Macromolecular non-specific clearance assay

Abstract

Herein is reported a method for determining non-specific 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 background level is indicative of non-specific clearance of the antibody.

Description

[0001] A novel method for estimating the clearance of therapeutic proteins in humans using a new in vitro assay based on human primary cells is reported herein. This macromolecule non-specific clearance assay (LUCA) provides an in vitro based approach to assess and predict key PK properties for therapeutic proteins. BACKGROUND

[0002] Class G human immunoglobulins (IgG) comprise two antigen binding (Fab) regions that convey specificity for a target antigen as well as a constant region (Fc region) that is responsible for interaction with Fc receptors (see e.g. Edelman, G. M., Scand. J. Immunol. 34 (1991) 1-22; Reff, M. E. and Heard, C, Crit. Rev. Oncol. Hematol. 40 (2001) 25-35). The average serum half-life of human IgG of the IgGl, IgG2 and IgG4 subclasses is 21 days, which is longer than the serum half-life of any other known serum protein (see e.g. Waldmann, T. A. and Strober, W., Prog. Allergy 13 (1969) 1-110). This long half-life is mainly mediated by the interaction between the Fc region and the neonatal Fc receptor (FcRn) (see e.g. Ghetie, V. and Ward, E. S., 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.

[0003] The neonatal Fc receptor, FcRn, is a membrane-associated receptor that is involved in IgG and albumin homeostasis, maternal IgG transport across the placenta, and antigen IgG immune complex phagocytosis (see, e.g., Brambell, F.W., et al., Nature 203 (1964) 1352-1354; Rouse, D.C., et al., J. Immunol. 170 (2003) 3528-3533). Human FcRn is a heterodimer composed of a glycosylated class I major histocompatibility complex-like protein (a-FcRn) and a β2 microglobulin (β2m) subunit (see, e.g., 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, e.g., Rouse, D.C. and Akilesh, S., Nat. Rev. Immunol. 7 (2007) 715-725; Martin, W.L., et al., Mol. Cell 7 (2001) 867-877; Goebl, N.A., et al., Mol. Biol. Cell 19 (2008) 5490-5505; Kim, J.K., et al., Eur. J. Immunol. 24 (1994) 542-548.), and two FcRn molecules can bind to the Fc region simultaneously (see, e.g., 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, showing nanomolar affinity at endosomal pH of 5-6, and rather weak binding at physiological pH of 7.4 (see, e.g., Goebl, N.A., et al., Mol. Biol. Cell 19 (2008) 5490-5505; Ober, R.J., et al., Proc. Natl. Acad. Sci. USA 101 (2004) 11076-11081; Ober, R.J., et al., J. Immunol. 172 (2004) 2021-2029). The potential mechanism for conferring long half-life to IgG can be explained by three basic steps.First, IgG is subject to non-specific pinocytosis by multiple cell types (see, e.g., Akilesh, S., et al., J. Immunol. 179 (2007) 4580-4588; Montoyo, H.P., et al., Proc. Natl. Acad. Sci. USA 106 (2009) 2788-2793.). Second, IgG encounters and binds FcRn in acidic endosomes at pH 5-6, thereby protecting IgG from lysosomal degradation (see, e.g., Ropeenian, D.C. 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 in the extracellular space (see, e.g., Ghetie, V. and Ward, E.S., Annu. Rev. Immunol. 18 (2000) 739-766). This strict pH-dependent binding and release mechanism is crucial for IgG recycling, and any deviation in the binding properties at different pH values can strongly influence the circulating half-life of IgG (see, e.g., Vaccaro, C., et al., Nat. Biotechnol. 23 (2005) 1283-1288).

[0004] Eigenmann, M.J. et al. summarize that cellular uptake of antibodies is thought to occur mainly in endothelial cells and hematopoietic cells. Once antibodies are taken up into endosomes, they can be protected from degradation by binding to the neonatal Fc receptor (FcRn). The neonatal Fc receptor binds antibodies in a pH-dependent manner, with higher affinity at pH 6 in endosomes than at physiological pH > 7.4 in plasma. Thus, antibodies bound to FcRn in endosomes are released into plasma at neutral pH, allowing antibody recycling rather than lysosomal degradation (MABS 9 (2017) 1007-1015).

[0005] Grevys, A. et al. report a human endothelial cell-based recycling assay for screening molecules targeting FcRn (Nat. Commun. 9 (2018) 621). Nath, N. et al. report a homogeneous plate-based antibody internalization assay using a pH sensor fluorescent dye (J. Immunol. Meth. 431 (2016) 11-21).

[0006] Fluorescent sensor reagents and methods of their use and manufacture are provided in WO 2013 / 134686. In particular, sensor reagents are provided that exhibit a detectable change in fluorescence (e.g., fluorescence intensity) upon a change in the pH of the surrounding environment (e.g., upon moving from one pH environment to another).

[0007] Based on the major biological factors of non-specific clearance of therapeutic antibodies in patients, i.e. non-specific uptake via pinocytosis and FcRn-mediated recycling, an in vitro method for predicting in vivo clearance (i.e. half-life) is needed. SUMMARY

[0008] Herein is reported a method for determining the level of non-specific clearance of a therapeutic protein, especially an antibody, by pinocytosis and lysosomal degradation.

[0009] The present invention is at least partially based on the finding that the uptake of an antibody into primary human endothelial cells in vitro can be used as a surrogate to assess non-specific clearance of said antibody in vivo, in particular in mice, cynomolgus monkeys and humans.

[0010] The present invention is at least partially based on the finding that only primary human endothelial cells can be used to determine in vivo clearance rates from in vitro experiments, as non-primary endothelial cells do not show the same correlation and thus are not suitable for this purpose. Using said non-primary endothelial cells, no differentiation between different antibodies can be achieved.

[0011] The present invention is at least partially based on the finding that the contribution of the uptake of an antibody by pinocytosis and its transport to the lysosomal compartment of primary endothelial cells is the largest and shows a good correlation with the fluorescence of primary endothelial cells.

