Large molecule 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

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202511775751.5 (22) Application Date 2021.04.06 (30) Priority Data 20168671.4 2020.04.08 EP (62) Divisional Application Data 202180027436.8 2021.04.06 (71) Applicant Hofmeister Roche Ltd. Address Switzerland (72) Inventors AL. Braunde, M. Duschmall, L. Ibler, J. Freding, T. Kraft (74) Patent Agency Beijing Zhongzi Law Firm 11247 Patent Attorneys Shi Wenjing, Huang Gesheng (51) Int.Cl. G01N 33 / 68 (2006.01) G01N 33 / 58(2006.01) C12Q 1 / 02(2006.01) G01N 21 / 64(2006.01) (54) Invention Title: Macromolecular Nonspecific Clearance Assay (57) Abstract: This paper reports a method for determining the nonspecific clearance of an antibody, the method comprising the steps of: incubating the antibody conjugated to a pH-sensitive fluorescent dye with primary human endothelial cells, and determining the fluorescence intensity of the primary human endothelial cells, wherein an increase in the fluorescence intensity of the primary human endothelial cells relative to a background level indicates the nonspecific clearance of the antibody. Claims 1 page, Description 24 pages, Sequence Listing (Electronic Publication), Drawings 10 pages, CN 121577897 A 2026.02.27 CN 1 21 57 78 97 A 1. A method for determining nonspecific clearance of an antibody, the method comprising the steps of: a) incubating the antibody conjugated to a pH-sensitive fluorescent dye with primary human endothelial cells, and b) determining the fluorescence intensity of the primary human endothelial cells in step a), wherein nonspecific clearance of the antibody is detected 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 in the absence of the antibody. 2. The method of claim 1, further comprising the step of: c) determining the fluorescence intensity of the primary human endothelial cells not incubated with the antibody / in the absence of the antibody. 3. The method of 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 the dye is determined at the same concentration and using the same excitation wavelength at a physiological pH of about 7 and at pH 4 to 5.5. The method of any one of claims 1 to 4, wherein the dye is a pHAb of formula I. 6. The method of any one of claims 1 to 5, wherein the dye is conjugated to the antibody at residue 297 (according to Kabat numbering). 7. The method of any one of claims 1 to 6, wherein the dye is conjugated to the antibody via a sulfonylDBCO-PEG4-amine linker of formula II. 8. The method of any one of claims 1 to 7, wherein the dye is conjugated to a linker, and the linker is conjugated to the antibody and has the structure of formula III. 9. The method of any one of claims 1 to 8, wherein the fluorescence intensity is determined by FACS to determine the shift of the fluorescence maximum. 10. The method of any one of claims 1 to 9, wherein the fluorescence intensity is the geometrically average fluorescence intensity determined by FACS. 11. The method of 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. Claims 1 / 1 page 2 CN 121577897 A Macromolecular Nonspecific Clearance Assay

[0001] This application is a divisional application of PCT application PCT / EP2021 / 058839, filed on April 6, 2021, entitled "Macromolecular Nonspecific Clearance Assay," which entered the Chinese national phase on October 8, 2022, with application number 202180027436.8.

[0002] This article reports a novel method for estimating the clearance of therapeutic proteins in the human body using a novel 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 characteristics of therapeutic proteins. Background Art

[0003] Class G human immunoglobulins (IgG) contain two antigen-binding (Fab) regions that deliver specificity to target antigens and a constant region (Fc region) responsible for interacting with Fc receptors (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 the serum half-life 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, T.T., 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. Cell 19 (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, L.M., et al., Biochemistry 38 (1999) 9471-9476; Huber, A.H., et al., J. Mol. Biol. 230 (1993) 1077-1083). The affinity between FcRn and the Fc region is pH-dependent, exhibiting nanomolar affinity at endosomal 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 for transferring the long half-life to IgG can be explained through three basic steps. First, IgG undergoes non-specific pinocytosis in multiple cell types (see, for example, Akilesh, S., et al., J. Immunol. 179 (2007) 4580–4588; Montoyo, H.P., et al., Proc. Natl. Acad. Sci. USA 106 (2009) 2788–2793). Secondly, at pH 5-6, IgG encounters and binds to FcRn in acidic endosomes, 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 is crucial, 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, thus allowing antibody recycling rather than lysosomal degradation (MABS 9 (2017) 1007-1015).

[0006] Grevys, A. et al. reported a human endothelial cell-based recycling 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 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 main biological factors of nonspecific clearance of therapeutic antibodies in patients, namely nonspecific uptake via endocytosis and FcRn-mediated recycling, there is a need for in vitro methods to predict in vivo clearance (i.e., half-life).

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

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

[0011] The invention is based at least in part on the finding that only primary human endothelial cells can be used to determine in vivo clearance based on in vitro experiments, as 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 said 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, this invention includes a method for determining or estimating the nonspecific (i.e., non-target-mediated) clearance (rate) of an antibody, the method comprising the steps of: a) incubating an antibody conjugated to a pH-sensitive fluorescent dye with primary human endothelial cells (for a specified time), and b) determining the (intracellular) fluorescence intensity of the primary human endothelial cells obtained in step a) (after the specified incubation time), wherein the presence of nonspecific clearance of the antibody (i.e., nonspecific clearance of the indicator antibody) is determined by an increase in the (intracellular) fluorescence intensity of the 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).

