Simple) was combined with treated antibodies to a final concentration of 0.25 mg / mL. Electropherograms were imaged at 280 nm with a light absorption detector.

[0182] Cell-binding assay by flow cytometry Cells were stained with test molecules at 100 μg / mL in FACS buffer (PBS / 1% BSA / 2 mM EDTA / 0.1% sodium azide) and incubated on ice for 1 h. After washing with FACS buffer, cells were stained with PE-conjugated mouse anti-human IgG Fc secondary antibody (Southern Biotech, Birmingham, AL) for 1 h on ice. Cells were washed and fixed with fixation buffer (BD Biosciences, San Jose, CA). Fluorescence intensity of stained cells was measured using a FACS Canto II flow cytometer (BD Biosciences, San Jose, CA) and data was analyzed using FlowJo software (Tree Star, Ashland, OR).

[0183] Mouse PK studies using X-Fc sialylated variants A single intravenous bolus dose of X-Fc variants (1, 5, 8, 12 or 15) was administered to CD1 mice at a dose of 1 mg / kg. Serum samples (n=4 / time point) were collected and analyzed for drug concentration at various time points up to 21 days after dosing. Drug concentrations in serum were measured using a validated ELISA assay. Serum concentration-time data from individual animals were used to estimate pharmacokinetic parameters using non-compartmental sparse analysis in Phoenix Winnonlin, version 6.4.0.768 (Winnonlin 6.4, Pharsight Corporation, Mountain View, CA).

[0184] Total antibody concentrations in mouse serum were measured by ELISA and universal PK assays using the Gyros technology platform (Gyros US Inc., Warren, NJ). The assay uses biotinylated sheep anti-human IgG antibody as capture and Alexa Fluor_647-conjugated sheep anti-human IgG as detection. The minimum dilution for the assay was 1:10. The assay had a standard curve range of 0.03-30 μg / mL for mouse serum.

[0185] Example 1: Development and characterization of a neutral pH FcRn-mediated transcytosis assay MDCK cell line was transfected with both human B2M (B2M) and FcRn heavy chain (FCGRT) genes. Stable cells expressing both genes were isolated by FACS sorting, and a clonal cell line (MDCK-hFcRn, 305-6) expressing high levels of FCGRT and B2M on the cell surface (Figure 2) was selected for further development of the FcRn-dependent transcytosis assay. To minimize potential assay bias / artifacts and better mimic physiological conditions, the molecules tested in the assay were label-free and the assay was performed in growth medium at physiological pH.

[0186] In the present assay, cells were grown to confluency on the filter membrane of a transwell, test molecules were added to the growth medium in the inner chamber, and after incubation, molecules transported through the cells and released into the medium in the outer chamber were quantified by ELISA. The assay was extensively optimized for seeding density, transwell plate format, loading concentration, assay medium, and assay duration. The assay medium contains bovine serum albumin (BSA), which has been shown to bind human FcRn and form a trimolecular complex in the presence of IgG (Chaudhury C. et al., 2003, J. Exp. Med. 197:315-322), so human FcRn in this assay is expected to simultaneously engage both the test antibody and BSA. Alternative assay media containing variable amounts of human serum albumin instead of BSA showed little difference in the transcytosis readout compared to media containing BSA. Due to operational considerations, the assay was performed in normal growth medium containing FBS / BSA.

[0187] The functional integrity of filter-grown MDCK-hFcRn cells was monitored by measuring the transepithelial electrical resistance (TEER; Srinivasan B. et al., 2015, J Lab Autom 20(2):107-126). The TEER of the monolayers prior to the assay was typically in the range of 250-300 Ω*cm, which is characteristic for polarized MDCK II cells. 2 (Tesar DB et al., 2006, Traffic 7(9):1127-1142). In addition, the barrier integrity (leakyness) of the monolayer during the assay was monitored by mixing Lucifer Yellow with the test molecule in the inner chamber as described in the methods.