[0012] The present invention thus comprises a method for determining or estimating the non-specific (i.e. non-target mediated) clearance (rate) of an antibody, the method comprising the steps of:

[0013] a) incubating an antibody conjugated to a pH-sensitive fluorescent dye with primary human endothelial cells (for a defined time), and

[0014] b) determining the (intracellular) fluorescence intensity of the primary human endothelial cells obtained in step a) (after the defined incubation time),

[0015] wherein the presence of non-specific clearance of the antibody (i.e. indicative of non-specific clearance of the antibody) is determined by an increase in the (intracellular) fluorescence intensity of the primary human endothelial cells determined in step b) over the background level (i.e. the (intracellular) fluorescence of the primary human endothelial cells not incubated with the antibody).

[0016] In certain embodiments, the method further comprises the step of:

[0017] - determining the (intracellular) fluorescence intensity of the primary human endothelial cells before incubation with / without the antibody,

[0018] and

[0019] determining the non-specific clearance (i.e. indicative for non-specific clearance of the antibody) in the presence of the antibody by the increase of the (intracellular) fluorescence intensity of the primary human endothelial cells determined in step b) over the (intracellular) fluorescence intensity determined for the primary human endothelial cells in the absence of the antibody.

[0020] Further, the present application comprises a method for selecting one or more antibodies with a low relative non-specific (non-target mediated) clearance (rate) from a multitude of antibodies, comprising the following steps:

[0021] a) incubating each of the multitude of antibodies separately 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;

[0022] b) selecting one or more antibodies from the multitude of antibodies which result in the lowest (intracellular) fluorescence intensity (change) of the primary human endothelial cells after incubation,

[0023] thereby selecting one or more antibodies with a low relative non-specific (non-target mediated) clearance (rate).

[0024] Further, the present application comprises a method for ranking a multitude of antibodies based on their non-specific (non-target mediated) clearance (rate), comprising the following steps:

[0025] a) incubating each of the multitude of antibodies separately 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;

[0026] b) ranking the antibodies based on the (intracellular) fluorescence intensity (change) from low to high or from high to low,

[0027] thereby ranking the antibodies based on their non-specific (non-target mediated) clearance (rate).

[0028] Further, the present application comprises a method for estimating or determining the (relative) in vivo clearance rate of an antibody in humans or cynomolgus monkeys or mice, comprising the following steps:

[0029] a) incubating the antibody conjugated to a pH sensitive fluorescent dye with the primary human endothelial cells for a defined time, and thereafter determining the (intracellular) fluorescence intensity (change) of the primary human endothelial cells;

[0030] b) incubating at least a first reference antibody with the primary human endothelial cells for the same defined time as in a), for which the human or cynomolgus or murine clearance is known, and which is conjugated to (in one preferred embodiment the same as in a)) a pH sensitive fluorescent dye, and thereafter determining the (intracellular) fluorescence intensity (change) of the primary human endothelial cells,

[0031] wherein the (relative) in vivo clearance of the antibody in humans or cynomolgus or mice is estimated or determined as the clearance of the first reference antibody in humans or cynomolgus or mice multiplied by the ratio of the fluorescence intensity (change) determined in a) to the (intracellular) fluorescence intensity (change) determined in b).

[0032] In certain embodiments, step b) is

[0033] b) i) incubating each member of a multitude of reference antibodies (i.e. at least two) with the primary human endothelial cells (separately) for the same defined time as in a), for which the human or cynomolgus or murine clearance is known, and which is conjugated to (in one preferred embodiment the same as in a)) a pH sensitive fluorescent dye,

[0034] ii) thereafter determining the (intracellular) fluorescence intensity (change) of the primary human endothelial cells for each of the reference antibodies, and

[0035] iii) calculating for the values obtained in ii) the best fitting straight line of the equation y = a*x + b, where y is the clearance in ml / day / kg and x corresponds to the fluorescence intensity (change).

[0036] In one embodiment of all aspects and embodiments, the (intracellular) fluorescence intensity (change) is the geometric mean (intracellular) fluorescence intensity (change).

[0037] In one embodiment of all aspects and embodiments, the (intracellular) fluorescence intensity (change) of the respective antibody in question is the relative normalized (intracellular) fluorescence intensity (change) rate obtained in a further step c) comprising:

[0038] 1) determining the (geometric mean) (intracellular) fluorescence intensity after two or more specified incubation times for the antibody in question and at least two reference antibodies, wherein in preferred embodiments the determination is at least for two time points after an incubation time of 2 hours and 4 hours;

[0039] 2) subtracting from each of the (geometric mean) (intracellular) fluorescence intensities determined in 1) for each of the antibody in question and the reference antibodies the (geometric mean) (intracellular) fluorescence intensity of the primary human endothelial cells (incubated for the same time but in the absence of antibodies) respectively, thereby obtaining corrected (geometric mean) (intracellular) fluorescence intensities;

[0040] 3) dividing the corrected (geometric mean) (intracellular) fluorescence intensities obtained in 2) for the antibody in question and the reference antibodies by the number of fluorescent dye molecules present in the respective antibody, to obtain normalized (geometric mean) (intracellular) fluorescence intensities (e.g. of at least two reference antibodies or of the antibody in question);

[0041] 4) determining the slope of the best fit straight line (i.e. the linear regression curve y = s*x + b, wherein y = normalized (geometric mean) (intracellular) fluorescence intensity, s = slope, x = time and b = y axis intersection) for each of the antibody in question and the reference antibodies based on the set of values consisting of the normalized (geometric mean) (intracellular) fluorescence intensities for at least two different incubation times of the antibody in question (i.e. each individual) as calculated in 3) and including the origin;

[0042] 5) normalizing the slope of the best fit straight line of the antibody in question as follows:

[0043]

[0044] In one embodiment of all aspects and embodiments, the incubated primary human endothelial cells are washed prior to determining the (intracellular) fluorescence (to remove non-specific / outer cell surface bound and unbound antibodies).

[0045] In one embodiment of all aspects and embodiments, the dye has a change in fluorescence intensity between a physiological pH of about 7 and an acidic pH in the range of pH 4 to 5 of about 10-fold, preferably of about 25-fold and most preferably of about 50-fold. In certain embodiments, the dye is of formula I / is a pHAb of formula I.

[0046]

[0047] Conjugation to the antibody or linker, if present, is at residue R of formula I.

[0048] In one embodiment of all aspects and embodiments, the dye is conjugated to the antibody at amino acid residue 297 in the Fc region (numbering according to Kabat).

[0049] In one embodiment of all aspects and embodiments, the dye is conjugated to the antibody by click chemistry.