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

[0015] Furthermore, the present invention includes a method for selecting one or more antibodies from a plurality of antibodies having a low relative nonspecific (non-target mediated) clearance (rate), the method comprising the steps of: a) incubating each of a plurality of antibodies with primary human endothelial cells for the same defined time, and thereafter determining the (intracellular) fluorescence intensity (change) of the primary human endothelial cells (i.e., determining the change in fluorescence intensity), wherein each antibody is conjugated to the same pH-sensitive fluorescent dye; b) selecting one or more antibodies from the plurality of antibodies that, after incubation, result in the lowest (intracellular) fluorescence intensity (change) of the primary human endothelial cells, thereby selecting one or more antibodies having a low relative nonspecific (non-target mediated) clearance (rate).

[0016] Furthermore, the present invention includes a method for ranking a plurality of antibodies based on nonspecific (non-target-mediated) clearance (rate), comprising the steps of: a) incubating each of the plurality of antibodies with primary human endothelial cells for the same defined time, and thereafter determining the (intracellular) fluorescence intensity (change) of the primary human endothelial cells, wherein each antibody is conjugated to the same pH-sensitive fluorescent dye; b) ranking the antibodies based on (intracellular) fluorescence intensity (change) 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 of an antibody in a human, cynomolgus monkey, or mouse, comprising the steps of: a) incubating an antibody conjugated to a pH-sensitive fluorescent dye with primary human endothelial cells for a predetermined time, and thereafter determining the (intracellular) fluorescence intensity (change) of the primary human endothelial cells; b) incubating at least a first reference antibody with primary human endothelial cells for the same defined time as in a), for which the clearance rate in humans, cynomolgus monkeys, or mice is known, and the at least first reference antibody is conjugated to a pH-sensitive fluorescent dye (in the same preferred embodiment as in a), and thereafter determining the (intracellular) fluorescence intensity (change) of the primary human endothelial cells, wherein the (relative) in vivo clearance of the antibody in a human, cynomolgus monkey, or mouse is estimated or determined as the clearance rate of the first reference antibody in a human, cynomolgus monkey, or mouse multiplied by the ratio of the fluorescence intensity (change) determined in a) to the (intracellular) fluorescence intensity (change) determined in b).

[0018] In some embodiments, step b) is

[0019] b) i) incubating each of a plurality of reference antibodies (i.e., at least two) with primary human endothelial cells for the same time defined in a), for which the clearance rate of human, cynomolgus monkey, or mouse is known, and the reference antibody is conjugated to a pH-sensitive fluorescent dye (in one preferred embodiment as in a), specification 3 / 24 page 5 CN 121577897 A ii) thereafter determining the (intracellular) fluorescence intensity (change) of primary human endothelial cells for each of the reference antibodies, and iii) calculating the best-fit line of the formula y = ax + b for the values ​​obtained in ii), 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 (intracellular) fluorescence intensity (change) is a geometric mean (intracellular) fluorescence intensity (change).

[0021] In all aspects and in one embodiment of each embodiment, 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) determining the (geometric mean) (intracellular) fluorescence intensity after two or more prescribed incubation times for the antibody under discussion and at least two reference antibodies, 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) from 1)1) 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, to obtain the corrected geometric mean (intracellular) fluorescence intensity; 2) Divide the corrected geometric mean (intracellular) fluorescence intensity of the antibodies 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); 3) Based on the set of values ​​consisting of the normalized geometric mean (intracellular) fluorescence intensities for at least two different incubation times for the antibody (i.e., for each individual) calculated as in 3), including the origin, determine the best-fit line (i.e., the linear regression curve y=sx + b, where y=normalized (geometric mean) (intracellular) fluorescence intensity, s=slope, x=time, and b=y 5) The slope of the best-fit line of the antibody under discussion is normalized as follows: Normalized slope (antibody under discussion) =

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

[0023] In one embodiment of all aspects and examples, the dye has 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 Formula I / is a pHAb of Formula I.

[0024] (Formula I). ​​Specification 4 / 24 pages 6 CN 121577897 A

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

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

[0027] In one embodiment of all aspects and various embodiments, the dye is conjugated to the antibody by click chemistry.

[0028] In one embodiment of all aspects and various embodiments, 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 is at the free amino group of Formula II.

[0031] In one embodiment of all aspects and various embodiments, the dye is conjugated to the linker and the linker is conjugated to the antibody andAnd the conjugate has the structure of Formula III.

[0032] (Formula III)

[0033] In one embodiment of all aspects and embodiments, the dye is chemically cross-linked to the antibody.

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

[0035] In one embodiment of all aspects and embodiments, fluorescence is the geometric mean fluorescence intensity determined by FACS.

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

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

[0038] In one embodiment of all aspects and embodiments, it is determined that the incubation is performed after a maximum duration of 24 hours, i.e., the specified time is at most 24 hours. In some embodiments, it is determined that the incubation is performed after a maximum duration of 16 hours. In a preferred embodiment, it is determined that the incubation is performed after an incubation period of up to 4 hours, i.e., the specified time is up to 4 hours. In some embodiments, it is determined that the incubation is performed after an incubation period 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 is performed after an incubation period 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 is performed after an incubation period 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 incubation is performed directly after incubation.

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

[0041] In one embodiment of all aspects and embodiments, 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 one embodiment of all aspects and embodiments, the antibody is a bispecific antibody.

[0043] In one embodiment of all aspects and embodiments, the first reference antibody is a muvizumab having the mutant M252Y / S254T / T256E, and / or a TCB.Bispecific antibodies in the form of

[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 = acimetab; 6 = anti-human α-synuclein antibody; 7 = anti-CD20 antibody (2).

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

[0047] Figure 4: Scheme of the method according to the invention.