[0188] Example 2: Correlation of in vitro transcytosis efficiency with in vivo clearance of mAbs in humans To evaluate the potential utility of this assay for determining or predicting PK behavior of mAbs in vivo, 53 mAbs, including 30 marketed therapeutic antibodies and 23 clinical candidates, were tested in the assay. The transcytosis output of mAbs was analyzed for potential correlation with their reported clearance rates in humans. These molecules were selected based on the availability of clinical grade material and human clearance data. In addition to the transcytosis assay, some of the molecules were also tested in two other in vitro assays: BV ELISA and Biolayer Interferometry (BLI)-based human FcRn binding assay. BV ELISA measures nonspecific binding to baculovirus particles as an indication of the general nonspecific binding properties of mAbs and has been reported to be able to identify antibodies with an increased risk of fast clearance in cynomolgus monkeys (Hoetzel et al., 2012, (2012), MAbs vol. 4(no. 6): pp. 753-760). A BLI-based FcRn binding assay has been used to generate combined FcRn association rates at pH 6.0 and dissociation rates at pH 7.4, which showed strong correlation with the half-lives of five mAbs in humans and human FcRn transgenic mice (Souders CA et al., 2015, MAbs 7(5):912-921).

[0189] The results of this study are presented in Table 1, which also includes the clearance rates of the test molecules in both humans and cynomolgus monkeys (cyno). The mean transcytosis output of the test panel was 5.4ng / mL with a median of 4.5 and a range of 2.6-14.9, reflecting an average of approximately 0.005% of the material (100μg / mL) loaded into the inner chamber. The respective mean and median / range of FcRn binding affinity (Kd, in μM) at pH 6.0 and BV ELISA scores are as follows: 0.63, 0.54 / 0.25-1.8, and 0.25, 0.06 / 0.038-3.916, respectively. Human clearance rates of marketed mAb drugs were obtained from the prescribing information or reports of the drugs, and clearance rates of clinical stage mAbs were generated from clinical studies or reports conducted by Genentech. The cyno clearance rates were generated from a cyno study performed by Genentech. The mean and median / range clearance rates in humans are 5.0, 4.1 / 1.4-14.1 mL / Kg / day and 6.8, 4.9 / 3.0-15.8 mL / Kg / day for cyno. The values ​​for all parameters seem to span at least a 5-fold range, indicating substantial diversity of the mAb panel tested in this study.

[0190] As shown in Figure 3A, a clear association trend was observed between transcytosis output and clearance values, with a fitted linear regression R-squared value of 0.8. Antibodies with reported faster clearance rates in humans generally showed higher transcytosis output in this assay. Notably, the observed correlation was not affected by subgrouping the tested molecules based on heavy and light chain subclasses, target nature (soluble or membrane-bound), mechanism of action (agonistic, antagonistic or toxic) or route of administration. On the other hand, clearance rates showed no clear correlation with FcRn binding at pH 6.0 via BLI or BV ELISA (Figures 3B, C).

[0191] Cyno PK generally provides the most translatable model for determining or predicting human PK of mAbs. Correlation between transcytosis output and cyno clearance may provide further confirmation to the usefulness of this assay as a determination or prediction tool for PK. Cyno clearance data were obtained for 25 mAbs in the panel and their correlation with either human clearance rates or transcytosis efficiency values ​​was analyzed. As shown in Figure D, a trend of association was observed between transcytosis efficiency and cyno clearance, and the degree of correlation as judged by the R-squared value appears to be similar to that between cyno and human clearance (Figure 3D). However, around 0.6, the R-squared values ​​were relatively low due to a small number of "outliers". Two of the most obvious outliers in the cyno clearance vs. transcytosis plot (Figure 3D) are aNRP1 and pertuzumab, the former known for its inconsistency with human clearance (Figure 3E) and the latter binding to MDCK cells likely due to cross-reactivity with canine epidermal growth factor receptor-2 (Fazekas, 2016). Removal of these two molecules increased the R-squared value of the linear regression fit to approximately 0.75 in Figure 2E and 0.76 in Figure 3E. [Table 1] TIFF0007631187000002.tif255170TIFF0007631187000003.tif250170TIFF0007631187000004.tif252170ND=not performed, NA=not measurable, ROA=route of administration, M=membrane bound, S=soluble, SC=subcutaneous, IV=intravenous, IM=intramuscular.

[0192] To demonstrate the dependence of the observed correlation between transcytosis output and clearance on FcRn interaction, 12 mAbs (10 from the panel with human clearance data added) were randomly selected based on their various transcytosis outputs observed in the assay and tested in a similar transcytosis assay using parental MDCK cells. The obtained transcytosis values ​​were analyzed for their tendency to correlate with clearance rates (Figure 4A) and compared to those using MDCK-hFcRn cells (Figure 4B). As shown in Table 3 and Figure 4A, the transcytosis values ​​in MDCK cells were generally less than 10% of those from the assay using FcRn-transfected cells and showed no clear correlation with clearance rates in humans. On the other hand, transcytosis in MDCK-hFcRn cells with the same set of mAbs shows a strong correlation with clearance (Figure 4B). The results of this study confirm that interactions between FcRn and the test antibodies are not only involved in promoting transcytosis in the assays of the present invention, but also directly contribute to their observed correlation with clearance in humans.