[0050] In one embodiment of all aspects and embodiments, the dye is conjugated to the antibody directly or via a linker. In certain embodiments, the linker is sulfoDBCO-PEG4-amine of Formula II.

[0051]

[0052] The conjugation to the antibody is at the free amino group of Formula II.

[0053] In one embodiment of all aspects and embodiments, the dye is conjugated to a linker and the linker is conjugated to the antibody and the conjugate has the structure of Formula III.

[0054]

[0055] In one embodiment of all aspects and embodiments, the dye is conjugated to the antibody by chemical cross-linking.

[0056] In one embodiment of all aspects and embodiments, the fluorescence is determined by FACS by determining the shift of the fluorescence maximum.

[0057] In one embodiment of all aspects and embodiments, the fluorescence is the geometric mean fluorescence intensity determined by FACS.

[0058] In one embodiment of all aspects and embodiments, the primary human endothelial cells are primary human liver endothelial cells.

[0059] In one embodiment of all aspects and embodiments, the determination is performed after an incubation of at least 0.5 hours, i.e. the defined time is at least 0.5 hours.

[0060] In one embodiment of all aspects and embodiments, the determination is made after an incubation lasting up to 24 hours, i.e. the prescribed time is up to 24 hours. In certain embodiments, the determination is made after an incubation lasting up to 16 hours. In a preferred embodiment, the determination is made after an incubation lasting up to 4 hours, i.e. the prescribed time is up to 4 hours. In certain embodiments, the determination is made after an incubation lasting 2 hours or / and 4 hours, i.e. the prescribed time is 2 hours or / and 4 hours. In certain embodiments, the determination is made after an incubation lasting between 4 and 24 hours, i.e. the prescribed time is between 4 hours and 24 hours inclusive. In certain embodiments, the determination is made after an incubation lasting 4 hours or / and 8 hours, i.e. the prescribed time is 4 hours or / and 8 hours.

[0061] In certain embodiments, the determination is made directly after the incubation.

[0062] In one embodiment of all aspects and embodiments, the antibody has a Fc region of human origin. In certain embodiments, the Fc region belongs to the human IgGl or IgG2 or IgG4 subclass. In certain embodiments, the Fc region comprises one or more mutations affecting binding to human FcRn.

[0063] In one embodiment of all aspects and embodiments, the antibody is a fusion of the antibody with a further polypeptide. In certain embodiments, the further polypeptide is a scFv, a Fab, a scFab or a non-antibody polypeptide. In certain embodiments, the fusion is at the C-terminus of one of the heavy chains of the antibody.

[0064] In one embodiment of all aspects and embodiments, the antibody is a bispecific antibody.

[0065] In one embodiment of all aspects and embodiments, the first reference antibody is moxidomycin with mutations M252Y / S254T / T256E, and / or a bispecific antibody in the form of a TCB. BRIEF DESCRIPTION OF DRAWINGS

[0066] Figure 1 Time course of fluorescence intensity of different antibodies, all 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 = olaratumab; 4 = anti-CD20 antibody (1); 5 = avdismab; 6 = anti-human alpha-synuclein antibody; 7 = anti-CD20 antibody (2).

[0067] Figure 2Time course of fluorescence intensity of different antibodies labeled with the same pH sensitive fluorescent dye during incubation with human primary liver endothelial cells; 1 = anti-human phosphorylated Tau 422 antibody; 2 = anti-CD44 antibody; 3 = olaratumab; 4 = anti-CD20 antibody (1); 5 = avutumab; 6 = anti-human alpha-synuclein antibody; 7 = anti-CD20 antibody (2).

[0068] Figure 3 Protocol for fluorescently labeled antibodies used in the method according to the application; pHAb dye conjugated to antibodies via a Sulfo-DBCO-PEG4-Amine linker.

[0069] Figure 4 Protocol for the method according to the application.

[0070] Figure 5 The corrected mean fluorescence intensity (MFI, more specifically the geometric mean) of internalized antibodies acquired using FACS is obtained by subtracting the negative control and then normalizing (dividing) with the dye to antibody ratio (DAR). The corrected and normalized geometric mean from each antibody is plotted as a linear regression curve and the slope is extracted (geometric mean MFI / minute for 120 and 240 minutes). Two standard antibodies are chosen to normalize the slope: moxetumab-YTE is set to 0 and TCB is set to 1. The final slope is plotted against in vivo human clearance values. If different clearance values are obtained, then the dose linear clearance describing non-specific clearance of the molecule is used.

[0071] Figure 6 The corrected mean fluorescence intensity (MFI, more specifically the geometric mean) of internalized antibodies acquired using FACS is obtained by subtracting the negative control and then normalizing (dividing) with the dye to antibody ratio (DAR). The corrected and normalized geometric mean from each antibody is plotted as a linear regression curve and the slope is extracted (geometric mean MFI / minute for 120 and 240 minutes). Two standard antibodies are chosen to normalize the slope: moxetumab-YTE is set to 0 and TCB is set to 1. The final slope is plotted against in vivo cynomolgus monkey clearance values. If different clearance values are obtained, then the dose linear clearance describing non-specific clearance of the molecule is used.

[0072] Figure 7The corrected mean fluorescence intensity (MFI, more specifically the geometric mean) of internalized antibodies acquired using FACS is 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 is plotted as a linear regression curve and the slope is extracted (geometric mean MFI / minute for 120 and 240 minutes). Two standard antibodies are chosen to normalize the slope: Movlitumab-YTE is set to 0 and TCB is set to 1. The final slope is plotted against the in vivo hFcRn Tg32+ / + mouse clearance value.

[0073] Figure 8 Fc variants of IgG show the same in vitro-in vivo correlation as wt Fc IgG.

[0074] Figure 9 Time course of mean fluorescence intensity of primary human endothelial cells incubated with monospecific bivalent antibodies.

[0075] Figure 10 Flow cytometry analysis of primary human liver-derived endothelial cells. Endothelial cells were incubated with antibodies, previously labeled with pHAb amine-reactive dye (532 nm): low clearance antibody Movlitumab-YTE (solid line), two medium clearance bispecific antibodies (dashed and dotted lines, respectively), and high clearance bispecific antibody (dotted line). After 4 hours, fluorescence intensity was recorded and cells were single-line, morphology, and viability gated;

[0076] The y-axis scaling is relative to the number of events,

[0077] The x-axis scaling shows the intensity in the PE channel. DETAILED DESCRIPTION

[0078] The present invention is based at least in part on the finding that a cell-based assay using primary human endothelial cells can be used to estimate the in vivo lysosomal degradation rate of therapeutic antibodies in vitro.

