Macromolecule non-specific clearance assay

Abstract

Herein is reported a method for determining non-specific clearance of an antibody comprising the steps of incubating the antibody conjugated to a pH-sensitive fluorescent dye with a primary human endothelial cell, and determining the fluorescence intensity of the primary human endothelial cell, wherein an increase in the fluorescence intensity of the primary human endothelial cell relative to a background level is indicative of non-specific clearance of the antibody.

Description

[0001] This application is a divisional application of PCT application PCT / EP2021 / 058839, filed on April 6, 2021, entitled "Determination of Non-specific Scavenging of Macromolecules". The date of entry into the national phase of the PCT application in China was October 8, 2022, and the application number was 202180027436.8.

[0002] This article reports a novel method for estimating the clearance of therapeutic proteins in the human body using a new in vitro assay based on primary human cells. This macromolecular nonspecific clearance assay (LUCA) provides an in vitro-based approach to assess and predict the major PK properties of therapeutic proteins. Background Technology

[0003] Human immunoglobulin G (IgG) comprises two antigen-binding (Fab) regions that deliver specificity to the target antigen, and a constant region (Fc region) responsible for interacting with the Fc receptor (see, for example, Edelman, GM, Scand. J. Immunol. 34 (1991) 1-22; Reff, ME and Heard, C., Crit. Rev. Oncol. Hematol. 40 (2001) 25-35). The mean serum half-life of human IgG subclasses IgG1, IgG2, and IgG4 is 21 days, which is longer than that of any other known serum protein (see, for example, Waldmann, TA and Strober, W., Prog. Allergy 13 (1969) 1-110). This long half-life is primarily mediated by the interaction between the Fc region and the neonatal Fc receptor (FcRn) (see, for example, Ghetie, V. and Ward, ES, Annu. Rev. Immunol. 18(2000) 739-766; Chaudhury, C., et al., J. Exp. Med. 197 (2003) 315-322). This is one of the reasons why IgG or Fc-containing fusion proteins are used as a broad class of therapeutic agents.

[0004] Neonatal Fc receptor FcRn is a membrane-associated receptor involved in IgG and albumin homeostasis, maternal IgG transplacental transport, and phagocytosis of antigen-IgG immune complexes (see, for example, Brambell, FW, et al., Nature 203 (1964) 1352-1354; Ropeenian, DC, et al., J. Immunol. 170 (2003) 3528-3533). Human FcRn is a heterodimer composed of a glycosylated major histocompatibility complex-like protein (α-FcRn) and a β2-microglobulin (β2m) subunit (see, for example, Kuo, TT, et al., J. Clin. Immunol. 30 (2010) 777-789). FcRn binds to sites in the CH2-CH3 region of the Fc region (see, for example, Ropeenian, DC and Akilesh, S., Nat. Rev. Immunol. 7 (2007) 715-725; Martin, WL, et al., Mol. Cell 7 (2001) 867-877; Goebl, NA, et al., Mol. Biol. Cell19 (2008) 5490-5505; Kim, JK, et al., Eur. J. Immunol. 24 (1994) 542-548), and two FcRn molecules can bind to the Fc region simultaneously (see, for example, Sanchez, LM, et al., Biochemistry 38 (1999) 9471-9476; Huber, AH, et al., J. Mol. Biol.). 230(1993) 1077-1083. ). The affinity between FcRn and Fc regions is pH-dependent, showing nanomolar affinity at endosome pH 5-6, while showing rather weak binding at physiological pH 7.4 (see, for example, Goebl, NA, et al., Mol. Biol. Cell 19 (2008) 5490-5505; Ober, RJ, et al., Proc. Natl. Acad. Sci. USA 101 (2004) 11076-11081; Ober, RJ, et al., J. Immunol. 172 (2004) 2021-2029).The potential mechanism by which long half-life is transferred to IgG can be explained through three basic steps. First, IgG undergoes non-specific pinocytosis across multiple cell types (see, for example, Akilesh, S., et al., J. Immunol. 179 (2007) 4580-4588; Montoyo, HP, et al., Proc. Natl. Acad. Sci. USA 106 (2009) 2788-2793). Second, IgG encounters and binds to FcRn in acidic endosomes at pH 5-6, thereby protecting IgG from lysosomal degradation (see, for example, Ropeenian, DC and Akilesh, S., Nat. Rev. Immunol. 7 (2007) 715-725; Rodewald, R., J. Cell Biol. 71 (1976) 666-669). Finally, at physiological pH 7.4, IgG is released into the extracellular space (see, for example, Ghetie, V. and Ward, ES, Annu. Rev. Immunol. 18 (2000) 739-766). This strictly pH-dependent binding and release mechanism is crucial for IgG recycling, and any deviation in binding properties at different pH values ​​can strongly affect the circulating half-life of IgG (see, for example, Vaccaro, C., et al., Nat. Biotechnol. 23 (2005) 1283-1288).

[0005] Eigenmann, MJ et al. outlined that cellular uptake of antibodies is thought to occur primarily in endothelial and hematopoietic cells. Once antibodies are absorbed into the endosome, they can be protected from degradation by binding to neonatal Fc receptors (FcRn). Neonatal Fc receptors bind antibodies in a pH-dependent manner, with higher affinity in the endosome at pH 6 than in the physiological plasma at pH 7.4. Therefore, antibodies bound to FcRn in the endosome are released into the plasma at neutral pH, allowing for antibody recycling rather than lysosomal degradation (MABS 9 (2017) 1007-1015).

[0006] Grevys, A. et al. reported a human endothelial cell-based recirculation assay for screening molecules targeting FcRn (Nat. Commun. 9 (2018) 621). Nath, N. et al. reported a homogenized plate-based antibody internalization assay using a pH sensor and fluorescent dye (J. Immunol. Meth. 431 (2016) 11-21).

[0007] Fluorescent sensor reagents and their use and manufacturing methods are provided in WO 2013 / 134686. In particular, sensor reagents that exhibit detectable changes in fluorescence (e.g., fluorescence intensity) when the pH of the surrounding environment is changed (e.g., when moving from one pH environment to another pH environment) are provided.

[0008] Based on the key biological factors of nonspecific clearance of therapeutic antibodies in patients, namely nonspecific uptake via endocytosis and FcRn-mediated recycling, in vitro methods are needed to predict in vivo clearance (i.e., half-life). Summary of the Invention

[0009] This article reports a method for determining the level of nonspecific clearance of therapeutic proteins, particularly antibodies, through pinocytosis and lysosomal degradation.

[0010] The present invention is based at least in part on the discovery that in vitro ingestion of antibodies into primary human endothelial cells can serve as an alternative for assessing the nonspecific clearance of said antibodies in vivo, particularly in mice, cynomolgus monkeys, and humans.

[0011] This invention is based at least in part on the finding that only primary human endothelial cells can be used to determine in vivo clearance rates based on in vitro experiments, because non-primary endothelial cells do not show the same correlation and are therefore unsuitable for this purpose. Differentiation between different antibodies cannot be achieved using the aforementioned non-primary endothelial cells.

[0012] This invention is based at least in part on the finding that the uptake and transport of antibodies to the lysosomal compartment of primary endothelial cells via pinocytosis contributes the most and shows a good correlation with the fluorescence of primary endothelial cells.

[0013] Therefore, the present invention includes a method for determining or estimating the nonspecific (i.e., non-target-mediated) clearance (rate) of an antibody, the method comprising the following steps: a) Incubate the antibody conjugated to a pH-sensitive fluorescent dye with primary human endothelial cells (for a specified time), and b) Determine the intracellular fluorescence intensity of the primary human endothelial cells obtained in step a) (after the specified incubation time). The presence of nonspecific clearance of the antibody (i.e., nonspecific clearance of the indicator antibody) is determined by the increase in the intracellular fluorescence intensity of primary human endothelial cells as determined in step b) relative to the background level (i.e., the intracellular fluorescence of primary human endothelial cells not incubated with the antibody).

[0014] In some embodiments, the method further includes the following steps: - Determine the intracellular fluorescence intensity of primary human endothelial cells before incubation with / without antibody. as well as The presence of antibody-indicating nonspecific clearance (i.e., nonspecific clearance of the indicator antibody) is determined by the increase in the (intracellular) fluorescence intensity of primary human endothelial cells as determined in step b) relative to the (intracellular) fluorescence intensity determined for primary human endothelial cells without antibodies.

[0015] Furthermore, the present invention includes a method for selecting one or more antibodies from a plurality of antibodies that have a low relative nonspecific (non-target-mediated) clearance (rate), the method comprising the following steps: a) Each of a plurality of antibodies was incubated with primary human endothelial cells for the same defined time, and thereafter the intracellular fluorescence intensity (change) of the primary human endothelial cells was determined (i.e. the change in fluorescence intensity was determined), wherein each antibody was conjugated to the same pH-sensitive fluorescent dye; b) Select one or more antibodies from a large number of antibodies that, after incubation, result in the lowest possible (intracellular) fluorescence intensity (change) in primary human endothelial cells. This allows for the selection of one or more antibodies with low relative nonspecific (non-target-mediated) clearance (rate).

[0016] Furthermore, the present invention includes a method for ranking numerous antibodies based on their nonspecific (non-target-mediated) clearance (rate), comprising the following steps: a) Each of a number of antibodies was incubated with primary human endothelial cells for the same defined time, and thereafter, the fluorescence intensity (change) of the primary human endothelial cells (intracellular) was determined, wherein each antibody was conjugated to the same pH-sensitive fluorescent dye; b) Sort antibodies based on changes in (intracellular) fluorescence intensity from low to high or from high to low. Antibodies are thus ranked based on their non-specific (non-target-mediated) clearance (rate).