[0048] Figure 5: Corrected mean fluorescence intensity (MFI, more specifically geometric mean) of internalized antibodies obtained using FACS was obtained by subtracting the negative control and then normalizing (dividing) by the dye-antibody ratio (DAR). The corrected and normalized geometric mean values ​​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 was set to 0, and TCB was 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) with dye-antibody ratio (DAR). The corrected and normalized geometric mean values ​​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 was set to 0, and TCB was set to 1. The final slopes were plotted against in vivo cynomolgus monkey clearance values. If different clearance values ​​are obtained, dose-linear clearance describing nonspecific clearance of the molecule is used.

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

[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. Specification 6 / 24 pages 8 CN 121577897 A

[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 cells were gated for singlet state, morphology, and viability; y-axis scaling is relative to the number of events, and x-axis scaling shows the intensity in the PE channel. Detailed Description

[0054] This invention is based at least in part on the finding 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 found a significant correlation between readouts according to the method of 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 further discovered that the method according to the invention is equally applicable to conventional bispecific antibodies and monoclonal antibodies that reflect the Y-shape of wild-type human antibodies, as well as unconventional bispecific antibody-type human antibodies that have a different form and more than two valences and antibody Fc region fusions. This provides evidence for the general applicability of the readout of the method according to the invention to the correlation with clearance rates in mice, cynomolgus monkeys, and humans.

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

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

[0058] - support the selection of suitable clinical lead molecules with respect to PK (pharmacokinetic) properties (clearance and half-life, respectively); - deselect antibodies from the library that have PK properties unsuitable for therapeutic applications, i.e., those with high clearance rates, respectively.Antibodies with short clearance or in vivo half-life; - Ranking members of a group of antibodies according to PK properties (clearance and half-life, respectively); - Determining the relative in vivo clearance of the antibody in question based solely on the in vivo clearance of a reference antibody (or many reference antibodies) with known PK properties, i.e., without in vivo testing; - Guiding antibody engineering with PK properties (by altering the FcRn affinity of the Fc region or by engineering charged plaques of Fab (the latter described in WO 2018 / 197533); - Determining the need for PK engineering and evaluating the results of PK engineering.

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

[0060] I. Definitions

[0061] As used herein, the amino acid positions of all constant regions and domains of the heavy and light chains are based on Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD The numbering system described in (1991) is used and is referred to herein as “according to the Kabat numbering system”. 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 κ and λ isotype light chain constant domain CL, and the Kabat 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 as “according to the Kabat EU index numbering system” in the case of page 7 / 24 of this specification, 9 CN 121577897 A).

[0062] The mortar structure dimer module and its use in antibody engineering are discussed in Carter P., Ridgway JBB, Presta LG: Immunotechnology, 1996. The description is found in the February issue of Volume 2, Issue 1, pp. 73-73(1).

[0063] General information on the nucleotide sequences of the light and heavy chains of human immunoglobulins is given in: Kabat, EA, et al.Human, Sequences of Proteins of Immunological Interest, 5th ed., 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, N.D. and Hames, BD (eds.), DNA Cloning: A Practical Approach, Volumes I and II (1985), Oxford University Press; Freshney, RI (ed.), Animal Cell Culture – a practical approach, IRL Press Limited (1986); Watson, J.D. et al., Recombinant DNA, 2nd edition, CHSL Press (1992); Winnacker, EL, From Genes to Clones; NY, VCH Publishers (1987); Celis, J. (ed.), Cell Biology, 2nd edition, Academic Press (1998); Freshney, RI, Culture of Animal Cells: A Manual of Basic Technique, 2nd Edition, 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 single 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, B.D. and Higgins, S.G.,Nucleic acid hybridization – a practical approach (1985) IRL Press, Oxford, England.

[0066] It must be noted that, as used herein and in the appended claims, the singular forms “an,” “a,” and “the” include plural references unless the context explicitly 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 “an,” “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 numerical value that follows it. In some embodiments, the term “about” indicates a range of + / - 10% of the numerical value that follows it. In some embodiments, the term “about” indicates a range of + / - 5% of the numerical value that follows it.

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

[0069] The term “comprising” also includes the term “composed of”.

[0070] The term “antibody” is used herein in the broadest sense and includes a variety of antibody structures, including but not limited to monoclonal antibodies, 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” means having binding specificity with respect to at least two different epitopes or two different antigens on the same antigen. Multispecific antibodies may be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibody) or combinations thereof (e.g., full-length antibody plus 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 / 0004587 specification, page 8 / 24, 10 CN 121577897 A A1).

[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 that, when in solution, can adjust the pH of a solution caused, for example, by the addition or release of acidic or alkaline substances.

[0074] The “class” of an antibody refers to the type of constant domain or constant region possessed by the heavy chain of the antibody. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these antibodies can be further subdivided 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 referred to as α, β, γ, γ, and µ, respectively.

[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 exhibiting 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 carboxyl 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 to form 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 pathways, thereby causing a decrease in clearance and an increase in 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 region of the Fc region of IgG. The interaction between IgG and FcRn is strictly pH-dependent and occurs in a 1:2 stoichiometric ratio, 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), and IgG is released at the neutral cell surface (pH approximately 7.4). This pH sensitivity of the 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 the recycling of IgG to the cell surface.Subsequently, the FcRn-IgG complex is released into the bloodstream when exposed to an 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 number 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, E258, V259, T260, C261, F275, N276, W277, Y278, V279, D280, V282, E283, V284, H285, N286, A287, K288, T289, K290, P291, R292, E293, V302, V303. S304, V305, L306, T307, V308, L309, H310, Q311, D312, W313, L314, N315, G316, K317, E318, Y319, I336, S337, K338, A339, K340, G341, Q342, P343, R344, E345, P346, Q347, V348, C367, V369, F372, Y373, P374, S375, D376, I377, A378, V379, E380, W381, E382, 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 number).