[0079] By using human primary cells, the inventors of the present application have found that the readout of the method according to the present invention correlates significantly with non-specific clearance in humans. This has been demonstrated for more than 20 therapeutic antibodies in clinical trials or already on the market. The inventors of the present application further found that the method according to the present invention is equally applicable to regular bispecific antibodies, monoclonal antibodies, reflecting the Y shape of wild-type human antibodies, as well as to unconventional bispecific antibody types of human antibodies having a different form than wild-type and more than two valences and antibody Fc region fusions. This provides evidence for the general applicability of the correlation of the readout of the method according to the present invention to mouse, cynomolgus monkey, and human clearance rates.

[0080] The method according to the present application can be used to estimate the pharmacokinetic (PK) properties of different antibody molecules (different in form, valency and specificity).

[0081] Thus, the method according to the present application can be used to

[0082] - support the selection of suitable clinical lead molecules with respect to PK (pharmacokinetic) properties (clearance and half-life, respectively);

[0083] - deselect antibodies from a library with PK properties that are not suitable for therapeutic applications, i.e. with high clearance or short half-life in vivo, respectively;

[0084] - rank members of a set of antibodies according to PK properties (clearance and half-life, respectively);

[0085] - determine the relative in vivo clearance of an antibody of interest based on the in vivo clearance of a reference antibody (or a number of reference antibodies) with known PK properties only, i.e. without the need to perform in vivo tests;

[0086] - guide the engineering of antibodies with respect to PK properties (by changing the FcRn affinity of the Fc region or by engineering the charge patch of the Fab (the latter is described in WO 2018 / 197533);

[0087] - determine the need for PK engineering and evaluate the results of PK engineering.

[0088] Thus, the assay according to the present application can reduce or even replace animal PK studies.

[0089] I. Definitions

[0090] As used herein, the amino acid positions of all constant regions and domains of heavy and light chains are numbered according to the Kabat numbering system described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991), and are referred to herein as "numbering according to Kabat". Specifically, the Kabat numbering system (see pages 647-660 of Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)) is used for the light chain constant domain CL of kappa and lambda isotypes, and the EU index numbering system of Kabat (see pages 661-723) is used for the constant heavy chain domains (CH1, hinge, CH2 and CH3, which are further classified herein by referring to "EU index numbering according to Kabat" in this case).

[0091] A knob-in-hole dimerization module and its use in antibody engineering is described in Carter P., Ridgway J. B. B., Presta L. G.: Immunotechnology, February 1996, Vol. 2, No. 1, pages 73-73(1 ).

[0092] General information on the nucleotide sequences of human immunoglobulin light and heavy chains is given in: Kabat, E. A. et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991 ).

[0093] Methods and techniques useful in practicing the present application are described, for example, in Ausubel, F. M. (ed.), Current Protocols in Molecular Biology, Volumes I to III (1997); Glover, N. D. and Hames, B. D. (eds.), DNA Cloning: A Practical Approach, Volumes I and II (1985), Oxford University Press; Freshney, R. I. (ed.), Animal Cell Culture - a practical approach, IRL Press Limited (1986); Watson, J. D. et al., Recombinant DNA, Second Edition, CHS L Press (1992); Winnacker, E. L., From Genes to Clones; N. Y., VCH Publishers (1987); Celis, J. (ed.), Cell Biology, Second Edition, Academic Press (1998); Freshney, R. I., Culture of Animal Cells: A Manual of Basic Technique, Second Edition, Alan R. Liss, Inc., N. Y. (1987).

[0094] Nucleic acid derivatives can be generated using recombinant DNA technology. Such derivatives can be modified, for example, at a single or few nucleotide positions by substitution, alteration, exchange, deletion or insertion. The modification or derivatization can be performed, for example, by means of site-directed mutagenesis. Such modifications can be readily performed by the person 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).

[0095] It must be noted that, as used herein and in the appended claims, singular articles such as "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth. Likewise, the terms "one or more", "one or the other", and "at least one" are used interchangeably. It is further noted that the terms "comprising", "including", and "having" can be used interchangeably.

[0096] The term "about" denotes a + / - 20% range of the numerical value that follows it. In certain embodiments, the term "about" denotes a + / - 10% range of the numerical value that follows it. In certain embodiments, the term "about" denotes a + / - 5% range of the numerical value that follows it.

[0097] As used herein, the term "determining" also includes the terms measuring and analyzing.

[0098] The term "comprising" also includes the term "consisting of".

[0099] The term "antibody" herein is used in the broadest sense and includes various antibody structures, including but not limited to monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies), so long as they are full-length antibodies and exhibit the desired antigen and / or FcRn binding activity.

[0100] "Multispecific antibodies" indicate having binding specificities for at least two non-identical epitopes on the same antigen or two different antigens. Multispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2bispecific antibodies) or combinations thereof (e.g., a full-length antibody plus an extra scFv or Fab fragment). Engineered antibodies with two, three or more (e.g., four) functional antigen binding sites have also been reported (see, e.g., US 2002 / 0004587 Al).

[0101] The term "binds (to an antigen)" denotes binding in an in vitro assay. In certain embodiments, binding is determined in a binding assay in which the antibody is bound to a surface and binding of antigen to the antibody is measured by surface plasmon resonance (SPR). The term "binds" also includes the term "specifically binds".

[0102] The term "buffering substance" denotes a substance which, when in solution, can adjust changes in the pH of the solution, e.g., due to the addition or release of acidic or basic substances.

[0103] The "class" of an antibody refers to the type of constant domains or constant regions of the heavy chains of the antibody. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes) e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, d, e, g, and m, respectively.

[0104] The term "Fc-fusion polypeptide" denotes a fusion of a binding domain (e.g., an antigen binding domain, such as a single chain antibody, or a polypeptide, such as a ligand of a receptor) to an antibody Fc region that exhibits the desired targetting and / or protein A and / or FcRn binding activity.