[0193] Example 3: Effect of cell binding on transcytosis of mAbs Non-limiting non-target binding is known to affect the PK of mAb drugs (Boswell et al., 2010, Bioconjug. Chem. vol. 21, pp. 2153-2163). Transcytosis of test molecules in the present assay involved binding and internalization by MDCK-hFcRn cells. Test molecules may bind to cells via non-specific interactions at physiological pH or specific binding to surface-bound FcRn, either of which may affect transcytosis output. To assess the effect of cell binding on transcytosis, selective mAbs were incubated with both transfected MDCK-hFcRn and non-transfected MDCK cells, and bound antibodies were detected by flow cytometry using fluorescently conjugated mouse anti-human IgG-Fc. The extent of cell binding was indicated by the geometric mean fluorescence intensity, and the results are presented in Table 2. With a few exceptions, the differences between mAbs in cell binding to either cell line were mostly small, which seemed to indicate that due to minimal binding to FcRn at physiological pH, FcRn expression in most cases did not dramatically alter the binding behavior of Abs to MDCK cells. No clear correlation was observed between cell binding and transcytosis output (Figure 4C).

[0194] Interestingly, both anti-HER2 antibodies, trastuzumab and pertuzumab, showed improved binding to MDCK cells with and without transfected human FcRn (Table 2). It has been reported that human HER2 shares high homology with its canine counterpart, and that both trastuzumab and pertuzumab can be used to treat breast cancer in dogs (Fazekas, 2016). Thus, the observed binding to cells by these two molecules can be explained by cross-reactivity with canine epidermal growth factor receptor-2 expressed in MDCK cells. In addition, anti-gD showed increased binding to MDCK-hFcRn as well as non-transfected MDCK cells, suggesting its specific binding to human FcRn on MDCK-FcRn cells at physiological pH. Furthermore, the improved binding to MDCK-FcRn cells did not translate to increased transcytosis in MDCK-FcRn cells. It is possible that the observed binding did not directly lead to internalization of the complex, or that internalized anti-gD was preferentially recycled back to the internal chamber or directed to lysosomes for degradation and therefore was not transported via the transcytosis pathway. Finally, two of the mAbs showing higher transcytosis and faster clearance among the 12 mAbs tested (baxiliximab and mAb12) also showed improved binding to both cell lines. This suggests that both molecules may exert non-specific interactions with MDCK cell membranes, leading to increased transcytosis in the assay and potentially playing a role in their reported faster clearance in humans. [Table 2]

[0195] Taken together, the results from this study suggest that while binding to cells through specific or non-specific interactions can affect transcytosis, the output from this assay is not determined by binding alone, and other contributing factors such as cellular uptake / internalization, interaction with FcRn in endosomes, and sorting / trafficking in cells were involved in the overall outcome. MFI: Geometric Mean Fluorescence Intensity

[0196] Example 4: Effect of charge on transcytosis of mAbs Charge is one of the major determinants of how mAbs interact with negatively charged moieties on surface binding. Changes in charge have been shown to alter the PK behavior of mAbs: mAbs carrying higher pI or more positive Fv charge have been shown to be cleared faster than their lower pI / less positively charged counterparts (Igawa, T. et al., 2010, Protein Eng Des Sel 23(5):385-392; Boswell et al., Chem. 21,2153-2163 (2010).10; Bumbaca Y. et al., 2015, J Biol Chem 290(50):29732-29741). It is hypothesized that a higher positive charge would lead to faster nonspecific clearance via increased nonspecific binding, possibly due to greater electrostatic interactions with the negatively charged extracellular matrix, whereas a lower positive charge would lead to decreased nonspecific binding and slower nonspecific clearance.