[0017] Furthermore, the present invention includes a method for estimating or determining the (relative) in vivo clearance rate of an antibody in a human, cynomolgus monkey, or mouse, comprising the following steps: a) Incubate primary human endothelial cells with an antibody conjugated to a pH-sensitive fluorescent dye for a specified time, and thereafter determine the (intracellular) fluorescence intensity (change) of the primary human endothelial cells. b) Incubate primary human endothelial cells with at least a first reference antibody for the same time defined in a), wherein the clearance rate of the at least first reference antibody in humans, cynomolgus monkeys, or mice is known, and the at least first reference antibody is conjugated to a pH-sensitive fluorescent dye (in one preferred embodiment as in a), and thereafter, determine the (intracellular) fluorescence intensity (change) of the primary human endothelial cells. The relative in vivo clearance of the antibody in humans, cynomolgus monkeys, or mice was estimated or determined as the clearance of the first reference antibody in humans, cynomolgus monkeys, or mice multiplied by the ratio of the fluorescence intensity (change) determined in a) to the fluorescence intensity (change) determined in b) (intracellular).

[0018] In some embodiments, step b) is

[0019] b) i) Incubate each member of a plurality of reference antibodies (i.e., at least two) with primary human endothelial cells for the same time defined in a), wherein the clearance rate of the reference antibody in humans, cynomolgus monkeys, or mice is known, and the reference antibody is conjugated to a pH-sensitive fluorescent dye (in one preferred embodiment as in a). ii) Subsequently, for each of the reference antibodies, the (intracellular) fluorescence intensity (changes) in primary human endothelial cells was determined, and iii) For the value obtained in ii), calculate the formula y = a The best-fit line is x + b, where y is the clearance rate in ml / day / kg and x corresponds to the fluorescence intensity (change).

[0020] In all aspects and in one embodiment of each embodiment, the fluorescence intensity (change) (intracellular) is the geometric mean fluorescence intensity (change).

[0021] In all aspects and in one embodiment of each example, the (intracellular) fluorescence intensity (change) of the corresponding antibody under discussion is a relatively normalized (intracellular) fluorescence intensity (change) rate obtained in a further step c), which includes: 1) For the antibody in question and at least two reference antibodies, determine the (geometric mean) (intracellular) fluorescence intensity after two or more specified incubation times, wherein in a preferred embodiment, the determination is made at least for two time points after incubation times of 2 hours and 4 hours; 2) Subtract the geometric mean (intracellular) fluorescence intensity of primary human endothelial cells (incubated for the same time but without the antibody) from each of the determined (geometric mean) (intracellular) fluorescence intensities for each of the antibodies in question and the reference antibody in 1) to obtain the corrected (geometric mean) (intracellular) fluorescence intensity. 3) Divide the corrected (geometric mean) (intracellular) fluorescence intensity of the antibody in question and the reference antibody obtained in 2) by the number of fluorescent dye molecules present in the respective antibody to obtain (e.g., the normalized (geometric mean) (intracellular) fluorescence intensity of at least two reference antibodies or the antibody in question). 4) Based on a set of normalized (geometric mean) (intracellular) fluorescence intensities calculated as in 3) for at least two different incubation times for the antibody (i.e., for each individual), including the origin, determine the best-fit line (i.e., the linear regression curve y=s) for each of the antibodies in question and the reference antibody. x + b, where y = normalized (geometric mean) fluorescence intensity (intracellular), s = slope, x = time, and b = slope of the y-axis intersection point; 5) The slope of the best-fit line for the antibody under discussion is normalized as follows: Normalized slope (antibody under discussion) =

[0022] In one embodiment of all aspects and examples, incubated primary human endothelial cells are washed (to remove nonspecific / extracellular surface-bound and unbound antibodies) before measuring (intracellular) fluorescence.

[0023] In all aspects and in one embodiment of each example, the dye exhibits a fluorescence intensity variation of about 10 times, preferably about 25 times, and most preferably about 50 times between a physiological pH of about 7 and an acidic pH in the range of pH 4 to 5. In some embodiments, the dye has a pHAb of Formula I / is Formula I.

[0024] (Formula I).

[0025] The conjugation with the antibody or adapter (if present) is located at residue R of Formula I.

[0026] In all aspects and in one embodiment of each example, the dye is conjugated to the antibody at amino acid residue 297 (according to Kabat number) in the Fc region.

[0027] In all aspects and in one embodiment of each example, the dye is chemically conjugated to the antibody via click chemistry.

[0028] In all aspects and in one embodiment of each example, the dye is conjugated to the antibody directly or via a linker. In some embodiments, the linker is a sulfonylDBCO-PEG4-amine of formula II.

[0029] (Formula II)

[0030] The conjugation with the antibody occurs at the free amino group of formula II.

[0031] In all aspects and in one embodiment of each example, the dye is conjugated to the adapter and the adapter is conjugated to the antibody and the conjugate has the structure of Formula III.

[0032] (Formula III)

[0033] In all aspects and in one embodiment of each example, the dye is chemically crosslinked to the antibody.

[0034] In all aspects and in one embodiment of each example, fluorescence is determined by means of FACS by determining the shift of the fluorescence maximum value.

[0035] In all aspects and in one embodiment of each example, fluorescence is the geometrically average fluorescence intensity determined by FACS.

[0036] In all aspects and in one embodiment of each example, the primary human endothelial cells are primary human liver endothelial cells.

[0037] In all aspects and in one embodiment of each embodiment, it is determined that the incubation is carried out after at least 0.5 hours, i.e., the specified time is at least 0.5 hours.

[0038] In all aspects and in one embodiment of each embodiment, it is determined that the incubation will proceed after a maximum duration of 24 hours, i.e., the specified time is a maximum of 24 hours. In some embodiments, it is determined that the incubation will proceed after a maximum duration of 16 hours. In a preferred embodiment, it is determined that the incubation will proceed after a maximum duration of 4 hours, i.e., the specified time is a maximum of 4 hours. In some embodiments, it is determined that the incubation will proceed after a duration of 2 hours and / or 4 hours, i.e., the specified time is 2 hours and / or 4 hours. In some embodiments, it is determined that the incubation will proceed after a duration of 4 to 24 hours, i.e., the specified time is between 4 hours and 24 hours and includes 4 hours to 24 hours. In some embodiments, it is determined that the incubation will proceed after a duration of 4 hours and / or 8 hours, i.e., the specified time is 4 hours and / or 8 hours.

[0039] In some embodiments, it is determined that the process is performed directly after incubation.

[0040] In all aspects and in one embodiment of each example, the antibody has a human-derived Fc region. In some embodiments, the Fc region belongs to a human IgG1, IgG2, or IgG4 subclass. In some embodiments, the Fc region contains one or more mutations that affect binding to human FcRn.

[0041] In all aspects and in one embodiment of each example, the antibody is a fusion of an antibody having an additional polypeptide. In some embodiments, the additional polypeptide is scFv, Fab, scFab, or a non-antibody polypeptide. In some embodiments, the fusion is located at the C-terminus of one of the heavy chains of the antibody.

[0042] In all aspects and in one embodiment of each example, the antibody is a bispecific antibody.

[0043] In all aspects and in one embodiment of each example, the first reference antibody is a movizumab with the mutant M252Y / S254T / T256E, and / or a bispecific antibody in the form of a TCB. Attached Figure Description

[0044] Figure 1. Time course of fluorescence intensity of different antibodies labeled with the same pH-sensitive fluorescent dye during incubation with human microvascular endothelial cells; 1 = Anti-human phosphorylated Tau 422 antibody; 2 = Anti-CD44 antibody; 3 = Olamumab; 4 = Anti-CD20 antibody (1); 5 = Acimenab; 6 = Anti-human α-synuclein antibody; 7 = Anti-CD20 antibody (2).

[0045] Figure 2. Time course of fluorescence intensity of different antibodies labeled with the same pH-sensitive fluorescent dye during incubation with human primary hepatic endothelial cells; 1 = Anti-human phosphorylated Tau 422 antibody; 2 = Anti-CD44 antibody; 3 = Olamumab; 4 = Anti-CD20 antibody (1); 5 = Acimenab; 6 = Anti-human α-synuclein antibody; 7 = Anti-CD20 antibody (2).

[0046] Figure 3 shows the scheme of fluorescently labeled antibody used in the method according to the invention; pHAb dye is conjugated to the antibody via a sulfonyl DBCO-PEG4-Amine linker.

[0047] Figure 4 shows a scheme according to the method of the present invention.

[0048] Figure 5 shows the corrected mean fluorescence intensity (MFI, more specifically, geometric mean) of internalized antibodies obtained using FACS, obtained by subtracting the negative control and then normalizing (dividing) by the dye-antibody ratio (DAR). The corrected and normalized geometric means from each antibody were plotted as linear regression curves, and the slopes were extracted (geometric mean MFI / min for 120 and 240 minutes). Two standard antibodies were selected to normalize the slopes: murvizumab-YTE set to 0 and TCB set to 1. The final slopes were plotted against in vivo human clearance values. If different clearance values ​​were obtained, dose-linear clearance describing molecular nonspecific clearance was used.

[0049] Figure 6 shows the corrected mean fluorescence intensity (MFI, more specifically, geometric mean) of internalized antibodies obtained using FACS, obtained by subtracting the negative control and then normalizing (dividing) by the dye-antibody ratio (DAR). The corrected and normalized geometric means from each antibody were plotted as linear regression curves, and the slopes were extracted (geometric mean MFI / min for 120 and 240 min). Two standard antibodies were selected to normalize the slopes: muvizumab-YTE set to 0 and TCB set to 1. The final slopes were plotted against in vivo cynomolgus monkey clearance values. If different clearance values ​​were obtained, dose-linear clearance describing molecular nonspecific clearance was used.

[0050] Figure 7 shows the corrected mean fluorescence intensity (MFI, more specifically the geometric mean) of internalized antibodies obtained using FACS, obtained by subtracting the negative control and then normalizing (dividing) by the dye-antibody ratio (DAR). The corrected and normalized geometric means from each antibody were plotted as linear regression curves, and the slopes were extracted (geometric mean MFI / min for 120 and 240 minutes). Two standard antibodies were selected to normalize the slopes: murvizumab-YTE set to 0 and TCB set to 1. The final slopes were plotted against in vivo hFcRn Tg32+ / + mouse clearance values.