[0105] The term "human-derived Fc region" denotes the C-terminal region of a human-derived immunoglobulin heavy chain, which contains at least part of the hinge region, the CH2 domain and the CH3 domain. In certain embodiments, the human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxy-terminus of the heavy chain. In certain embodiments, the Fc region has the amino acid sequence of SEQ ID NO: 05. However, the C-terminal lysine (Lys447) of the Fc region can or can not be present. The Fc region consists of two heavy chain Fc region polypeptides, which can be covalently linked to each other via the hinge region cysteine residues, forming an interchain disulfide bond.

[0106] The term "FcRn" denotes the human neonatal Fc receptor. The function of FcRn is to salvage IgG from the lysosomal degradation pathway, resulting in decreased clearance and increased half-life. FcRn is a heterodimeric protein consisting of a 50 kDa class I major histocompatibility complex-like protein (a-FcRn) and a 15 kDa b2-microglobulin (b2m). FcRn binds with high affinity to the CH2-CH3 portion of the Fc region of IgG. The interaction between IgG and FcRn is strictly pH-dependent and occurs with a stoichiometry of 1 :2, in which one IgG binds via its two heavy chains to two FcRn molecules (Huber, A. H. et al., J. Mol. Biol. 230 (1993) 1077-1083). FcRn binding occurs in endosomes at acidic pH (pH < 6.5) and IgG is released at the cell surface at neutral pH (pH of about 7.4). The pH sensitivity of this interaction facilitates FcRn-mediated protection of IgG from intracellular degradation by binding to the receptor within the acidic environment of the endosome. FcRn then facilitates recycling of IgG to the cell surface, whereupon it is released into the bloodstream upon exposure of the FcRn-IgG complex to the extracellular neutral pH environment.

[0107] The term“FcRn binding portion of an Fc region” refers to the portion of an antibody heavy chain polypeptide that extends generally from EU position 243 to EU position 261, generally from EU position 275 to EU position 293, generally from EU position 302 to EU position 319, generally from EU position 336 to EU position 348, generally from EU position 367 to EU position 393 and EU position 408, and generally from EU position 424 to EU position 440. In certain embodiments, one or more of the following amino acid residues according to EU numbering of Kabat are altered: 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 numbering).

[0108] The term“full length antibody” refers to an antibody having a structure substantially similar to that of a native antibody. A full length antibody comprises two full length antibody light chains comprising a light chain variable domain and a light chain constant domain, and two full length antibody heavy chains comprising a heavy chain variable domain, a first constant domain, a hinge region, a second constant domain, and a third constant domain. A full length antibody can comprise additional domains such as, for example, an additional scFv or scFab conjugated to one or more chains of the full length antibody. These conjugates are also encompassed by the term full length antibody.

[0109] The term "derived from" means that an amino acid sequence is derived from a parent amino acid sequence by the introduction of changes at at least one position. Thus, a 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 Fc region of an antibody). In certain embodiments, an amino acid sequence derived from a parent amino acid sequence differs by 1 to 15 amino acid residues at the corresponding positions. In certain embodiments, an amino acid sequence derived from a parent amino acid sequence differs by 1 to 10 amino acid residues at the corresponding positions. In certain embodiments, an amino acid sequence derived from a parent amino acid sequence differs by 1 to 6 amino acid residues at the corresponding positions. Likewise, a derived amino acid sequence has a high amino acid sequence identity to its parent amino acid sequence. In certain embodiments, an amino acid sequence derived from a parent amino acid sequence has 80% or more amino acid sequence identity. In certain embodiments, an amino acid sequence derived from a parent amino acid sequence has 90% or more amino acid sequence identity. In certain embodiments, an amino acid sequence derived from a parent amino acid sequence has 95% or more amino acid sequence identity.

[0110] The term "human Fc region polypeptide" means an amino acid sequence identical to a "native" or "wild type" human Fc region polypeptide. The term "variant (human) Fc region polypeptide" means an amino acid sequence derived from a "native" or "wild type" human Fc polypeptide that differs by at least one "amino acid change." A "human Fc region" is comprised of two human Fc region polypeptides. A "variant (human) Fc region" is comprised of two Fc region polypeptides, either of which can be a variant (human) Fc region polypeptide, or one is a human Fc region polypeptide and the other is a variant (human) Fc region polypeptide.

[0111] A "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally can comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0112] An “isolated” antibody is one which has been separated from a component of its natural environment. In some embodiments, an antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., size-exclusion, ion-exchange, or reverse-phase HPLC) methods. See, e.g., Flatman, S. et al., J. Chrom. B 848 (2007) 79-87 for a review of methods for assessing, e.g., antibody purity.

[0113] An “isolated” nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location different from that of normal existence.

[0114] The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind the same epitope, except for possible variants that can arise during production of the monoclonal antibody preparation, such variants are typically present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present application can be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein.

[0115] “Native antibodies” refer to naturally occurring immunoglobulin molecules with varying structures. For example, natural IgG antibodies are heterotetrameric glycoproteins of about 150,000 Daltons, composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From N- to C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from N- to C-terminus, each light chain has a variable region (VL), also called a variable light domain or a light chain variable domain, followed by a constant light (CL) domain. The light chains of antibodies can be assigned to one of two types, called kappa (K) and lambda (l), based on the amino acid sequence of their constant domains.

[0116] The term "pharmaceutical formulation" refers to a preparation which results in the bioavailability of the active ingredient(s) contained therein commensurate with its dosage as intended, and which is acceptable for administration to a subject in need to be administered the formulation.

[0117] A "pharmaceutically acceptable carrier" refers to a non-toxic component of a pharmaceutical formulation other than the active ingredients. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0118] The term "recombinant antibody" as used herein denotes all antibodies (chimeric, humanized and human antibodies) that are prepared, expressed, created or isolated by recombinant means. This includes antibodies isolated from a host cell such as a NS0, HEK, BHK or CHO cell or from a transgenic animal (e.g., mouse) of human immunoglobulin genes, or antibodies expressed using a recombinant expression plasmid transfected into a host cell. Such recombinant antibodies have variable and constant regions in a rearranged form. The recombinant antibodies as reported herein can be subject to in vivo somatic hypermutation. Thus, the amino acid sequences of the VH and VL regions of the recombinant antibodies are those that, although derived from and related to human germline VH and VL sequences, can not naturally exist within the human antibody germline repertoire in vivo.