[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 comprising a light chain variable domain and a light chain constant domain, the two full-length antibody heavy chains comprising a heavy chain variable domain, a first constant domain, a hinge region, and a secondConstant domain and 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. Thus, 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" means an amino acid sequence identical to that of a "natural" or "wild-type" human Fc region polypeptide. The term "variant (human) Fc region polypeptide" means an amino acid sequence derived from a "natural" or "wild-type" human Fc region polypeptide that 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, wherein both can be variant (human) Fc region polypeptides, or one is a human Fc region polypeptide and the other is a variant (human) Fc region polypeptide.

[0082] A "humanized" antibody refers to a chimeric antibody that contains amino acid residues from non-human HVR and amino acid residues from human FR. In some embodiments, the humanized antibody will substantially contain at least one of all, typically two, variable domains, wherein all or substantially all HVRs (e.g., CDRs) correspond to the HVRs of a nonhuman antibody, and all or substantially all FRs correspond to the FRs of a human antibody. The humanized antibody may optionally contain at least a portion of the antibody constant region derived from a human antibody. An antibody in a “humanized form,” such as a nonhuman antibody, refers to an antibody that has been humanized.

[0083] An “isolated” antibody is an antibody that has been isolated from components of its natural environment. In some embodiments, the antibody is purified to greater than 95%.Or a purity of 99%, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., size exclusion chromatography or ion exchange or reversed-phase HPLC). For a review of methods for assessing, for example, antibody purity, see Flatman, S. et al., J. Chrom. B 848 (2007) 79-87.

[0084] "Isolated" nucleic acid means a nucleic acid molecule that has been isolated from components of its natural environment. Isolated nucleic acids include nucleic acid molecules that are contained in cells that normally contain nucleic acid molecules, but which are present outside chromosomes or at chromosomal locations different from their natural chromosomal locations.

[0085] The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous group of antibodies, i.e., the individual antibodies comprising the group are identical and / or bind the same epitopes, except for possible variant antibodies (e.g., containing naturally occurring mutations or generated during the production of monoclonal antibody formulations, such variants are typically presented in small quantities). Unlike polyclonal antibody preparations, which typically include different antibodies targeting different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation targets a single determinant on the antigen. Therefore, the modifier "monoclonal" indicates that the antibody is obtained from a substantially homogeneous group of antibodies and should not be interpreted 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 using 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 antibody" refers to a naturally occurring immunoglobulin molecule with a different structure. For example, a natural IgG antibody is a heterotetrameric glycoprotein of about 150,000 Daltons, consisting 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 known as 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 known as a variable light chain domain or light chain variable domain, followed by a constant light chain (CL) domain. The light chains of an antibody, based on the amino acid sequence of their constant domains, can be classified into one of two types, referred to as Kappa (κ) and Lambda (λ).

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

[0088] “Pharmaceutical carrier” refers to a component of a pharmaceutical formulation that is non-toxic to the subject other than 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) with 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 the recombinant antibody are as follows, although derived from and associated with human germline VH and VL sequences, but 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 may have, for example, the form described in WO 2013 / 026831. These molecules can simultaneously bind to CD3 on T cells (first specificity) and antigens on target (e.g., tumor) cells (second specificity), thereby inducing the killing of target cells. A TCB is a trivalent bispecific antibody composed of four polypeptides or polypeptide chains: a light chain, which is a full-length light chain; another light chain, which is a domain-exchanged full-length light chain; a heavy chain, which is a full-length heavy chain; and another heavy chain, which is an extension of the heavy chain containing additional domain-exchanged heavy chains or light chain Fab fragments.

[0091] In a preferred embodiment, the TCB comprises: a) a first Fab fragment and a second Fab fragment, each binding to a first antigen; b) a domain-exchange Fab fragment specifically binding to a second antigen, wherein the CH1 domain and the CL domain are exchanged with each other in the domain-exchange Fab fragment; c) an Fc region comprising a first heavy chain Fc region polypeptide and a second heavy chain Fc-region polypeptide, wherein 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 polypeptides, and the C-terminus of the CL domain of the domain-exchange Fab fragment is attached to the N-terminus of the other heavy chain Fc region polypeptide, and wherein the C-terminus of the CH1 domain of the second Fab fragment is attached to the N-terminus of the VH domain of the first Fab fragment, or to the N-terminus of the VH domain of the domain-exchange Fab fragment, and wherein the first antigen or the second antigen is human CD3. (Description)Page 11 / 24 13 CN 121577897 A

[0092] In another equally preferred embodiment, the TCB comprises: a) a first Fab fragment and a second Fab fragment, each binding to a first antigen; b) a domain-exchange Fab fragment specifically binding to a second antigen, wherein the VH domain and the VL domain are exchanged with each other in the domain-exchange Fab fragment; c) an Fc region comprising a first heavy chain Fc region polypeptide and a second heavy chain Fc-region polypeptide, wherein 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 polypeptides, and the C-terminus of the CH1 domain of the domain-exchange Fab fragment is attached to the N-terminus of the other heavy chain Fc region polypeptide, and wherein the C-terminus of the CH1 domain of the second Fab fragment is attached to the N-terminus of the VH domain of the first Fab fragment, or to the N-terminus of the VL domain of the domain-exchange Fab fragment, and wherein the first antigen or the 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” indicate the presence of two, four, and six binding sites in the (antibody) molecule, respectively. 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 (FR) and three hypervariable regions (HVR) (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 specific antigens can be separated using either the VH or VL domains of the antibody binding to that antigen, to screen libraries with complementary VL or VH domains. See, for example: Portolano, S. et al., J. Immunol. 150 (1993) 880-887; Clackson, T. et al., Nature 352 (1991) 624-628).