[0051] Figure 8. The Fc variant of IgG shows the same in vitro-in vivo correlation as wt Fc IgG.

[0052] Figure 9. Time course of mean fluorescence intensity of primary human endothelial cells incubated with monospecific bivalent antibody.

[0053] Figure 10. Flow cytometry analysis of primary human liver-derived endothelial cells. Endothelial cells were incubated with antibodies and pre-labeled with pHAb amine-reactive dye (532 nm): low-clearance antibody movizumab-YTE (solid line), two medium-clearance bispecific antibodies (dotted and dashed lines, respectively), and high-clearance bispecific antibody (dotted and dashed lines). After 4 hours, fluorescence intensity was recorded and cell singlet state, morphology, and viability were gated. y-axis scaling is relative to the number of events. The x-axis scaling displays the intensity in the PE channel. Detailed Implementation

[0054] This invention is based in at least part on the discovery that cell-based assays using primary human endothelial cells can be used to estimate the in vivo lysosomal degradation rate of therapeutic antibodies in vitro.

[0055] By using primary human cells, the inventors of this application have discovered a significant correlation between readout of the method according to the invention and nonspecific clearance in the human body. This has been demonstrated for more than 20 therapeutic antibodies that are in clinical trials or already on the market. The inventors of this application have further discovered that the method according to the invention is equally applicable to conventional bispecific antibodies, monoclonal antibodies that reflect the Y-shape of wild-type human antibodies, and to unconventional bispecific antibody-type human antibodies that have a different form from the wild-type and more than two valences and antibody Fc region fusions. This provides evidence for the general applicability of the method according to the invention to the correlation between readout and clearance rates in mice, cynomolgus monkeys, and humans.

[0056] The method according to the invention can be used to estimate the pharmacokinetic (PK) characteristics of different antibody molecules (with different forms, valences, and specificities).

[0057] Therefore, the method according to the present invention can be used

[0058] - Supports the selection of appropriate clinical lead molecules based on PK (pharmacokinetic) properties (clearance and half-life, respectively); - Remove antibodies from the library that have PK properties that are unsuitable for therapeutic applications, i.e., antibodies with high clearance or short in vivo half-life, respectively. - The members of a group of antibodies are sorted according to their PK characteristics (clearance rate and half-life, respectively); - The relative in vivo clearance of the antibody in question is determined solely based on the in vivo clearance of a reference antibody (or many reference antibodies) with known PK properties, i.e., no in vivo testing is required; - Guide antibody engineering with PK properties (by altering the affinity of FcRn in the Fc region or by engineering charged plaques in the Fab (the latter described in WO 2018 / 197533). - Identify the requirements for PK engineering and evaluate the results of PK engineering.

[0059] Therefore, the assays according to the present invention can reduce or even replace animal PK studies.

[0060] I. Definition

[0061] As used herein, the amino acid positions of all constant regions and domains of the heavy and light chains are numbered according to the Kabat numbering system described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991), and are referred to herein as “according to Kabat numbering”. Specifically, the Kabat numbering system (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991), pp. 647–660) is used for the constant domains CL of the κ and λ isoforms of the light chain, and Kabat’s EU index numbering system (see pp. 661–723) is used for the constant heavy chain domains (CH1, hinge, CH2, and CH3, which are further classified herein by way of “according to Kabat’s EU index numbering”).

[0062] The mortar-and-pot structure dimer module and its application in antibody engineering are described in Carter P., Ridgway JBB, Presta LG: Immunotechnology, February 1996, Vol. 2, No. 1, pp. 73-73(1).

[0063] General information about the nucleotide sequences of the light and heavy chains of human immunoglobulins is given in: Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0064] Methods and techniques that can be used to carry out the present invention are described, for example, in the following literature: Ausubel, FM (ed.), Current Protocols in Molecular Biology, Volumes I through III (1997); Glover, ND and Hames, BD (eds.), DNA Cloning: A Practical Approach, Volumes I and II (1985), Oxford University Press; Freshney, RI (ed.), Animal Cell Culture – apractical approach, IRL Press Limited (1986); Watson, JD et al., RecombinantDNA, 2nd ed., CHSL Press (1992); Winnacker, EL, From Genes to Clones; NY, VCHPublishers (1987); Celis, J. (ed.), Cell Biology, 2nd ed., Academic Press (1998); Freshney, RI, Culture of Animal Cells: A Manual of Basic Technique, 2nd ed., Alan R. Liss, Inc. NY (1987).

[0065] Recombinant DNA technology can be used to generate nucleic acid derivatives. Such derivatives can be modified, for example, at one or several nucleotide positions by substitution, alteration, exchange, deletion, or insertion. Modification or derivatization can be performed, for example, by site-directed mutagenesis. Such modifications can be readily performed by those skilled in the art (see, for example, Sambrook, J. et al., Molecular Cloning: A laboratory manual (1999) Cold Spring Harbor Laboratory Press, New York, USA; Hames, BD and Higgins, SG, Nucleic acid hybridization – a practical approach (1985) IRL Press, Oxford, England).

[0066] It is important to note that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly specifies otherwise. Thus, for example, reference to “a cell” includes a plurality of such cells and their equivalents known to those skilled in the art, and so on. Similarly, the terms “a,” “one or more,” and “at least one” are used interchangeably herein. It should also be noted that the terms “comprising,” “including,” and “having” are used interchangeably.

[0067] The term "about" indicates a range of + / - 20% of the value that follows it. In some embodiments, the term "about" indicates a range of + / - 10% of the value that follows it. In some embodiments, the term "about" indicates a range of + / - 5% of the value that follows it.

[0068] As used in this article, the term “determine” also includes term measurement and analysis.

[0069] The term "contains" also includes the term "composes of".

[0070] The term “antibody” is used in the broadest sense and includes a variety of antibody structures, including but not limited to monoclonal antibodies and multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies), as long as they are full-length antibodies and exhibit the desired antigen and / or FcRn binding activity.

[0071] "Multispecific antibody" refers to an antibody that has binding specificity for at least two different epitopes on the same antigen or for two different antigens. Multispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibody) or combinations thereof (e.g., a full-length antibody with an additional scFv or Fab fragment). Engineered antibodies having two, three, or more (e.g., four) functional antigen-binding sites have also been reported (see, for example, US 2002 / 0004587A1).

[0072] The term "binding (to antigen)" refers to the binding of an antibody in an in vitro assay. In some embodiments, binding is determined in a binding assay, wherein the antibody binds to a surface, and the binding of the antigen to the antibody is measured by surface plasmon resonance (SPR). The term "binding" also includes the term "specific binding".

[0073] The term "buffer substance" refers to a substance in solution that can regulate the pH value of a solution, for example, due to the addition or release of acidic or alkaline substances.

[0074] An antibody's "class" refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these antibodies can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The constant domains of the heavy chain corresponding to different classes of immunoglobulins are respectively called α, β, γ ... , , and µ.

[0075] The term "Fc-fusion polypeptide" refers to a fusion of a binding domain (e.g., an antigen-binding domain, such as a single-chain antibody, or a polypeptide, such as a receptor ligand) with an antibody Fc region that exhibits the desired targeting and / or protein A and / or FcRn binding activity.

[0076] The term "human Fc region" refers to the C-terminal region of a human immunoglobulin heavy chain, containing at least a portion of a hinge region, a CH2 domain, and a CH3 domain. In some embodiments, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the C-terminus of the heavy chain. In some embodiments, the Fc region has the amino acid sequence of SEQ ID NO: 05. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. The Fc region consists of two heavy chain Fc region polypeptides that can be covalently linked to each other via hinge region cysteine ​​residues, forming interchain disulfide bonds.

[0077] The term "FcRn" refers to the human neonatal Fc receptor. The function of FcRn is to rescue IgG from lysosomal degradation, resulting in decreased clearance and increased half-life. FcRn is a heterodimeric protein composed of two polypeptides: a 50 kDa class I major histocompatibility complex-like protein (α-FcRn) and a 15 kDa β2-microglobulin (β2m). FcRn binds with high affinity to the CH2-CH3 portion of the Fc region of IgG. The interaction between IgG and FcRn is strictly pH-dependent and occurs in a stoichiometric ratio of 1:2, with one IgG molecule binding to two FcRn molecules via its two heavy chains (Huber, AH et al., J. Mol. Biol. 230 (1993) 1077-1083). FcRn binding occurs in the endosome at acidic pH (pH < 6.5), while IgG is released at the neutral cell surface (pH approximately 7.4). This pH-sensitive interaction promotes FcRn-mediated protection of endocytosed IgG from intracellular degradation by binding to the receptor in the acidic environment of the endosome. FcRn then promotes IgG recycling to the cell surface, subsequently releasing it into the bloodstream when the FcRn-IgG complex is exposed to the extracellular neutral pH environment.

[0078] The term "FcRn binding portion of the Fc region" refers to the following portions of the antibody heavy chain polypeptide: approximately from EU position 243 to EU position 261, approximately from EU position 275 to EU position 293, approximately from EU position 302 to EU position 319, approximately from EU position 336 to EU position 348, approximately from EU position 367 to EU position 393 and EU position 408, and approximately from EU position 424 to EU position 440. In some embodiments, according to the EU numbering of Kabat, one or more of the following amino acid residues are modified F243, P244, P245. P, K246, P247, K248, D249, T250, L251, M252, I253, S254, R255, T256, P257, E25 8. V259, T260, C261, F275, N276, W277, Y278, V279, D280, V282, E283, V284, H28 5. N286, A287, K288, T289, K290, P291, R292, E293, V302, V303, S304, V305, L30 6. T307, V308, L309, H310, Q311, D312, W313, L314, N315, G316, K317, E318, Y31 9. I336, S337, K338, A339, K340, G341, Q342, P343, R344, E345, P346, Q347, V34 8. C367, V369, F372, Y373, P374, S375, D376, I377, A378, V379, E380, W381, E38 2. S383, N384, G385, Q386, P387, E388, N389, Y391, T393, S408, S424, C425, S426, V427, M428, H429, E430, A431, L432, H433, N434, H435, Y436, T437, Q438, K439 and S440 (EU numbers).