[0119] The term "TCB" as used herein denotes a T cell bispecific antibody. Such antibodies can have the format described in WO2013 / 026831, for example. These molecules can simultaneously bind CD3 on a T cell (first specificity) and an antigen on a target (e.g., tumor) cell (second specificity), thereby inducing killing of the target cell. A TCB is a trivalent bispecific antibody composed of four polypeptides or polypeptide chains: one light chain which is a full length light chain; another light chain which is a domain exchanged full length light chain; one heavy chain which is a full length heavy chain; and another heavy chain which is an extended heavy chain comprising an additional domain exchanged heavy chain or light chain Fab fragment.

[0120] In a preferred embodiment, the TCB comprises:

[0121] a) a first Fab fragment and a second Fab fragment, each binding to a first antigen,

[0122] b) one domain exchanged Fab fragment specifically binding to a second antigen, in which the CH1 domain and the CL domain are exchanged with each other,

[0123] c) one Fc region comprising a first heavy chain Fc region polypeptide and a second heavy chain Fc region polypeptide,

[0124] wherein the C-terminus of the CHI domain of the first Fab fragment is connected to the N- terminus of one of the heavy chain Fc-region polypeptides and the C-terminus of the CHI domain of the domain exchanged Fab fragment is connected to the N-terminus of the other heavy chain Fc-region polypeptide, and

[0125] wherein the C-terminus of the CHI domain of the second Fab fragment is connected 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 exchanged Fab fragment, and

[0126] wherein the first antigen or the second antigen is human CD3.

[0127] In another equally preferred embodiment, the TCB comprises:

[0128] a) a first Fab fragment and a second Fab fragment, each binding to a first antigen,

[0129] b) one domain exchanged Fab fragment specifically binding to a second antigen, in which the VH domain and the VL domain are exchanged with each other,

[0130] c) one Fc region comprising a first heavy chain Fc-region polypeptide and a second heavy chain Fc-region polypeptide,

[0131] wherein the C-terminus of the CHI domain of the first Fab fragment is connected to the N- terminus of one of the heavy chain Fc-region polypeptides and the C-terminus of the CHI domain of the domain exchanged Fab fragment is connected to the N-terminus of the other heavy chain Fc-region polypeptide, and

[0132] wherein the C-terminus of the CHI domain of the second Fab fragment is connected 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 exchanged Fab fragment, and

[0133] wherein the first antigen or the second antigen is human CD3.

[0134] The term "valency" as used in the present application denotes the presence of a specified number of binding sites in a (antibody) molecule. Thus, the terms "bivalent", "tetravalent" and "hexavalent" denote the presence of two binding sites, four binding sites and six binding sites, respectively, in a (antibody) molecule. A preferred embodiment of the bispecific antibodies as reported herein is "bivalent".

[0135] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to its antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of an antibody generally have similar structures, with each domain comprising four framework regions (FRs) and three hypervariable regions (HVRs) (see, e.g., Kindt, T.J. et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., N.Y. (2007), page 91). A single VHor VLdomain can be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen can be isolated using a VHor VLdomain from an antibody that binds the antigen, to screen libraries of complementary VLor VHdomains, respectively. See, e.g., Portolano, S. et al., J. Immunol. 150 (1993) 880-887; Clackson, T. et al., Nature 352 (1991) 624-628).

[0136] The terms "variant," "modified antibody," and "modified fusion polypeptide" refer to molecules having an amino acid sequence that differs from that of a parent molecule. Typically, such molecules have one or more alterations, insertions, or deletions. In certain embodiments, a modified antibody or modified fusion polypeptide comprises an amino acid sequence comprising at least a portion of a non-naturally occurring Fc region. Such molecules have less than 100% sequence identity with the parent antibody or parent fusion polypeptide. In certain embodiments, the amino acid sequence of a variant antibody or variant fusion polypeptide has from about 75% to less than 100% amino acid sequence identity with the amino acid sequence of a parent antibody or parent fusion polypeptide, especially from about 80% to less than 100%, especially from about 85% to less than 100%, especially from about 90% to less than 100%, especially from about 95% to less than 100%. In certain embodiments, a parent antibody or parent fusion polypeptide and a variant antibody or variant fusion polypeptide differ by one (single), two, or three amino acid residues.

[0137] A "primary human endothelial cell" is a human cell isolated directly from its 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 a plastic or glass container, in a specialized medium containing essential nutrients and growth factors to support proliferation. Primary cells can be of two types: adherent cells or cells that grow in suspension. Adherent cells require attachment to grow and are known as anchorage-dependent cells. Adherent cells are usually derived from organ tissues. Suspension cells do not require attachment to grow and are known as anchorage-independent cells. Most suspension cells are isolated from blood.

[0138] The term "pH sensitive fluorescent dye" denotes a dye having different fluorescence intensity or emission wavelength at physiological pH of about pH 7.4 and at lysosomal pH of about pH 4.5.

[0139] II. In vivo antibodies

[0140] Since IgG molecules are bivalent, a single IgG molecule can neutralize up to two antigen molecules. There are two types of target antigens for neutralizing antibodies: soluble antigens present in the plasma and membrane-bound antigens expressed on the surface of cells.

[0141] In case the antigen is a membrane-bound antigen, the administered therapeutic antibody binds to the membrane-bound antigen on the surface of the cell. Subsequently, the antibody is taken up into the endosome inside the cell together with the membrane-bound antigen to which it is bound by internalization. Thereafter, the antibody still bound to the antigen moves to the lysosome, where it is degraded together with the antigen. The elimination of the antibody from the plasma mediated by internalization of the membrane-bound antigen is called antigen-dependent elimination. This has been reported for different antibody molecules (see for example Drug Discov. Today, 11 (2006) 81-88). Since a single IgG antibody molecule binds to two antigen molecules when it binds bivalently to the antigen, and is then internalized into the lysosome and directly degraded, a single ordinary IgG antibody cannot neutralize two or more antigen molecules.

[0142] The reason for the long retention (slow elimination) of IgG molecules in the plasma is the FcRn, called the salvage receptor for IgG molecules (see for example Nat. Rev. Immunol. 7 (2007) 715-725). IgG molecules that have been taken up into the endosome by pinocytosis bind to the FcRn expressed in the endosome under the acidic conditions in the endosome. The IgG molecules bound to the FcRn move to the cell surface, where they dissociate from the FcRn under the neutral conditions of the plasma. IgG molecules that cannot bind to the FcRn enter the lysosome, where they are degraded.