[0197] To understand the effect of charge on mAb transcytosis, two sets of mAbs with engineered Fv charge variants previously evaluated for PK behavior in several preclinical species (Bumbaca Y. et al., 2015, J Biol Chem 290(50):29732-29741) were tested in our assay. These charge variants were based on anti-lymphotoxin alpha (anti-LTα) and humAb4D5-8 (anti-HER2), with one more positively charged Fv for each parent antibody (anti-LTα+3 and anti-HER2+3) and the other less positively charged Fv (anti-LTα-4 and anti-HER2-4). In the reported study, both variants with more positive Fv charge showed faster nonspecific clearance, whereas the less positively charged one showed similar or lower clearance compared to the parent molecule (Bumbaca Y. et al., 2015, J Biol Chem vol. 290(50):29732-29741). The correlation between Fc charge and clearance was observed in all preclinical species tested, including cynomolgus monkeys, mice and rats, and the lack of species dependency supports the hypothesis of nonspecific electrostatic interactions between positively charged mAbs and negatively charged cellular components. As shown in Table 3, the order of transcytosis values ​​for both sets of charge variants follows the same trend as the degree of Fv charge and the clearance rate observed in cynomolgus monkeys. Compared to the parent molecules, both more positively charged variants (anti-LTα+3 and anti-HER2+3) showed higher transcytosis output, whereas the two less charged molecules showed lower transcytosis output. The transcytosis output appeared to correctly reflect the observed ranking of the clearance rates of both sets of charge variants. The results of this study indicate that changes in the charge of the mAbs affect the transcytosis readout in our assay in the same way that they do for their PK, and that our assay can reflect the effect of charge on clearance. [Table 3]

[0198] To better understand the general impact of electrostatic interactions on mAb clearance in humans, the combined Fv (vL+vH) charges of 19 mAbs (out of 50 mAbs tested) were calculated and their correlation with clearance was analyzed. In addition, the pI of these molecules was measured by icIEF and a similar analysis was performed. As shown in Figures 5A and 5B, a trend for association was observed between the clearance rate in humans and either the pI or the combined Fv charge values, but the strength of the correlation appeared to be weak, as evidenced by the relatively low R-squared values ​​(less than 0.25). On the other hand, the correlation between clearance and transcytosis remains strong in this subset of mAbs (R-squared values ​​greater than 0.8; Figure 5C). Although substantial changes in charge would be expected to modulate the transcytosis behavior of mAbs, the output of this assay appears to be determined by a combined effect contributed by multiple factors in addition to charge.

[0199] Example 5: Effect of glycosylation on transcytosis of Fc-containing molecules The glycosylation pattern of mAbs or Fc fusion proteins can significantly affect their PK and PD behavior (Higel F. et al., 2016, Eur J Pharm Biopharm 100:94-100). It has been shown that mAbs carrying more than 99% high mannose N-glycans (Man5 or Man8 / 9) exhibit faster clearance in preclinical species compared to controls with typical glycosylation patterns (Yu M et al., 2012, MAbs 4(4):475-487), and that Fc fusion proteins with extensive sialylation in the fusion partner exhibit reduced clearance compared to their less sialylated counterparts (Stefanich EG et al., 2008 J Pharmacol Exp Ther 327(2):308-315). It is hypothesized that mAbs carrying high mannose glycans are bound and cleared by the mannose receptor, which is most prominently expressed on immune cells (Lee SJ et al., 2002, Science 295:1898-1901), whereas Fc fusion proteins with exposed terminal galactose (no sialylation) are bound and cleared by the asialoglycoprotein receptor, which is expressed in the liver (Stockert RJ, 1995, Physiol. Rev. 75:591-609).

[0200] To investigate the effect of glycosylation on the transcytosis of mAbs and Fc fusion proteins, two sets of molecules with varying glycosylation patterns were tested. The ocrelizumab (2H7) glycoform variants include one lacking Fc N-linked glycans by in vitro deglycosylation (2H7-DG) and the other carrying more than 99% mannose 5 glycans (2H7-Man5). The other set of variants is based on X-Fc, a fusion protein consisting of human glycoprotein X and human IgG1 Fc, with five variants of X-Fc carrying different levels of sialylation representing 1, 5, 8, 12 and 15 moles of sialic acid per mole of X-Fc (X-Fc1, X-Fc5, X-Fc8, X-Fc12 and X-Fc15, respectively). The clearance rates of 2H7 and 2H7-Man5 were taken from a previously published study (Yu M et al., 2012, MAbs 4(4):475-487), and the clearance rates of the X-Fc sialylated variants were generated from an in-house mouse study. No clearance data was available for 2H7-DG, but it was reported that removal of the Fc N-glycans did not induce any discernible change in their half-life (Tao MH and Morrison SL, 1989, J Immunol 143(8):2595-2601). In addition, atezolizumab, which does not carry Fc N-glycans due to the N297G mutation, exhibits a normal PK profile in humans (Prescribing Information and Table 1). As shown in Table 4, compared to 2H7, 2H7-DG exhibited similar transcytosis, whereas 2H7-Man5 exhibited a two-fold increase in transcytosis, with the former predicting similar clearance and the latter predicting faster clearance, both of which are consistent with their expected / observed ranking of clearance rates in vivo. Similarly, the five X-Fc variants exhibit decreasing levels of transcytosis in an order consistent with the decreasing amount of exposed terminal galactose as a result of increased sialylation, and the predicted ranking of clearance rates of these sialylated variants is consistent with the observed clearance data from mouse studies.