[0079] The term "full-length antibody" refers to an antibody having a structure substantially similar to that of a natural antibody. A full-length antibody comprises two full-length antibody light chains and two full-length antibody heavy chains. The two full-length antibody light chains contain a light chain variable domain and a light chain constant domain. The two full-length antibody heavy chains contain a heavy chain variable domain, a first constant domain, a hinge region, a second constant domain, and a third constant domain. Full-length antibodies may contain other domains, such as, for example, additional scFv or scFab conjugated to one or more chains of the full-length antibody. These conjugates are also covered by the term full-length antibody.

[0080] The term "derived from" means that the amino acid sequence is derived from the parent amino acid sequence by introducing a change at at least one position. Therefore, the derived amino acid sequence differs from the corresponding parent amino acid sequence at at least one corresponding position (numbered according to the Kabat EU index of the antibody Fc region). In some embodiments, the amino acid sequence derived from the parent amino acid sequence differs from the corresponding position by 1 to 15 amino acid residues. In some embodiments, the amino acid sequence derived from the parent amino acid sequence differs from the corresponding position by 1 to 10 amino acid residues. In some embodiments, the amino acid sequence derived from the parent amino acid sequence differs from the corresponding position by 1 to 6 amino acid residues. Similarly, the derived amino acid sequence has high amino acid sequence identity with its parent amino acid sequence. In some embodiments, the amino acid sequence derived from the parent amino acid sequence has 80% or more amino acid sequence identity. In some embodiments, the amino acid sequence derived from the parent amino acid sequence has 90% or more amino acid sequence identity. In some embodiments, the amino acid sequence derived from the parent amino acid sequence has 95% or more amino acid sequence identity.

[0081] The term "human Fc region polypeptide" refers to a polypeptide with the same amino acid sequence as a "natural" or "wild-type" human Fc region polypeptide. The term "variant (human) Fc region polypeptide" refers to a polypeptide derived from a "natural" or "wild-type" human Fc region polypeptide whose amino acid sequence differs by at least one "amino acid alteration." A "human Fc region" consists of two Fc region polypeptides. A "variant (human) Fc region" consists of two Fc region polypeptides, where both can be variant (human) Fc region polypeptides, or one can be a human Fc region polypeptide and the other a variant (human) Fc region polypeptide.

[0082] "Humanized" antibodies refer to chimeric antibodies that contain amino acid residues from non-human HVRs and amino acid residues from human FRs. In some embodiments, the humanized antibody will substantially contain at least one of these variable domains, typically two variable domains, wherein all or substantially all HVRs (e.g., CDRs) correspond to the HVRs of the non-human antibody, and all or substantially all FRs correspond to the FRs of the human antibody. Optionally, the humanized antibody may contain at least a portion of the antibody constant region derived from the human antibody. "Humanized form" antibodies, such as non-human antibodies, refer to antibodies that have already been humanized.

[0083] "Isolated" antibodies are antibodies that have been separated from components of their natural environment. In some embodiments, antibodies are purified to a purity greater than 95% or 99%, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., size exclusion chromatography, ion exchange, or reversed-phase HPLC). For a review of methods used to assess, for example, antibody purity, see Flatman, S. et al., J. Chrom. B 848 (2007) 79-87.

[0084] "Isolated" nucleic acids refer to nucleic acid molecules that have been isolated from components of their natural environment. Isolated nucleic acids include nucleic acid molecules that are contained in cells that normally contain nucleic acid molecules, but which are located outside chromosomes or at chromosomal locations different from their natural chromosomal locations.

[0085] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous group of antibodies, meaning that, apart from possible variant antibodies (e.g., those containing naturally occurring mutations or generated during the production of a monoclonal antibody formulation, such variants are typically presented in small quantities), the individual antibodies comprising this group are identical and / or bind to the same epitopes. In contrast to polyclonal antibody formulations, which typically comprise different antibodies targeting different determinants (epitaxes), each monoclonal antibody in a monoclonal antibody formulation targets a single determinant on the antigen. Therefore, the modifier "monoclonal" indicates that the antibody is characterized by being obtained from a substantially homogeneous group of antibodies and should not be construed as requiring the antibody to be produced by any particular method. For example, monoclonal antibodies used according to the invention can be prepared by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for preparing monoclonal antibodies are described herein.

[0086] "Natural antibodies" refer to naturally occurring immunoglobulin molecules with different structures. For example, natural IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 Daltons, composed of two identical light chains and two identical heavy chains bonded by disulfides. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called a variable heavy chain domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called a variable light chain domain or light chain variable domain, followed by a constant light chain (CL) domain. The light chains of antibodies can be classified into one of two types based on the amino acid sequence of their constant domains, called kappa (κ) and lamuda (λ).

[0087] The term "pharmaceutical formulation" refers to a formulation in which the bioactive ingredient contained therein is in a form in which the activity is effective and which does not contain any additional components that would have unacceptable toxicity to a subject to whom the formulation will be administered.

[0088] "Pharmaceutical carriers" refer to components in a pharmaceutical preparation that are non-toxic to the test subject, excluding the active ingredient. Pharmaceutical carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0089] As used herein, the term "recombinant antibody" refers to all antibodies (chimeric antibodies, humanized antibodies, and human antibodies) prepared, expressed, created, or isolated by recombinant means. This includes antibodies isolated from host cells such as NSO, HEK, BHK, or CHO cells, or from transgenic animals (e.g., mice) carrying human immunoglobulin genes, or antibodies expressed using recombinant expression plasmids transfected into host cells. Such recombinant antibodies have variable and constant regions in rearranged form. Recombinant antibodies, as reported herein, may be subject to in vivo somatic hypermutation. Therefore, the amino acid sequences of the VH and VL regions of recombinant antibodies are the sequences described below, which, although derived from and associated with human germline VH and VL sequences, may not be present in the in vivo human antibody germline under natural conditions.

[0090] As used herein, the term "TCB" refers to a T-cell bispecific antibody. Such antibodies can have, for example, the forms described in WO2013 / 026831. These molecules can simultaneously bind to CD3 on T cells (primary specificity) and antigens on target (e.g., tumor) cells (secondary specificity), thereby inducing the killing of target cells. A TCB is a trivalent bispecific antibody composed of four polypeptides or polypeptide chains: a full-length light chain; another light chain, a domain-exchanged full-length light chain; a full-length heavy chain; and a third heavy chain, an extension of the heavy chain containing additional domain-exchanged heavy chains or light chain Fab fragments.

[0091] In a preferred embodiment, the TCB includes: a) The first Fab fragment and the second Fab fragment, each binding to the first antigen, b) A domain-exchange Fab fragment that specifically binds to a second antigen, in which the CH1 and CL domains exchange with each other. c) An Fc region comprising a first-heavy-chain Fc region polypeptide and a second-heavy-chain Fc-region polypeptide. The C-terminus of the CH1 domain of the first Fab fragment is attached to the N-terminus of one of the heavy chain Fc region peptides, and the C-terminus of the CL domain of the domain-exchange Fab fragment is attached to the N-terminus of another heavy chain Fc region peptide. The C-terminus of the CH1 domain of the second Fab segment connects to the N-terminus of the VH domain of the first Fab segment, or connects to the N-terminus of the VH domain of the domain-swapping Fab segment. The first or second antigen is human CD3.

[0092] In another equally preferred embodiment, the TCB includes: a) The first Fab fragment and the second Fab fragment, each binding to the first antigen, b) A domain-exchange Fab fragment that specifically binds to a second antigen, in which the VH and VL domains exchange with each other. c) An Fc region comprising a first-heavy-chain Fc region polypeptide and a second-heavy-chain Fc-region polypeptide. The C-terminus of the CH1 domain of the first Fab fragment is attached to the N-terminus of one of the heavy chain Fc region peptides, and the C-terminus of the CH1 domain of the domain-exchange Fab fragment is attached to the N-terminus of another heavy chain Fc region peptide. The C-terminus of the CH1 domain of the second Fab segment connects to the N-terminus of the VH domain of the first Fab segment, or connects to the N-terminus of the VL domain of the domain-exchanged Fab segment. The first or second antigen is human CD3.

[0093] As used herein, the term "valence" indicates the presence of a specified number of binding sites in the (antibody) molecule. Therefore, the terms "bivalent," "tetravalent," and "hexavalent" respectively indicate the presence of two, four, and six binding sites in the (antibody) molecule. The bispecific antibody reported herein is a preferred embodiment of "bivalent."

[0094] The term "variable region" or "variable domain" refers to a domain of the antibody heavy or light chain involved in the binding of the antibody to its antigen. The variable domains (VH and VL, respectively) of the antibody heavy and light chains typically have similar structures, with each domain containing four frame regions (FRs) and three hypervariable regions (HVRs) (see, for example, Kindt, TJ et al., Kuby Immunology, 6th ed., WH Freeman and Co., NY (2007), p. 91). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies binding to a specific antigen can be isolated using either the VH or VL domain from the antibody binding to that antigen to screen libraries containing complementary VL or VH domains. See, for example: Portolano, S. et al., J. Immunology. . 150 (1993) 880-887; Clackson, T. et al., Nature 352 (1991) 624-628.