[0143] If the IgG antibody dissociates from the antigen under the acidic conditions in the endosome when the IgG antibody is taken up into the endosome inside the cell by internalization, the dissociated antibody can bind to the FcRn also present in the endosome. Thus, the IgG molecule that dissociated from the antigen and was bound by the FcRn is transferred to the cell surface and released from the FcRn into the plasma under the neutral conditions of the pH. The antibody is thereby recirculated into the plasma. The IgG molecule back in the plasma is able to bind to a new antigen again. The repetition of this process allows a single IgG molecule to repeatedly bind to an antigen, thereby enabling the neutralization of multiple antigens with a single IgG molecule.

[0144] In the case of soluble antigen, the administered therapeutic antibody binds to the antigen in the plasma and remains in the plasma in the form of an antigen-antibody complex. As in the case of IgG molecules that do not bind to the antigen, IgG molecules that bind to the antigen in the plasma are taken up into endosomes by pinocytosis. In the endosomes, they can bind to FcRn expressed in the endosomes under the acidic conditions in the endosomes. IgG molecules that bind to FcRn move to the cell surface and then dissociate from FcRn under the neutral conditions in the plasma. If the IgG molecules can dissociate from the antigen under the acidic conditions in the endosomes, the dissociated antigen cannot bind to FcRn and can be degraded by lysosomes. Since the IgG molecules that have returned to the plasma have dissociated from the antigen in the endosomes, they are able to bind to new antigens in the plasma again. Repetition of this process allows a single IgG molecule to repeatedly bind to soluble antigens. This enables a single IgG molecule to neutralize multiple antigens.

[0145] Therefore, regardless of whether the antigen is a membrane-bound antigen or a soluble antigen, if dissociation of the IgG antibody from the antigen under the acidic conditions in the endosomes is possible, a single IgG molecule can repeatedly neutralize the antigen.

[0146] More specifically, a single IgG molecule can be able to neutralize multiple antigens, improve pharmacokinetics, and the like, by strongly binding to the antibody at the cell surface pH of 7.4 and weakly binding to the antigen at the pH of 5.5 to 6.0 in the endosomes, since it has been reported that the pH in the endosomes is generally pH 5.5 to 6.0 (see, for example, Nat. Rev. Mol. Cell. Biol. 5 (2004) 121-132).

[0147] In general, protein-protein interactions are composed of hydrophobic interactions, electrostatic interactions, and hydrogen bonding, and the binding strength is generally expressed as a binding constant (affinity) or an apparent binding constant (avidity). The binding strength of pH-dependent binding, which is present in naturally occurring protein-protein interactions, changes between neutral conditions (pH 7.4) and acidic conditions (pH 5.5 to 6.0). For example, the binding between the above-described IgG molecule and FcRn, which is called a salvage receptor for IgG molecules, 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 above-described IgG-FcRn interaction is associated with histidine residues present in IgG (see, for example, Mol. Cell. 7 (2001) 867-877).

[0148] III. Methods according to the application

[0149] A new method for estimating therapeutic antibody clearance in humans using a novel in vitro assay based on human primary cells is reported herein. This macromolecule non-specific clearance assay (LUCA) provides an in vitro based method to assess and predict the PK characteristics of therapeutic antibodies.

[0150] The present invention is based at least in part on the finding that the sum of antibody uptake and recycling into primary human endothelial cells in vitro can be used as a surrogate to estimate the non-specific clearance of said antibody in vivo.

[0151] The present invention is based at least in part on the finding that only primary human endothelial cells can be used to predict clearance rates in vivo from in vitro experiments, as non-primary endothelial cells do not show the same correlation and thus are not suitable for this purpose. Using said non-primary endothelial cells, differentiation between different antibodies cannot be achieved (compare Figure 1 and Figure 2 ). Figure 4 A scheme of the method according to the present invention is depicted.

[0152] The present invention thus comprises a method for determining or estimating the non-specific (non-target mediated) clearance (rate) of an antibody, comprising the following steps:

[0153] a) incubating an antibody conjugated to a pH sensitive fluorescent dye with primary human endothelial cells, and

[0154] b) determining the fluorescence intensity (intracellularly) of the primary human endothelial cells of step a) after a defined incubation time,

[0155] wherein an increase of the fluorescence intensity (intracellularly) of the primary human endothelial cells determined in step b) relative to the background level (i.e. the fluorescence (intracellularly) of the primary human endothelial cells not incubated with the antibody) is indicative for the non-specific clearance of the antibody.

[0156] The present invention is based at least in part on the finding that the sum of antibody uptake and recycling into primary human endothelial cells in vitro can be used as a surrogate to estimate the non-specific clearance of said antibody in vivo.

[0157] The present invention is based at least in part on the finding that only primary human endothelial cells can be used to predict clearance rates in vivo from in vitro experiments, as non-primary endothelial cells do not show the same correlation and thus are not suitable for this purpose. Using said non-primary endothelial cells, differentiation between different antibodies cannot be achieved (see Figure 1 and 2 ).

[0158] The present invention is based at least in part on the finding that the contribution to the primary endothelial cell fluorescence is greatest by pinocytosis of the antibody and its transport to the lysosomal compartment without recycling by the FcRn of the primary endothelial cells.

[0159] In Figure 1 and Figure 2 , the time course of the fluorescence intensity during incubation with endothelial cells (human microvascular endothelial cells, HMEC1 ; Figure 1 ) and during incubation with primary endothelial cells (human primary liver endothelial cells; Figure 2 ) was compared for different antibodies which have been labeled with the same pH sensitive fluorescent dye. It can be seen from Figure 1 that for five of the seven antibodies, no differentiation was possible when using simple endothelial cells. In contrast, all seven antibodies could be differentiated when using primary endothelial cells (see Figure 2 ).

[0160] The labeled antibodies have been analyzed by heparin and FcRn chromatography. Exemplary retention times for non-labeled and labeled antibodies are shown in the table below. It can be seen that the labeling does not change the heparin and FcRn binding properties of the antibodies. For antibodies which are reliable in the assay according to the application, a difference from the geometric mean below 15% is expected.