[0095] The terms “variant,” “modified antibody,” and “modified fusion polypeptide” refer to molecules having a different structure from the parent molecule.A molecule with an amino acid sequence. Typically, such molecules have one or more alterations, insertions, or deletions. In some embodiments, the modified antibody or modified fusion polypeptide 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 parent fusion polypeptide. In some embodiments, the amino acid sequence of the variant antibody or variant fusion polypeptide has from about 75% to less than 100% amino acid sequence identity with the amino acid sequence of the parent antibody or parent fusion polypeptide, 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 parent fusion polypeptide and the variant antibody or variant fusion polypeptide 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-growing 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 non-anchorage-dependent cells. Most suspension cells are isolated from blood.

[0097] The term “pH-sensitive fluorescent dye” refers to a dye that has different fluorescence intensities or emission wavelengths at a physiological pH of about 7.4 and a lysosomal pH of about 4.5. Specification 12 / 24 pages 14 CN 121577897 A

[0098] II. In vivo antibodies

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

[0100] In the case where the antigen is a membrane-bound antigen, the administered 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 membrane-bound antigen bound to the antibody. 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 plasma mediated by the 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 binds to a bivalent antigen, and is then incorporated into the lysosome and directly degraded, a single ordinary IgG antibody cannot neutralize two or more antigen molecules.

[0101] The reason for the prolonged retention (slow elimination) of IgG molecules in plasma is 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 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, the IgG antibodies dissociate from the antigen under acidic conditions within the endosome, and the dissociated antibodies can bind to FcRn, which is also present in the endosome. Therefore, IgG molecules dissociated from the antigen and bound by FcRn are transferred to the cell surface and released from FcRn into the plasma under neutral pH conditions. This recirculates the antibody into the plasma. IgG molecules returning to the plasma can bind to new antigens again. Repeating this 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 in the plasma as an antigen-antibody complex. Similar to the case of IgG molecules that do not bind to the antigen, IgG molecules bound to the antigen in the plasma are absorbed into the endosome via endocytosis. In the endosome, they can bind to FcRn expressed in the endosome under acidic conditions within the endosome. IgG molecules bound to FcRn move 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 body, the dissociated antigens will not be able to 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 body, they are able to bind to new antigens in the plasma again. The repetition of this process allows a single IgG molecule to repeatedly bind to soluble antigens. This makes it possible for a single IgG molecule to neutralize multiple antigens.

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

[0105] More specifically, a single IgG molecule on the cell surfaceProteins bind strongly to antibodies at pH 7.4 and weakly to antigens at pH 5.5 to 6.0 in vivo, potentially neutralizing multiple antigens and thus improving pharmacokinetics (it has been reported that the in vivo pH is typically between pH 5.5 and 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). The binding strength of pH-dependent binding varies between neutral conditions (pH 7.4) and acidic conditions (pH 5.5 to 6.0) and exists in naturally occurring protein-protein interactions. For example, the binding between the aforementioned IgG molecules and FcRn, the 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). It has been reported that the pH-dependent binding of the aforementioned IgG-FcRn interaction is associated with histidine residues present in IgG (see, for example, Mol. Cell. 7 (2001) 867-877).

[0107] III. Method according to the invention

[0108] This paper reports a novel method for estimating the clearance of therapeutic antibodies in the human body using a novel in vitro assay based on primary human cells. This macromolecular nonspecific clearance assay (LUCA) provides an in vitro-based method for assessing and predicting the PK characteristics of therapeutic antibodies.

[0109] The invention is based at least in part on the finding that the sum of antibodies taken up in vitro and recycled to 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. Differentiation between different antibodies cannot be achieved using the aforementioned non-primary endothelial cells (compare Figures 1 and 2). Figure 4 depicts a scheme of the method according to the invention.

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

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

[0113] The present 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 not suitable for this purpose. Differentiation between different antibodies cannot be achieved using said non-primary endothelial cells (see Figures 1 and 2).

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

[0115] In Figures 1 and 2, the time course of fluorescence intensity of different antibodies during incubation with endothelial cells (human microvascular endothelial cells, HMEC1; Figure 1) and with primary endothelial cells (human primary liver endothelial cells; Figure 2) labeled with the same pH-sensitive fluorescent dye is compared. As can be seen from Figure 1, differentiation was not possible for five of the seven antibodies when using simple endothelial cells. In contrast, all seven antibodies were able to differentiate 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 antibodies. 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. Specification 14 / 24 pages 16 CN 121577897 A

[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 pH range of 4 to 5.

[0120] An exemplary suitable dye is pHAb dyes 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 at 560 nm.The emission maximum (Em) is located at nm. pHAb dyes have 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 pH decreases 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 linker is sulfoDBCO-PEG4-Amine, sold by ClickChemistryTools. SulfoDBCO-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 linker. Conjugation is achieved by activating the antibody with an azide and reacting it with the DBCO moiety using click chemistry.