[0095] The terms "variant," "modified antibody," and "modified fusion peptide" refer to molecules having an amino acid sequence that differs from that of the parent molecule. Typically, such molecules have one or more alterations, insertions, or deletions. In some embodiments, the modified antibody or modified fusion peptide comprises an amino acid sequence that includes at least a portion of a non-naturally present Fc region. Such molecules have less than 100% sequence identity with the parent antibody or parental fusion peptide. In some embodiments, the amino acid sequence of the variant antibody or variant fusion peptide has from about 75% to less than 100% amino acid sequence identity with the amino acid sequence of the parent antibody or parental fusion peptide, particularly from about 80% to less than 100%, particularly from about 85% to less than 100%, particularly from about 90% to less than 100%, particularly from about 95% to less than 100%. In some embodiments, the parent antibody or parental fusion peptide and the variant antibody or variant fusion peptide differ by one (single), two, or three amino acid residues.

[0096] Primary human endothelial cells are human cells isolated directly from their source, organ, tissue, or blood using enzymatic or mechanical methods. Primary cells are not immortal. Once isolated, they are placed in an artificial environment, such as, for example, in plastic or glass containers in a specialized culture medium containing essential nutrients and growth factors to support proliferation. Primary cells can be of two types: adherent cells or suspension cells. Adhesive cells require attachment to grow and are called anchorage-dependent cells. Adhesive cells are typically derived from organ tissues. Suspension cells do not require attachment to grow and are called anchorage-independent cells. Most suspension cells are isolated from blood.

[0097] The term "pH-sensitive fluorescent dye" refers to a dye that exhibits different fluorescence intensities or emission wavelengths at physiological pH (approximately 7.4) and lysosomal pH (approximately 4.5).

[0098] II. Antibodies in vivo

[0099] Because IgG molecules are divalent, a single IgG molecule can neutralize up to two antigen molecules. There are two types of target antigens for neutralizing antibodies: soluble antigens present in plasma and membrane-bound antigens expressed on the cell surface.

[0100] In cases where the antigen is a membrane-bound antigen, the applied therapeutic antibody binds to the membrane-bound antigen on the cell surface. Subsequently, the antibody is absorbed into the endosome within the cell by internalizing the antibody-bound membrane-bound antigen. Thereafter, the antibody, still bound to the antigen, moves to the lysosome, where it is degraded along with the antigen. The elimination of antibodies from the plasma mediated by internalization of membrane-bound antigens is called antigen-dependent elimination. This has been reported for different antibody molecules (see, for example, Drug Discov. Today, 11 (2006) 81-88). Because a single IgG antibody molecule binds to two antigen molecules when it is bivalent to the antigen, and is then internalized by the lysosome and directly degraded, a single ordinary IgG antibody cannot neutralize two or more antigen molecules.

[0101] The reason IgG molecules are retained in plasma for a long time (slowly eliminated) is due to FcRn, called the IgG molecule rescue receptor (see, for example, Nat. Rev. Immunol. 7 (2007) 715-725). IgG molecules that have been absorbed into the endosome via endocytosis bind to FcRn expressed in the endosome under acidic conditions. IgG molecules bound to FcRn move to the cell surface, where they dissociate from FcRn under neutral conditions in the plasma. IgG molecules that cannot bind to FcRn enter the lysosome, where they are degraded.

[0102] When IgG antibodies are absorbed into endosomes within cells via internalization, they dissociate from the antigen under acidic conditions within the endosome. The dissociated antibody can then bind to FcRn, which is also present in the endosome. Therefore, IgG molecules dissociated from the antigen and bound to FcRn are transferred to the cell surface and released from FcRn into the plasma under neutral pH conditions. This recirculates the antibody back into the plasma. The IgG molecules returning to the plasma can then bind to new antigens again. This repeated process allows a single IgG molecule to repeatedly bind to the antigen, thus enabling the neutralization of multiple antigens with a single IgG molecule.

[0103] In the case of soluble antigens, the administered therapeutic antibody binds to the antigen in the plasma and remains there as an antigen-antibody complex. Similar to the case of IgG molecules that do not bind to antigens, IgG molecules bound to antigens in the plasma are absorbed into the endosome via pinocytosis. In the endosome, they can bind to FcRn expressed in the endosome under acidic conditions. IgG molecules bound to FcRn migrate to the cell surface and then dissociate from FcRn under neutral conditions in the plasma. If IgG molecules can dissociate from antigens under acidic conditions in the endosome, the dissociated antigen cannot bind to FcRn and can thus be degraded by lysosomes. Since the IgG molecules that have returned to the plasma have dissociated from the antigens in the endosome, they are able to bind to new antigens in the plasma again. This repetition of the process allows a single IgG molecule to repeatedly bind to soluble antigens. This enables a single IgG molecule to neutralize multiple antigens.

[0104] Therefore, regardless of whether the antigen is membrane-bound or soluble, if the dissociation of IgG antibodies from the antigen is possible under acidic conditions in the body, a single IgG molecule can repeatedly neutralize the antigen.

[0105] More specifically, a single IgG molecule binds strongly to antibodies at the cell surface at pH 7.4, but weakly to antigens at the in vivo pH of 5.5 to 6.0, which may neutralize multiple antigens and thus improve pharmacokinetics (it has been reported that the in vivo pH is usually pH 5.5 to 6.0 (see, for example, Nat. Rev. Mol. Cell. Biol. 5(2004) 121-132)).

[0106] Generally, protein-protein interactions consist of hydrophobic interactions, electrostatic interactions, and hydrogen bonding, and the binding strength is usually expressed as a binding constant (affinity) or an apparent binding constant (affinity). pH-dependent binding, where the binding strength varies between neutral (pH 7.4) and acidic (pH 5.5 to 6.0) conditions, is present in naturally occurring protein-protein interactions. For example, the binding between the aforementioned IgG molecule and the FcRn, a rescue receptor known as the IgG molecule, is strong under acidic conditions (pH 5.5 to 6.0) but significantly weak under neutral conditions (pH 7.4). The pH-dependent binding of the aforementioned IgG-FcRn interaction has been reported to be associated with histidine residues present in IgG (see, for example, Mol. Cell. 7 (2001) 867-877).

[0107] III. The method according to the present invention

[0108] This article reports a novel method for estimating the clearance of therapeutic antibodies in humans using a new in vitro assay based on primary human cells. This macromolecular nonspecific clearance assay (LUCA) provides an in vitro-based approach to assess and predict the pharmacokinetic (PK) properties of therapeutic antibodies.

[0109] The present invention is based at least in part on the discovery that the sum of antibodies taken up in vitro and recycled into primary human endothelial cells can be used as a substitute for estimating the nonspecific clearance of said antibodies in vivo.

[0110] This invention is based at least in part on the finding that only primary human endothelial cells can be used to predict in vivo clearance rates based on in vitro experiments, because non-primary endothelial cells do not show the same correlation and are therefore unsuitable for this purpose. Using the aforementioned non-primary endothelial cells, differentiation between different antibodies cannot be achieved (compare Figures 1 and 2). Figure 4 depicts a scheme of the method according to the invention.

[0111] Therefore, the present invention includes a method for determining or estimating the nonspecific (non-target-mediated) clearance (rate) of an antibody, comprising the following steps: a) Incubate antibodies conjugated to pH-sensitive fluorescent dyes with primary human endothelial cells, and b) Determine the intracellular fluorescence intensity of the primary human endothelial cells from step a) (after the specified incubation time). The increase in the intracellular fluorescence intensity of primary human endothelial cells determined in step b) relative to the background level (i.e., the intracellular fluorescence of primary human endothelial cells not incubated with the antibody) indicates nonspecific clearance by the antibody.

[0112] The present invention is based at least in part on the discovery that the sum of antibodies taken up in vitro and recycled into primary human endothelial cells can be used as a substitute for estimating the nonspecific clearance of said antibodies in vivo.

[0113] This invention is based, at least in part, on the finding that only primary human endothelial cells can be used to predict in vivo clearance from in vitro experiments, as non-primary endothelial cells do not show the same correlation and are therefore unsuitable for this purpose. Using the non-primary endothelial cells, differentiation between different antibodies cannot be achieved (see Figures 1 and 2).

[0114] This invention is based at least in part on the discovery that the uptake of antibodies by pinocytosis and their transport to the lysosomal compartment without being recycled by the FcRn of primary endothelial cells contributes most to the fluorescence of primary endothelial cells.

[0115] Figures 1 and 2 compare the effects of different antibodies on endothelial cells (human microvascular endothelial cells, HMEC1). Figure 1 The time course of fluorescence intensity during incubation with simple endothelial cells and with primary endothelial cells (human primary liver endothelial cells; Figure 2) shows the different antibodies labeled with the same pH-sensitive fluorescent dye. As can be seen from Figure 1, differentiation was impossible for five of the seven antibodies when using simple endothelial cells. Conversely, all seven antibodies differentiated when using primary endothelial cells (see Figure 2).

[0116] Labeled antibodies have been analyzed by heparin and FcRn chromatography. Exemplary retention times for unlabeled and labeled antibodies are shown in the table below. It can be seen that labeling does not alter the heparin and FcRn binding properties of the antibody. For antibodies reliable within the assays according to the invention, the expected difference from the geometric mean is less than 15%.

[0117] Table 1: Retention times of unlabeled and labeled antibodies on human heparin and human FcRn columns.

[0118]

[0119] The fluorescent label used in the method according to the invention can be any pH-dependent fluorescent dye with a fluorescence intensity shift of about 10 times, preferably about 25 times, and most preferably about 50 times between a physiological pH of about 7 and an acidic pH in the range of pH 4 to 5.

[0120] An exemplary suitable dye is the pHAb dye sold by Promega. These dyes are pH sensor dyes, exhibiting very low fluorescence at pH > 7, and a sharp increase in fluorescence as the pH of the solution becomes acidic. pHAb dyes have an excitation maximum (Ex) at 532 nm and an emission maximum (Em) at 560 nm. pHAb dyes are available in two reactive forms suitable for antibody conjugation: pHAb amine-reactive dyes and pHAb thiol-reactive dyes. pHAb amine-reactive dyes have a succinimide ester group that reacts with the primary amine on the lysine amino acid of the antibody. pHAb thiol-reactive dyes have a maleimide group that reacts with thiols. This maleimide group is expected to conjugate to the antibody after the cysteine ​​disulfide bond in the hinge region of the antibody is reduced to a thiol using a reducing agent such as DTT or TCEP. pHAb dyes retain their fluorescence response to decreasing pH after conjugation to the antibody.