[0201] To investigate the potential effect of glycosylation on FcRn binding and / or non-specific interactions with cells, both sets of glycosylation variants were tested for FcRn binding affinity and relative binding to MDCK-hFcRn cells. Compared to 2H7, the deglycosylated molecule (2H7-DG) showed no discernible difference in binding to FcRn and cells. On the other hand, the mannose-5 glycoform (2H7-Man5) showed a slight increase in binding to MDCK cells and a moderate increase in binding to FcRn at pH 6.0, but no increase in transcytosis in MDCK cells and no further increase in binding to MDCK-FcRn cells. Similar to the 2H7 man-5 glycoform, the X-Fc sialylated variants showed no clear correlation between transcytosis in MDCK-hFcRn cells and either cell binding or transcytosis in MDCK cells. However, in contrast to 2H7-Man5, which showed increased FcRn binding and increased transcytosis, X-Fc sialylated variants showed a trend towards increased FcRn binding that inversely correlated with transcytosis output in MDCK-FcRn cells, suggesting that glycosylation may affect FcRn binding and transcytosis differently depending on the glycan species. Of note, the lack of a notable increase in binding by 2H7-Man5 and X-Fc-SA1 to MDCK cells suggests that the increased transcytosis was due to binding to specific receptors such as the mannose receptor or asialoglycoprotein receptor, which are known to be involved in the catabolism of these IgG glycoforms, although we have no direct evidence to confirm the presence or absence of such receptors in MDCK cells. [Table 4]

[0202] Example 6: Effect of extreme FcRn binding on transcytosis of mAbs Modification of binding affinity to FcRn via genetic engineering has been shown to affect the half-life of therapeutic antibodies and Fc fusion proteins (Ward ES et al., 2015, Mol Immunol 67(part 2A):131-141). It is therefore of interest to investigate the transcytosis behavior of mAbs carrying engineered mutations with substantially altered FcRn binding affinity. Two sets of molecules were tested for transcytosis in both parental MDCK and MDCK-hFcRn cells. The first set includes anti-VEGF (humanized IgG1 mAb) and anti-VEGF-QA carrying the T307Q / N434A mutations that increase FcRn binding by nearly 10-fold (Yeung YA et al., 2010, Cancer Res 70(8):3269-3277). The second set includes a humanized herpes simplex virus glycoprotein D-specific IgG1 antibody, anti-gD(WT), and two of its FcRn-binding variants, anti-gD-HAHQ, carrying H310A / H435Q mutations that reduce its FcRn-binding activity to undetectable levels (Kenanova V. et al., 2005, Cancer Res 65(2):622-631), and anti-gD-YTE, carrying M252Y / S254T / T256E mutations that increase its FcRn-binding by more than 10-fold (Dall'Acqua, 2006). As shown in Table 5, apparent transcytosis of all five molecules was clearly observed in MDCK-hFcRn cells with anti-gD-HAHQ, which showed the lowest transcytosis value, likely due to its inability to bind FcRn. However, in contrast to results from the mAb panel where increased transcytotic output correlated with increased clearance rate (fast clearance), anti-VEGF-QA and anti-gD-YTE, which show slow clearance in vivo, both showed increased transcytosis compared to their wild-type counterparts. It is conceivable that molecules with extreme FcRn binding affinity may behave differently in this assay due to their differential response to sorting mechanisms that govern recycling and transcytosis pathways.