[0123] In the following, the invention is illustrated by example using pHAb dyes and conjugation using sulfoDBCO-PEG4-amine linkers. Any other dye exhibiting the above-described features or linker or binding properties that do not interfere with antibody binding, as well as the pH-dependent fluorescence properties of dye specification page 15 / 24, CN 121577897 A, can also be used. This is presented only 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 internalized antibody was obtained using FACS with an excitation wavelength of 488 nm and a detection wavelength 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). GraphPad Prism was used to analyze the data from each antibody.The normalized geometric mean was plotted as a linear regression curve to extract the slope (geometric mean MFI / min for 120 and 240 min, including the origin, i.e., 0 / 0). Two antibodies were used to normalize the slope: murvizumab with 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 slope was plotted against the clearance values ​​of human, cynomolgus monkey, and hFcRn Tg32+ / + mice in the corresponding in vivo using TIBCO Spotfire software. The corresponding plots for human, cynomolgus monkey, and human FcRn transgenic mice with different antibodies including those 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 steps of: a) incubating 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 determining the geometric mean intracellular fluorescence intensity of the primary human endothelial cells for each incubation time, optionally washing the cells to remove adhering fluorescently labeled antibodies before determining the intracellular fluorescence intensity; b) at the same time point as a), determining the geometric mean intracellular fluorescence intensity of primary human endothelial cells not incubated with any labeled antibody, optionally washing the cells to remove adhering fluorescent compounds before determining the intracellular fluorescence intensity; c) determining a relatively normalized intracellular fluorescence intensity rate by: i) subtracting the geometric mean intracellular fluorescence intensity of the primary human endothelial cells determined at the same time point from each of the geometric mean intracellular fluorescence intensities determined in a) for the antibody and reference antibody in question, to obtain a corrected geometric mean intracellular fluorescence intensity; ii) The corrected geometric mean intracellular fluorescence intensity obtained in step 2) of the antibody under discussion and the reference antibody is divided by the number of fluorescent dye molecules present in the respective 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 step ii) as in step a) and the origin, the best-fit line (i.e., the linear regression curve y = sx + b) is determined for each of the antibodies under discussion and the reference antibody.y = normalized geometric mean (intracellular) fluorescence intensity, s = slope, x = time and b = slope of the y-axis intersection point; iv) normalize the slope of the best-fit line of the antibody in question as follows: normalized slope (antibody in question) = specification 16 / 24 page 18 CN 121577897 A

[0129] where the in vivo clearance rate of the antibody in humans or cynomolgus monkeys or mice is the clearance rate of the first reference antibody in humans or 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 the relatively normalized (intracellular) fluorescence intensity (rate).

[0131] An in vitro-in vivo correlation has been established by comparing the relatively normalized intracellular fluorescence intensity rate according to the invention with the clearance rate determined in vivo. 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 antibodies with 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 present invention, the true scope of which is set forth in the appended claims. It should be understood that modifications may be made to the described procedures without departing from the spirit of the invention.

[0135] Examples

[0136] I Materials and Methods

[0137] Antibodies

[0138] The reference antibodies used in the experiments were an anti-pTau antibody having the heavy chain amino acid sequence of SEQ ID NO: 01 and the light chain amino acid sequence of SEQ ID NO: 02, and an anti-Her 3 antibody having 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 herein 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) using a BioAnalyzer 2100 (Agilent Technologies).(Technologies, Santa Clara, CA, USA) molecular weight distribution was analyzed.

[0142] Expression plasmid

[0143] To express the above-mentioned antibody, 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 genomic tissues with the CMV promoter.

[0144] In addition to the antibody expression cassette, the plasmid also contains: - an origin of replication that allows the plasmid to be replicated in E. coli, - a β-lactamase gene that confers ampicillin resistance in E. coli, and - a dihydrofolate reductase gene from Mus musculus as a selection marker in eukaryotic cells.

[0145] The transcriptional unit of the antibody gene consists of the following elements: - a unique restriction site at the 5' end, - an immediate early enhancer and promoter from human cytomegalovirus, - in the case of cDNA organization, followed by an intron A sequence, 19 CN 121577897 A (page 17 / 24 of specification), - the 5' untranslated region of the human antibody gene, - an immunoglobulin heavy chain signal sequence, - the human antibody chain, which is either cDNA or a genome organization with immunoglobulin exon-intron organization, - a 3' untranslated region with a polyadenylation signal sequence, and - a unique restriction site at the 3' end.

[0146] A fusion gene containing the antibody chain is generated by PCR and / or gene synthesis, and the fusion gene is assembled by known recombination methods and techniques, for example by linking the corresponding nucleic acid segments using unique restriction sites in the corresponding plasmid. The nucleic acid sequence of the subcloned gene is verified by DNA sequencing. For transient transfection, a large quantity of plasmids (Nucleobond AX, Macherey-Nagel) was prepared from the transformed E. coli culture via plasmid preparation.

[0147] Cell Culture Techniques

[0148] Standard cell culture techniques were used as described in Current Protocols in Cell Biology (2000), Bonifacino, JS, Dasso, M., Harford, JB, Lippincott-Schwartz, J., and Yamada, KM (ed.), John Wiley & Sons, Inc.