[0121] An unsuitable dye is Invitrogen's Click-iT™ pHrodo™ iFL Red sDIBO alkyne. The fluorescence intensity of this dye changes only slightly when the pH value changes by a factor of 2 to 3.

[0122] A suitable connector is sulfonated DBCO-PEG4-Amine, sold by ClickChemistryTools. Sulfo DBCO-PEG4-Amine is a water-soluble reagent used to derivatize carboxyl-containing molecules or activated esters (e.g., NHS esters) with the DBCO moiety via a stable amide bond. The hydrophilic sulfonated spacer arm enhances the water solubility of the DBCO-derived molecule, making it completely soluble in aqueous media in many cases. The PEG spacer arm provides a long and flexible connection. Conjugation is achieved by activation of the antibody with an azide and reaction with the DBCO moiety using click chemistry.

[0123] In the following description, the invention is illustrated by example using pHAb dyes and conjugations using sulfoDBCO-PEG4-amine linkers. Any other dye exhibiting the aforementioned characteristics, linker, or conjugation chemistry that does not interfere with antibody binding properties, as well as the pH-dependent fluorescence properties of the dye, can also be used. This is presented merely as an example of the invention and should not be construed as limiting. The true scope is set forth in the appended claims.

[0124] The structure of the exemplary labeled antibody is shown in Figure 3.

[0125] The mean fluorescence intensity (MFI, more specifically, geometric mean fluorescence intensity) of the internalizing antibodies was obtained using FACS with an excitation wavelength of 488 nm and detection wavelengths of 585 / 540 nm. The exact same conditions, gain, and gate were used for all time points (i.e., 2 and 4 hours). Data extraction was performed using FloJo_V10 software. The negative control values ​​were subtracted from all geometric means and then normalized to the dye-antibody ratio (DAR). The normalized geometric means from each antibody were plotted as linear regression curves using GraphPad Prism to extract the slope (geometric mean MFI / min for 120 and 240 minutes, including the origin, i.e., 0 / 0). Two antibodies were used for normalizing the slope: murvizumab with the mutant M252Y / S254T / T256E was set to 0, and TCB was set to 1. These antibodies were chosen because they spanned a sufficient rate range. The final slopes were plotted using TIBCO Spotfire software for clearance values ​​in human, cynomolgus monkey, and hFcRn Tg32+ / + mice in the corresponding in vivo samples. Corresponding plots for human, cynomolgus monkey, and human FcRn transgenic mice with different antibodies, including those listed in Table 1, are shown in Figures 5 through 7.

[0126] Figure 8 shows that the method according to the invention can also be used to determine the in vivo clearance of Fc region variants of IgG. This further indicates that FcRn recycling was appropriately captured in the method according to the invention.

[0127] Figure 9 shows the dependence of fluorescence on incubation time. It can be seen that the linear range is at least as long as 24 hours.

[0128] In some embodiments, the method according to the invention is a method for estimating or determining the in vivo clearance rate of an antibody in a human, cynomolgus monkey, or mouse, the method comprising the following steps: a) Incubate primary human endothelial cells with an antibody conjugated to the same pH-sensitive fluorescent dye and at least first and second reference antibodies for at least 2 and 4 hours, respectively, and determine the geometric mean intracellular fluorescence intensity of the primary human endothelial cells for each incubation time. Optionally, wash the cells to remove adhering fluorescently labeled antibodies before determining the intracellular fluorescence intensity. b) At the same time point as a), determine the geometric mean intracellular fluorescence intensity of primary human endothelial cells that were not incubated with any labeled antibody, optionally washing the cells to remove adhering fluorescent compounds before determining the intracellular fluorescence intensity. c) Determine the relatively normalized intracellular fluorescence intensity rate using the following: i) For each of the geometric mean intracellular fluorescence intensities determined in a), subtract the geometric mean intracellular fluorescence intensity of primary human endothelial cells determined at the same time point from each of the geometric mean intracellular fluorescence intensities determined in a), to obtain the corrected geometric mean intracellular fluorescence intensity. ii) Divide the corrected geometric mean intracellular fluorescence intensity obtained in step 2) of the antibody under discussion and the reference antibody by the number of fluorescent dye molecules present in the corresponding antibody to obtain the normalized geometric mean intracellular fluorescence intensity. iii) Based on a set of values ​​consisting of the normalized geometric mean intracellular fluorescence intensity for each incubation time determined in a) as in ii), and the origin, determine the best-fit line (i.e., the linear regression curve y=s) for each of the antibodies in question and the reference antibody. x + b, where y = normalized geometric mean (intracellular) fluorescence intensity, s = slope, x = time and b = slope of the y-axis intersection point; iv) The slope of the best-fit line for the antibody under discussion is normalized as follows: Normalized slope (antibody under discussion) =

[0129] The in vivo clearance rate of the antibody in humans, cynomolgus monkeys, or mice is the clearance rate of the first reference antibody in humans, cynomolgus monkeys, or mice multiplied by the relatively normalized intracellular fluorescence intensity rate.

[0130] It has been found that intra- and intra-day biases can be minimized by using relative normalization rates (intracellular) fluorescence intensity (rates).

[0131] An in vitro-in vivo correlation has been established by comparing the fluorescence intensity rate in relatively normalized cells with the clearance rate determined in vivo according to the invention. This correlation does not depend on the specific antibody used during its generation. Similarly, other antibodies with known in vivo clearance rates can be used.

[0132] For antibodies with unknown in vivo clearance, the in vivo clearance of the antibody with an undetermined in vivo clearance can be estimated as a y value by using a determined relative normalized intracellular fluorescence intensity rate as the x value in the in vivo-in vitro correlation according to the present invention.

[0133]

[0134] The following examples, sequences, and figures are provided to aid in understanding the invention, and the true scope of the invention is set forth in the appended claims. It should be understood that modifications can be made to the described procedures without departing from the spirit of the invention.

[0135] Example

[0136] I. Materials and Methods

[0137] Antibody

[0138] The reference antibodies used in the experiment were anti-pTau antibody with the heavy chain amino acid sequence of SEQ ID NO: 01 and the light chain amino acid sequence of SEQ ID NO: 02, and anti-Her 3 antibody with the heavy chain amino acid sequence of SEQ ID NO: 03 and the light chain amino acid sequence of SEQ ID NO: 04.

[0139] The synthetic gene was produced at Geneart (Life Technologies GmbH, Carlsbad, CA, USA).

[0140] The monoclonal antibody used in this article was transiently expressed in HEK293 cells (see below) and purified by protein A chromatography using a standard procedure (see below).

[0141] Biochemical characterization included size exclusion chromatography (Waters BioSuite™ 250 7.8 x 300 mm, eluent: 200 mM KH2PO4, 250 mM KCl, pH 7.0) and molecular weight distribution was analyzed using a BioAnalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA).

[0142] Expression plasmids

[0143] To express the aforementioned antibodies, variants of expression plasmids for transient expression (e.g., in HEK293-F) were applied, based on cDNA tissues with or without the CMV-intron A promoter or on genomic tissues with the CMV promoter.

[0144] In addition to the antibody expression cassette, the plasmid also contains: - Copy starting point, which allows in E. coli The plasmid is then replicated. - β-lactamase gene, which confers E. coli Ampicillin resistance in, and - From Mus musculus The dihydrofolate reductase gene serves as a selection marker in eukaryotic cells.

[0145] The transcription unit of an antibody gene consists of the following elements: - A unique restriction site at the 5' end, - Immediate early enhancer and promoter from human cytomegalovirus, In the case of cDNA organization, the subsequent sequence is intron A. - The 5' untranslated region of the human antibody gene, - Immunoglobulin heavy chain signaling sequence, - Human antibody chains, which can be used as cDNA or as genomic tissue with immunoglobulin exon-intron structures. - The 3' untranslated region containing the polyadenylation signal sequence, and - A unique restriction site at the 3' end.

[0146] pass PCR and / or gene synthesis produce a fusion gene containing an antibody chain, and the fusion gene is assembled using known recombination methods and techniques, for example, by ligating corresponding nucleic acid segments using unique restriction sites in the appropriate plasmid. The nucleic acid sequence of the subcloned DNA is verified by DNA sequencing. For transient transfection, plasmids are prepared from the transformed... E. coli Large quantities of plasmids (Nucleobond AX, Macherey-Nagel) were prepared from the culture.

[0147] Cell culture technology

[0148] Use standard cell culture techniques as described in Current Protocols in Cell Biology (2000), Bonifacino, JS, Dasso, M., Harford, JB, Lippincott-Schwartz, J., and Yamada, KM (eds.), JohnWiley & Sons, Inc.

[0149] Transient transfection in the HEK293-F system

[0150] According to the manufacturer's instructions, antibodies are generated using the HEK293-F system (Invitrogen) by transiently transfecting the appropriate plasmid (e.g., encoding the heavy chain and the corresponding light chain). In short, HEK293-F cells (Invitrogen) grown in suspension in serum-free FreeStyle™ 293 expression medium (Invitrogen) in shake flasks or stirred fermentation tubes are transfected with the appropriate expression plasmid and a mixture of 293fectin™ or fectin (Invitrogen). For 2 L shake flasks (Corning), HEK293-F cells are added at 1... 10 6 Cells were seeded at a density of [number] cells / mL in 600 mL of water and incubated at 120 rpm with 8% CO2. Cells were grown at approximately 1.5 [units unspecified]. 10 6 Cells at a density of 1 cell / mL were transfected the day after inoculation with approximately 42 mL of the following mixtures, encoding the heavy chain and the corresponding light chain in equimolar ratios: A) 20 mL Opti-MEM (Invitrogen) containing 600 µg total plasmid DNA (1 µg / mL) and B) 20 mL Opti-MEM + 1.2 mL 293 fectin or fectin (2 µL / mL). Glucose solution was added during fermentation according to glucose consumption. The supernatant containing the secreted antibodies was harvested after 5–10 days, and the antibodies were either purified directly from the supernatant or frozen and stored. Some antibodies thus produced include:

[0151] purification

[0152] By using MabSelectSure-Sepharose TMAntibodies were purified from cell culture supernatant using affinity chromatography (GE Healthcare, Sweden), hydrophobic interaction chromatography using butyl agarose (GE Healthcare, Sweden), and size exclusion chromatography (GE Healthcare, Sweden).