[0203] Interestingly, most of the molecules showed low levels of transcytosis in MDCK cells, either below or barely above the detection limit of the ELISA at 0.3 ng / mL, while anti-gD-YTE showed notable transcytosis levels above 1 ng / mL, which are likely independent of FcRn. The substantial difference in transcytosis output observed between the two cell lines confirms that the transcytosis observed in MDCK-hFcRn cells is primarily driven by FcRn and that the assay is specific for molecules containing intact Fc domains of IgG that can interact with FcRn. Notably, both anti-VEGF-QA and anti-gD-YTE show an 8- to 10-fold increase in FcRn binding activity compared to their wild-type counterparts, but the increase in transcytosis was somewhat modest, less than 2-fold for anti-VEGF-QA and slightly more than 2-fold for anti-gD-YTE. [Table 5]

[0204] All publications, patents, and other references cited herein are incorporated by reference in their entirety into this disclosure.

Claims

1. 1. A method for determining the in vivo clearance of a molecule, comprising: a) introducing the molecule into a first chamber, wherein the first chamber is separated from a second chamber by a cell or a plurality of cells, and each of the first and second chambers has a physiological pH value; b) measuring the number of molecules transcytosed from the first chamber to the second chamber; and c) determining an in vivo clearance of the molecule based on the number of the molecules measured; The method, wherein the cells are MDCK cells and the physiological pH value is 7.

4.

2. The method of claim 1 , wherein the first chamber comprises a monolayer of cells.

3. The method of claim 1 or 2, wherein the cell comprises at least one heterologous gene.

4. 4. The method of claim 3, wherein the at least one heterologous gene is selected from the group consisting of the FCGRT gene and the B2M gene.

5. The method of any one of claims 1 to 4, wherein the cells express a heterologous cell surface protein.

6. The method of claim 5 , wherein the heterologous cell surface protein comprises a neonatal Fc receptor (FcRn).

7. 7. The method of any one of claims 1 to 6, wherein measuring the number of transcytosed molecules comprises the use of enzyme-linked immunosorbent assay (ELISA), liquid scintillation counting (LSC), quantitative PCR or a fluorescence reader system.

8. The method of any one of claims 1 to 7, wherein the molecule is a molecule that naturally binds to FcRn.

9. The method of any one of claims 1 to 7, wherein the molecule is engineered to bind to FcRn.

10. The method of any one of claims 1 to 9, wherein the molecule is an Fc-containing molecule.

11. The method of claim 10, wherein the Fc-containing molecule is a receptor-Fc fusion molecule.

12. The method of any one of claims 1 to 11, wherein the molecule is an antibody.

13. The method of claim 12 , wherein the antibody is a monoclonal antibody.

14. The method of any one of claims 1 to 13, wherein the molecule is an albumin-containing molecule.

15. 1. An assay for determining in vivo clearance of molecules by measuring transcytosis of a plurality of molecules, the assay comprising a first chamber and a second chamber, each of the first and second chambers having a physiological pH value, wherein the first chamber is configured to receive a plurality of molecules and transcytosis of the molecules to the second chamber, the transcytosis of the plurality of molecules being measured, the first chamber comprising a monolayer of cells, the cells being MDCK cells, and the physiological pH value being 7.

4.

16. The assay of claim 15 , wherein the cells contain at least one heterologous gene.

17. 17. The assay of claim 16, wherein the at least one heterologous gene is selected from the group consisting of the FCGRT gene and the B2M gene.

18. The assay of any one of claims 15 to 17, wherein the cells express a heterologous cell surface protein.

19. 20. The assay of claim 18, wherein the heterologous cell surface protein comprises a neonatal Fc receptor (FcRn).

20. 20. The assay of any one of claims 15 to 19, wherein measuring the number of transcytosed molecules comprises the use of an enzyme-linked immunosorbent assay (ELISA), liquid scintillation counting (LSC), quantitative PCR or a fluorescence reader system.

21. The assay of any one of claims 15 to 20, further comprising determining the in vivo clearance of said molecules based on the number of said molecules measured.

22. The assay of any one of claims 15 to 21, wherein the molecule is a molecule that naturally binds to FcRn.

23. The assay of any one of claims 15 to 22, wherein the molecule is engineered to bind to FcRn.

24. The assay of any one of claims 15 to 23, wherein the molecule is an Fc-containing molecule.

25. 25. The assay of claim 24, wherein the Fc-containing molecule is a receptor-Fc fusion molecule.

26. The assay of any one of claims 15 to 25, wherein the molecule is an antibody.

27. 27. The assay of claim 26, wherein the antibody is a monoclonal antibody.

28. The assay of any one of claims 15 to 27, wherein the molecule is an albumin-containing molecule.

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