[0149] Transient Transfection in the HEK293-F System

[0150] The corresponding plasmids were transiently transfected using the HEK293-F system (Invitrogen) according to the manufacturer's instructions.Antibodies are generated (e.g., encoding heavy chains and corresponding light chains). 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 seeded at a density of 1106 cells / mL in 600 mL and incubated at 120 rpm and 8% CO2. Cells were transfected at a density of approximately 1.5 × 10⁶ cells / mL using approximately 42 mL of the following mixtures on the second day, encoding the heavy and corresponding light chains in equimolar ratios: A) 20 mL of Opti-MEM (Invitrogen) containing 600 µg total plasmid DNA (1 µg / mL) and B) 20 mL of Opti-MEM + 1.2 mL of 293 fectin or fectin (2 µL / mL). Glucose solution was added during fermentation according to glucose consumption. The supernatant containing secreted antibodies was harvested after 5–10 days, and the antibodies were either purified directly from the supernatant or frozen and stored. Therefore, some antibodies have been produced: Instructions for Use, Pages 18 / 24, 20 CN 121577897 A

[0151] Purification

[0152] Antibodies were purified from cell culture supernatants using affinity chromatography with MabSelectSure-Sepharose™ (GE Healthcare, Sweden), hydrophobic interaction chromatography with butyl agarose (GE Healthcare, Sweden), and size exclusion chromatography with Superdex 200 (GE Healthcare, Sweden).

[0153] Briefly, sterile filtered cell culture supernatants were 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, pH 9.0. Antibody pools for hydrophobic interaction chromatography were prepared 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 using acetic acid.After equilibrating butyl agarose resin with 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 the antibody 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 the antibody were combined, concentrated to the desired concentration using a Vivaspin ultrafiltration device (Sartorius Stedim Biotech S.A., France), and stored at -80°C.

[0154] After each purification step, purity and antibody integrity were analyzed by CE-SDS using microfluidic Labchip technology (Caliper Life Science, 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 using the HT Protein Express chip on the LabChip instruction manual page 19 / 24, 21 CN 121577897 A GXII system. Data were analyzed using LabChip GX software.

[0155] Mice

[0156] B6 .Cg-Fcgrttm1Dcr Tg(FCGRT)276Dcr mice lacked 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 (Bar Harbor, ME, USA) (female, 4–10 weeks old, weight 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 divided into 3 groups of 6 mice each, covering 9 serum collection time points (0.08, 2, 8, 24, 48, 168, 336, 504 and 672 after administration).(hours). Each mouse underwent two retroorbital blood draws 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 by 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 reagent diluted in assay buffer (Roche Diagnostics, Penzberg, Germany) for 1 hour. After washing, serum samples of different dilutions were added, followed by incubation for another 1 hour. After repeated washing, the bound antibody was detected by subsequent incubation with the detection antibody, followed by the anti-digoxin antibody 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 out 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 a reference standard.

[0162] PK Analysis

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

[0164] Briefly, due to the non-linear reduction of the antibody, the area under the curve (AUC0-inf) value was calculated using the logarithmic trapezoidal method and extrapolated to infinity using the apparent terminal rate constant λz, extrapolated from the concentration observed at the last time point.

[0165] Plasma clearance was calculated as dose rate (D) divided by AUC0-inf. Apparent terminal half-life (T1 / 2) is derived from the equation T1 / 2 = ln2 / λz.

[0166] Example 1

[0167] Cynomolgus monkey SDPK study

[0168] The pharmacokinetics of the test compound were determined in cynomolgus monkeys after 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. Under linear pharmacokinetic conditions, pharmacokinetic parameters were determined by standard non-compartmental methods. The clearance rate 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 rate was determined by the following alternative methods: either estimate the clearance value after IV administration at a high dose level, at which additional nonlinear clearance pathways are practically saturated; or, establish a PK model that includes both linear and nonlinear, saturable clearance terms. In these cases, the linear clearance rate fraction determined by the model is used for correlation.

[0169] Example 2

[0170] Preparation of FcRn affinity column

[0171] Expression of FcRn in HEK293 cells

[0172] FcRn was transiently expressed by transfecting HEK293 cells with two plasmids containing coding sequences for FcRn and β-2-microglobulin. The 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 a density of 3 to 4 × 10⁵ cells / ml every 2–3 days.

[0173] For transient expression, a 14 L stainless steel bioreactor was started at 36.5 °C, pH 7.0 ± 0.2, pO₂ 35% (aerated with N₂ and air, total gas flow rate 200 ml min⁻¹), with a culture volume of 8.1 L and a stirrer speed of 100–400 rpm. When the cell density reached 20 × 10⁵ cells / ml, 10 mg of plasmid DNA (equimolar amounts of the two plasmids) was diluted in 400 ml of Opti-MEM (Invitrogen). 20 ml of 293fectin (Invitrogen) was added to the mixture, and the mixture was incubated at room temperature for 15 minutes, followed by transfer to a fermenter. Starting the next day, the cells were fed in a continuous mode: feed solution was added at a rate of 500 ml per day, and glucose was added as needed.Glucose levels were maintained above 2 g / L. Seven days after transfection, the supernatant was collected at 4000 rpm for 90 minutes using a swing centrifuge with a 1 L bucket. 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 neonatal Fc receptor

[0175] 3 mg of FcRn β-2-microglobulin complex was dissolved / diluted in 5.3 mL of 20 mM sodium dihydrogen phosphate buffer containing 150 mM sodium chloride and added to 250 μL of PBS and 1 tablet of complete protease inhibitor (Complete ULTRA tablet, Roche Diagnostics GmbH). FcRn was biotinylated using a biotinylation kit from Avidity according to the manufacturer's instructions (Bulk BIRA, Avidity LLC). The biotinylated reactants were incubated overnight at room temperature.

[0176] The biotinylated FcRn was dialyzed overnight at 4°C against 20 mM MES buffer (containing 140 mM NaCl, pH 5.5) (Buffer A) to remove excess biotin.