[0153] In summary, sterile filtered cell culture supernatant was captured on MabSelectSuRe resin equilibrated with PBS buffer (10 mM Na₂HPO₄, 1 mM KH₂PO₄, 137 mM NaCl, and 2.7 mM KCl, pH 7.4), washed with equilibration buffer, and eluted with 25 mM sodium citrate at pH 3.0. The eluted antibody fractions were combined and neutralized with 2 M Tris at pH 9.0. Antibody pools were prepared for hydrophobic interaction chromatography by adding 1.6 M ammonium sulfate solution until a final concentration of 0.8 M ammonium sulfate was achieved and the pH was adjusted to 5.0 with acetic acid. After equilibrating butyl agarose resin with 35 mM sodium acetate and 0.8 M ammonium sulfate (pH 5.0), the antibody was applied to the resin, washed with equilibration buffer, and eluted with a linear gradient to 35 mM sodium acetate at pH 5.0. The fractions containing antibodies were combined and further purified by size exclusion chromatography using a Superdex 200 26 / 60 GL (GE Healthcare, Sweden) column equilibrated with 20 mM histidine and 140 mM NaCl (pH 6.0). The fractions containing antibodies were then concentrated to the desired concentration using a Vivaspin ultrafiltration system (Sartorius Stedim Biotech S.A., France) and stored at -80°C.

[0154] Following each purification step, purity and antibody integrity were analyzed by CE-SDS using microfluidic Labchip technology (Caliper Life Sciences, USA). 5 µl protein solutions were prepared according to the manufacturer's instructions using the HT Protein Express kit for CE-SDS analysis, and analyzed on the LabChip GXII system using the HT Protein Express chip. Data were analyzed using LabChip GX software.

[0155] mice

[0156] B6.Cg- Fcgrt tm1Dcr Tg(FCGRT)276Dcr mice lack the mouse FcRn α-chain gene, but a hemizygous transgene targeting the human FcRn α-chain gene (muFcRn- / - huFcRn tg + / -, line 276) was used for pharmacokinetic studies. Mice were housed under specific pathogen-free conditions. Mice were obtained from Jackson Laboratory (BarHarbor, ME, USA) (female, 4–10 weeks old, weighing 17–22 g at administration). All animal experiments were approved by the Government of Upper Bavaria, Germany (License No. 55.2-1-54-2532.2-28-10) and conducted in AAALAC-accredited animal facilities in accordance with EU guidelines for the care and use of laboratory animals. Animals were housed in standard cages and had free access to food and water throughout the study.

[0157] Pharmacokinetic studies

[0158] A single dose of the antibody was administered intravenously via the lateral tail vein at a dose level of 5 mg / kg. Mice were randomly divided into three groups of six mice each, covering nine serum collection time points (0.08, 2, 8, 24, 48, 168, 336, 504, and 672 hours post-administration). Each mouse underwent two retroorbital blood samplings under mild anesthesia with isoflurane™ (CP-Pharma GmbH, Burgdorf, Germany); a third blood sample was collected at euthanasia. Blood was collected into serum tubes (Microvette 500Z-Gel, Sarstedt, Nümbrecht, Germany). After incubation for 2 hours, the samples were centrifuged at 9.300 g for 3 minutes to obtain serum. After centrifugation, serum samples were frozen at -20°C until analysis.

[0159] Determination of human antibody serum concentration

[0160] The concentration of antibodies in mouse serum was determined using a specific enzyme-linked immunosorbent assay (ELISA). Biotinylated capture reagents specific to each antibody and anti-human Fc mouse monoclonal antibodies labeled with isohydroxydigitoxin (Roche Diagnostics, Penzberg, Germany) were used for capture and detection, respectively. Streptavidin-coated microtiter plates (Roche Diagnostics, Penzberg, Germany) were coated with biotinylated capture reagents diluted in assay buffer (Roche Diagnostics, Penzberg, Germany) for 1 hour. After washing, serum samples at different dilutions were added, followed by incubation for another 1 hour. After repeated washing, the bound antibodies were detected by subsequent incubation with detection antibodies, followed by anti-isohydroxydigitoxin antibodies conjugated to horseradish peroxidase (HRP; Roche Diagnostics, Penzberg, Germany). ABTS (2,2'-Azino-di[3-ethylbenzthiazoline sulfonate]; Roche Diagnostics, Germany) was used as the HRP substrate to form a colored reaction product. The absorbance of the resulting reaction product was read at 405 nm using a Tecan daytime plate reader (Männedorf, Switzerland), with a reference wavelength of 490 nm.

[0161] All serum samples, positive and negative control samples were analyzed in duplicate and calibrated against reference standards.

[0162] PK Analysis

[0163] Pharmacokinetic parameters were calculated using WinNonlin™ 1.1.1 (Pharsight, CA, USA) via non-compartmental analysis.

[0164] In short, due to the non-linear reduction of the antibody, the area under the curve (AUC) 0-inf The values ​​were calculated using the logarithmic trapezoidal method and extrapolated to infinity using the apparent terminal rate constant λz, from the concentration observed at the last time point.

[0165] Plasma clearance is calculated as dose rate (D) divided by AUC. 0-inf The apparent terminal half-life (T1 / 2) is derived from the equation T1 / 2 = ln2 / λz.

[0166] Example 1

[0167] Crab-eating macaque SDPK research

[0168] Pharmacokinetics of the test compound were determined in cynomolgus monkeys following a single intravenous administration at dose levels ranging from 0.3 mg / kg to 150 mg / kg. Serial blood samples were collected from monkeys over several weeks, and serum / plasma were prepared from the collected blood samples. Serum / plasma levels of the test compound were determined by ELISA. In the case of linear pharmacokinetics, pharmacokinetic parameters were determined using standard non-compartmental methods. Clearance was calculated according to the following formula: Clearance rate = dose / concentration - area under the time curve In the case of nonlinear pharmacokinetics, the linear fraction of clearance is determined by either estimating clearance after intravenous administration at a high dose level, at which additional nonlinear clearance pathways are effectively saturated, or by constructing a PK model that includes both linear and nonlinear, saturable clearance terms. In these cases, the linear clearance fraction determined by the model is used for correlation.

[0169] Example 2

[0170] Preparation of FcRn affinity columns

[0171] FcRn expression in HEK293 cells

[0172] Transient expression of FcRn was achieved by transfecting HEK293 cells with two plasmids containing coding sequences for FcRn and β-2-microglobulin. Transfected cells were cultured in shake flasks at 36.5°C, 120 rpm (shake amplitude 5 cm), 80% humidity, and 7% CO2. Cells were diluted to 3-4 every 2-3 days. 10 5 Density of cells / ml.

[0173] For transient expression, at 36.5℃, pH 7.0 ± 0.2, pO2 35% (aerated with N2 and air, total gas flow rate 200 ml / min) -1 Start the 14 L stainless steel bioreactor, with a culture volume of 8.1 L and a stirrer speed of 100-400 rpm. When the cell density reaches 20... 10 5At a cell / ml concentration, 10 mg of plasmid DNA (equimolar amounts of both plasmids) was diluted in 400 ml of Opti-MEM (Invitrogen). 20 ml of 293fectin (Invitrogen) was added to this mixture, and the mixture was incubated at room temperature for 15 minutes before being transferred to a fermenter. Starting the next day, cells were fed continuously: feed solution was added at a rate of 500 ml per day, with glucose added as needed to maintain a level above 2 g / L. Seven days after transfection, the supernatant was collected at 4000 rpm for 90 minutes using a swing-head centrifuge with a 1 L drum. The supernatant (13 L) was removed by a Sartobran P filter (0.45 µm + 0.2 µm, Sartorius) and the FcRn β-2-microglobulin complex was purified from it.

[0174] Biotinylation of Fc receptors in newborns

[0175] Dissolve / dilute 3 mg of FcRn β-2-microglobulin complex in 5.3 mL of 20 mM sodium dihydrogen phosphate buffer containing 150 mM sodium chloride, and add to 250 μL of PBS and one tablet of complete protease inhibitor (ULTRA tablet, Roche Diagnostics GmbH). Biotinylate FcRn using a biotinylation kit from Avidity, following the manufacturer's instructions (Bulk BIRA, Avidity LLC). Allow the biotinylation reaction to proceed overnight at room temperature.

[0176] To remove excess biotinylated FcRn, dialyze it overnight at 4°C against 20 mM MES buffer (containing 140 mM NaCl, pH 5.5) (buffer A).

[0177] Coupling with streptavidin agarose

[0178] To couple with streptavidin agarose, 1 mL of streptavidin agarose (GE Healthcare, United Kingdom) was added to the biotinylated and dialyzed FcRn β-2-microglobulin complex and incubated overnight at 4°C. The derivatized FcRn β-2-microglobulin complex agarose was packed into a 4.6 mm x 50 mm column (Repligen). The column was stored in 80% buffer A and 20% buffer B (20 mM Tris(hydroxymethyl)aminomethane, pH 8.8, 140 mM NaCl).

[0179] Example 3

[0180] Chromatography using FcRn affinity columns and pH gradients

[0181] condition: Column dimensions: 50 mm x 4.6 mm Sample loading: 30 µg sample Buffer A: 20 mM MES containing 140 mM NaCl, adjusted to pH 5.5. Buffer B: 20 ​​mM Tris / HCl, containing 140 mM NaCl, adjusted to pH 8.8. Apply 30 µg of sample to an FcRn affinity column equilibrated with buffer A. After a 10-minute wash in 20% buffer B at a flow rate of 0.5 mL / min, elute for over 70 minutes using a linear gradient of 20% to 70% buffer B. Detect using UV absorption at 280 nm. Regenerate the column with 20% buffer B for 10 minutes after each run.