[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 instructions using FcRn affinity column and pH gradient, pages 21 / 24, CN 121577897 A

[0181] Conditions: Column size: 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 30 µg of sample was applied to the FcRn affinity column equilibrated with buffer A. In 20% buffer BAfter a 10-minute wash at a flow rate of 0.5 mL / min, elution was performed for over 70 minutes using a linear gradient of 20% to 70% buffer B. Detection was performed using UV absorption at a wavelength of 280 nm. The column was regenerated with 20% buffer B for 10 minutes after each run.

[0182] To calculate relative retention times, according to Bertoletti-Ciarlet, A. et al. (Mol. Immunol. 46 (2009) 1878-1882), standard samples (anti-Her3 antibody (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] Briefly, the antibody (9 mg / mL) in 10 mM sodium phosphate 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-cooled 10 mM sodium acetate buffer, pH 5.0.

[0184] Example 4

[0185] Chromatography using a heparin affinity column and pH gradient

[0186] Conditions: Column size: 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), which was 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 ultraviolet light absorption at a wavelength of 280 nm.

[0187] Example 5

[0188] Examination of antibody internalization

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

[0190] Briefly, the corresponding antibody was conjugated to a pHAb amine-reactive dye and then diluted with cell culture medium. Meanwhile, cells were seeded into 6-well plates (each well)1 × 10⁵ cells), 100 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] The antibody was labeled using the SiteClick™ Antibody Azido Modification Kit (Thermo Fisher Scientific) according to the manufacturer's instructions. Briefly, the N-linked galactose residue in the Fc region was removed by β-galactosidase and replaced by azide-containing galactose (GalNaz) via β-1,4-galactosyltransferase (GalT). This azide modification enables copper-free conjugation of sDIBO-modified dyes. A pH-sensitive amine-reactive dye (523 nm) was purchased from Promega and conjugated with sulfoDBCO PEG4 amine. The antibody was labeled with an excess of 2 molar dye. Excess dye was removed using an Amicon® Ultra-2 centrifuge filter with a MWCO of 50 kDa (EMD Millipore, # UFC200324), and the antibody was reburied in 20 mM histidine buffer (pH 5.5). The concentration of labeled antibody[1] and the dye-to-antibody ratio (DAR) were determined using a Nanodrop spectrometer at 280 nm (A280 nm) and 532 nm (A532 nm)[2].

[0194] CAB = [A280nm - [A280nm CFDye]] / εmAb [1]

[0195] DAR = [A532nm MWmAb] / [cmAb εDye] [2]

[0196] εDye = 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 EBMTM-2 endothelial cell growth basal medium-2 (Lonza, #CC-3156) supplemented with EGMTM-2 MV microvascular endothelial cell growth medium SingleQuotsTM (Lonza, #CC-4176). Before antibody treatmentFive days later, 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 10⁴ cells / well to allow adhesion for 48 hours. The culture medium was changed after 24 hours, and 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, and then incubated in the culture medium with 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 the 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. The trypsin was inactivated by adding 100 µl of FACS buffer (20% FCS, 1 mM EDTA in DPBS).

[0202] Example 8

[0203] Flow Cytometry and Pharmacokinetic Analysis

[0204] The mean fluorescence intensity (MFI, more specifically the geometric mean) of the internalized antibody was obtained using a MACSQuant® Analyzer 10 (Miltenyi Biotec), which is equipped with a laser excitation at 488 nm and filters for collecting the emitted light at 585 nm / 540 nm. The exact same conditions, gain, and gate were used at both time points (2 hours and 4 hours). Data extraction was performed using FloJo_V10 software. The negative control value was 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 to normalize the slope: murvizumab-YTE was set to 0 and TCB was set to 1. The final slope was plotted using TIBCO Spotfire software against 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 evaluate the antibody's specification 23 / 24 pages 25 CN 121577897 AWhether binding affinity changes during labeling. Heparin chromatography and neonatal Fc receptor binding have previously been shown to predict in vitro antibody clearance (Kraft, TE, et al., MABS 12 (2020) e1683432). Here, this method is used to illustrate the potential 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 antibody. 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 antibody was 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 that are reliable within the assays according to the invention, the difference from the geometric mean is expected to be less than 15%. Instruction manual, page 24 / 24; 26 CN 121577897 A; Figure 1; Instruction manual, Figure 1 / 10; 27 CN 121577897 A; Figure 2; Instruction manual, Figure 2 / 10; 28 CN 121577897 A; Figure 3; Instruction manual, Figure 3 / 10; 29 CN 121577897 A; Figure 4; Instruction manual, Figure 4 / 10; 30 CN 121577897 A; Figure 5; Instruction manual, Figure 5 / 10; 31 CN 121577897 A; Figure 6; Instruction manual, Figure 6 / 10; 32 CN 121577897 A; Figure 7; Instruction manual, Figure 7 / 10; 33 CN 121577897 A; Figure 8; Instruction manual, Figure 8 / 10; 34 CN 121577897 A; Figure 9; Instruction manual, Figure 9 / 10; 35 CN 121577897 A; Figure 10 Specification Figures 10 / 10 Page 36 CN 121577897 A PH 260169 CN 202511775751.5 PH 260169 Title: LARGE MOLECULE NON-SPECIFIC CLEARANCE ASSAY Invention Name: Macromolecular Non-Specific Scavenging Assay Abstract Herein isThis paper reports a method for determining the non-specific clearance of an antibody, comprising the steps of incubating the antibody, conjugated to a pH-sensitive fluorescent dye, with primary human endothelial cells, and determining the fluorescence intensity of the primary human endothelial cells, whereby an increase in the fluorescence intensity of the primary human endothelial cells above background level is indicative of the non-specific clearance of the antibody.

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 numbering).

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.

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