[0182] To calculate the relative retention time, according to Bertoletti-Ciarlet, A. et al. (Mol. Immunol.46 (2009) 1878-1882), standard samples (anti-Her3 antibodies (SEQ ID NO: 03 and 04)) oxidized with 0.02% peroxide for 18 hours were run at the beginning of the sequence and after every 10 sample injections.

[0183] In short, the antibody (9 mg / mL) in 10 mM sodium phosphate buffer (pH 7.0) was mixed with H2O2 to a final concentration of 0.02% and incubated at room temperature for 18 hours. To quench the reaction, the sample was thoroughly dialyzed into pre-chilled 10 mM sodium acetate buffer (pH 5.0).

[0184] Example 4

[0185] Chromatography using heparin affinity columns and pH gradients

[0186] condition: Column dimensions: 50 mm x 5.0 mm Sample loading: 20-50 µg sample Buffer A: 50 mM Tris, pH 7.4 Buffer B: 50 mM Tris, pH 7.4, 1000 mM NaCl Protein samples in 20–50 µg of low-salt buffer (≤ 25 mM ionic strength) were applied to a 5.0 x 50 mm TSKgel Heparin-5PW glass column (Tosoh Bioscience, Tokyo / Japan), pre-equilibrated with buffer A at room temperature. Elution was performed over 32 minutes with a linear gradient of 0–100% buffer B at a flow rate of 0.8 mg / mL. Detection was performed using UV absorption at 280 nm.

[0187] Example 5

[0188] Check antibody internalization

[0189] This method is based on a previously reported approach that uses uniform fluorescence imaging of pH-activated probes to detect internalized antibodies. This method achieves maximum fluorescence signal of antibodies under acidic conditions inside cells without detecting any fluorescence signal in the extracellular environment (Li, Z., et al., Int. Immunopharm.62 (2018) 299-308).

[0190] In short, the corresponding antibody was conjugated to a pHAb amine-reactive dye and then diluted with cell culture medium. Simultaneously, cells were seeded into 6-well plates (1 × 10⁶ cells per well). 5100 μL of culture medium containing pHAb amine-reactive dye conjugated antibody (final concentration 10 μg / mL) was added to each well. After incubation at 37°C, antibody internalization was measured by flow cytometry at different time points (0 h, 1.5 h, 2 h, 4 h, 5.5 h and / or 24 h).

[0191] Example 6

[0192] Antibody labeling

[0193] According to the manufacturer's instructions, the antibody was labeled using the SiteClick™ Antibody Azido Modification Kit (Thermo Fisher Scientific). In short, the N-linked galactose residue in the Fc region was removed by β-galactosidase and replaced by an azide-containing galactose (GalNaz) via β-1,4-galactosyltransferase (GalT). This azide modification enables copper-free conjugation of the sDIBO-modified dye. A pH-sensitive amine-reactive dye (523 nm) was purchased from Promega and conjugated with sulfoDBCO PEG4 amine. The antibody was labeled with a 2% excess of the dye. Excess dye was removed using an Amicon® Ultra-2 centrifuge filter with a MWCO rating of 50 kDa (EMD Millipore, # UFC200324), and the antibody was reburied in 20 mM histidine buffer (pH 5.5). The antibody was then analyzed using a Nanodrop spectrometer at 280 nm (A 280nm ) and 532 nm (A 532nm The concentration of the labeled antibody[1] and the dye-to-antibody ratio (DAR)[2] were determined.

[0194] CAB = [A 280nm - [A 280nm CF Dye ]] / ε mAb [1]

[0195] DAR = [A 532nm MW mAb ] / [c mAb ε Dye [2]

[0196] ε Dye = 47225

[0197] CFDye = 0.36

[0198] Example 7

[0199] Cell maintenance and preparation

[0200] Cryopreserved human liver-derived endothelial cells (HLEC-P2) were purchased from Lonza (Lonza, #HLECP2). Cells were maintained in an environment supplemented with EGM. TM -2 MV Microvascular Endothelial Cell Growth Medium SingleQuots TM EBM of (Lonza, #CC-4176) TM -2 Endothelial cell growth basal medium-2 (Lonza, #CC-3156). Five days before antibody treatment, cells were seeded into 100 mm collagen I-coated culture dishes (Corning® BioCoat™, #354450), and two days before treatment, cells were passaged into 96-well collagen I-coated plates (Corning® BioCoat™, #354407) at a cell density of 4 x 10⁻²⁶ cells / well. 4 Cells per well were allowed to adhere for 48 hours. The culture medium was changed after 24 hours, and the cells were maintained at 37 °C and 5% CO2.

[0201] On the day of the experiment, cells were washed twice with 200 µl of preheated culture medium, followed by incubation in the medium with either 400 nM labeled antibody or 20 mM histidine buffer (pH 5.5) as a negative control. After 2 and 4 hours, the antibody solution was removed, and cells were washed once with 200 µl of ice-cold DPBS (Mg and Ca-free), and separated by applying 100 µl of trypsin (containing EDTA) at 37 °C for 2.5 min. Trypsin was inactivated by adding 100 µl of FACS buffer (20% FCS, 1 mM EDTA in DPBS).

[0202] Example 8

[0203] Flow cytometry and pharmacokinetics analysis

[0204] The mean fluorescence intensity (MFI, more specifically the geometric mean) of the internalized antibodies was obtained using a MACSQuant® Analyzer 10 (Miltenyi Biotec), equipped with a laser excitation at 488 nm and filters at 585 nm / 540 nm for collecting the emitted light. Identical conditions, gain, and gates were used at both time points (2 hours and 4 hours). Data extraction was performed using FloJo_V10 software. The negative control values ​​were subtracted from all geometric means, and then DAR was normalized. The normalized geometric means from each antibody were plotted as linear regression curves using GraphPad Prism to extract the slope (geometric mean MFI / min for 120 and 240 minutes). Two standard antibodies were selected for normalization: murvizumab-YTE was set to 0, and TCB was set to 1. The final slope was plotted using TIBCO Spotfire software for the published in vivo clearance values ​​in humans, cynomolgus monkeys, and hFcRn Tg32 + / + mice.

[0205] Example 9

[0206] Quality control

[0207] Biophysical binding properties are a key determinant of clearance mechanisms. Therefore, it is important to assess whether antibody binding affinity changes during labeling. Heparin chromatography and neonatal Fc receptor binding have previously been shown to predict in vitro antibody clearance rates (Kraft, TE, et al., MABS 12 (2020) e1683432). Here, this method is used to illustrate potentially aberrant binding properties introduced by click-tag. Details of the method are provided in Examples 3 and 4.

[0208] To confirm the absence of unbound dye and to verify the concentration measured on the spectrometer, size exclusion chromatography was performed on the labeled antibodies. Samples were separated using a BioSuite Diol (OH) column (Waters, 186002165) with potassium dihydrogen phosphate buffer (pH 6.2) as the mobile phase at a flow rate of 0.5 ml / min. The labeled antibodies were quantified and analyzed using detectors at 280 nm and 532 nm. The area under the curve (AUC) at 280 nm and 532 nm was extracted to calculate the concentration. The geometric mean of the AUC for all antibodies was calculated, and the deviation of each antibody from this geometric mean was determined. For antibodies reliable within the assay according to the invention, the difference from the geometric mean is expected to be less than 15%.

Claims

1. A method for determining nonspecific clearance of an antibody, the method comprising the following steps: a) Incubate the antibody conjugated to a pH-sensitive fluorescent dye with primary human endothelial cells, and b) Determine the fluorescence intensity of the primary human endothelial cells obtained in step a). If the fluorescence intensity of the primary human endothelial cells determined in step b) is higher than the fluorescence intensity of the primary human endothelial cells determined in the absence of the antibody, then nonspecific clearance of the antibody is detected.

2. The method according to claim 1, further comprising the following steps: c) Determine the fluorescence intensity of the primary human endothelial cells when they are not incubated with the antibody or in the absence of the antibody.

3. The method according to any one of claims 1 to 2, wherein the primary human endothelial cells are washed prior to the determination of the fluorescence intensity.

4. The method according to any one of claims 1 to 3, wherein, as determined by the same concentration of the dye and the same excitation wavelength, the dye exhibits a fluorescence intensity variation of about 10-fold between a physiological pH of about 7 and an acidic pH in the range of pH 4 to 5.

5. The method according to any one of claims 1 to 4, wherein the dye is a pHAb of formula I.

6. The method according to any one of claims 1 to 5, wherein the dye is conjugated to the antibody at residue 297 (according to Kabat number).

7. The method according to any one of claims 1 to 6, wherein the dye is conjugated to the antibody via a sulfoDBCO-PEG4-amine linker of formula II.

8. The method according to any one of claims 1 to 7, wherein the dye is conjugated to the adapter, and the adapter is conjugated to the antibody and has the structure of formula III.

9. The method according to any one of claims 1 to 8, wherein the fluorescence intensity is determined by FACS to determine the shift of the fluorescence maximum value.

10. The method according to any one of claims 1 to 9, wherein the fluorescence intensity is the geometric mean fluorescence intensity determined by FACS.

11. The method according to any one of claims 1 to 10, wherein the primary human endothelial cells are primary human liver endothelial cells.

12. The method according to any one of claims 1 to 11, wherein the incubation lasts for a maximum of 4 hours.

13. The method according to any one of claims 1 to 12, wherein the incubation lasts for at least 0.5 hours.

14. The method according to any one of claims 1 to 13, wherein the antibody has an Fc region of a human IgG1 or IgG4 subclass.

15. The method according to any one of claims 1 to 14, wherein the antibody is a bispecific antibody.

Images