[0161] Table 1 : Retention times of non-labeled and labeled antibodies on human heparin and human FcRn chromatography columns.

[0162]

[0163] The fluorescent label used in the method according to the application can be any pH dependent fluorescent dye which has a shift in fluorescence intensity of about 10-fold, preferably about 25-fold and most preferably about 50-fold between physiological pH of about 7 and an acidic pH in the range of pH 4 to 5.

[0164] Exemplary suitable dyes are the pHAb dyes sold by Promega. These dyes are pH sensor dyes which have very low fluorescence at pH > 7 and the fluorescence increases sharply as the pH of the solution becomes acidic. The pHAb dyes have an excitation maximum (Ex) at 532 nm and an emission maximum (Em) at 560 nm. There are two reactive forms of the pHAb dyes which are suitable for conjugation to antibodies: the pHAb amine-reactive dye and the pHAb thiol-reactive dye. The pHAb amine-reactive dye has a succinimidyl ester group which can react with the primary amines on lysine amino acids on the antibody. The pHAb thiol-reactive dye has a maleimide group which reacts with thiols. This maleimide group is expected to conjugate to the antibody after reduction of the disulfide bond between cysteines in the hinge region of the antibody using a reducing agent such as DTT or TCEP. The pHAb dyes retain their fluorescence response to a decrease in pH after conjugation to the antibody.

[0165] The unsuitable dye is Invitrogen's Click-iT. TM pHrodo TM iFL Red sDIBO alkynes. The fluorescence intensity of this dye changes only slightly when the pH value changes by a factor of 2 to 3.

[0166] A suitable linker is sulfonated DBCO-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 connection. Conjugation is achieved by activation of the antibody with an azide and reaction with the DBCO moiety using click chemistry.

[0167] In the following description, the invention is illustrated by example using pHAb dyes and conjugations using sulfoDBCO-PEG4-amine linkers. Any other dye exhibiting the aforementioned characteristics, linker, or conjugation chemistry that does not interfere with antibody binding properties, as well as the pH-dependent fluorescence properties of the dye, can also be used. This is presented merely as an example of the invention and should not be construed as limiting. The true scope is set forth in the appended claims.

[0168] The structure of the exemplary labeled antibody is as follows: Figure 3 As shown.

[0169] Mean fluorescence intensity (MFI, more specifically geometric mean fluorescence intensity) of internalized antibodies was obtained using FACS at an excitation wavelength of 488 nm and detection wavelengths of 585 / 540 nm. The exact same conditions, gain, and gate were used for all time points (i.e., 2 and 4 hours). Data extraction was performed using FloJo_V10 software. The negative control values ​​were subtracted from all geometric means and then normalized to the dye-antibody ratio (DAR). The normalized geometric means from each antibody were plotted as linear regression curves using GraphPad Prism to extract the slope (geometric mean MFI / min for 120 and 240 minutes, including the origin, i.e., 0 / 0). Two antibodies were used for normalizing the slope: murvizumab with the mutant M252Y / S254T / T256E was set to 0, and TCB was set to 1. These antibodies were chosen because they spanned a sufficient rate range. The final slope was plotted using TIBCO Spotfire software against clearance values ​​in corresponding in vivo human, cynomolgus monkey, and hFcRn Tg32+ / + mouse models. The corresponding figures for human, cynomolgus monkey, and human FcRn transgenic mice with different antibodies, including those listed in Table 1, are shown in the figure. Figures 5 to 7 middle.

[0170] Figure 8 The display according to the method of the application can also be used to determine the in vivo clearance of Fc region variants of IgG. This further indicates that the FcRn recycling is appropriately captured in the method according to the application.

[0171] Figure 9 The fluorescence is shown to be dependent on the incubation time. It can be seen that the linear range is at least up to 24 hours.

[0172] In certain embodiments, the method according to the application is a method for estimating or determining the in vivo clearance of an antibody in a human or cynomolgus monkey or mouse, the method comprising the steps of:

[0173] a) incubating the antibody conjugated to the same pH sensitive fluorescent dye and at least a first and a second reference antibody separately with primary human endothelial cells 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 prior to determining the intracellular fluorescence intensity,

[0174] b) determining the geometric mean intracellular fluorescence intensity of the primary human endothelial cells not incubated with any labeled antibody at the same time points as a), optionally washing the cells to remove adhering fluorescent compounds prior to determining the intracellular fluorescence intensity,

[0175] c) determining the relative normalized intracellular fluorescence intensity rate by:

[0176] 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 in question and the reference antibodies to obtain corrected geometric mean intracellular fluorescence intensities,

[0177] ii) dividing the corrected geometric mean intracellular fluorescence intensities obtained in 2) for the antibody in question and the reference antibodies by the number of fluorescent dye molecules present in the respective antibody to obtain normalized geometric mean intracellular fluorescence intensities,

[0178] iii) determining the slope of the best fit line (i.e. linear regression curve y = s*x + b, where y = normalized geometric mean (intracellular) fluorescence intensity, s = slope, x = time and b = y axis intersection point) for each of the antibody in question and the reference antibodies based on the group of values consisting of aa) the normalized geometric mean intracellular fluorescence intensities determined in ii) for each incubation time of a) and bb) the origin point;

[0179] iv) the slope of the best fit line of the antibody in question is normalized as follows:

[0180]

[0181] where the in vivo clearance of the antibody in humans or cynomolgus monkeys or mice is the clearance of the first reference antibody in humans or cynomolgus monkeys or mice multiplied by the relative normalized rate of intracellular fluorescence intensity.

[0182] It has been found that by using the relative normalized rate of intracellular fluorescence intensity (rate), the determination of intra- and inter-day deviations can be minimized.

[0183] By using the correlation between the relative normalized rate of intracellular fluorescence intensity and the in vivo determined clearance according to the present application, an in vitro-in vivo correlation has been established. This correlation is independent of the specific antibody used during its generation. Likewise, other antibodies with known in vivo clearance can be used.

[0184] For antibodies with unknown in vivo clearance, the in vivo clearance of an antibody with an undetermined in vivo clearance can be estimated as the y-value by using the determined relative normalized rate of intracellular fluorescence intensity as the x-value in the in vivo-in vitro correlation according to the present application.

[0185] ***

[0186] The following examples, sequences and figures are provided to assist in understanding the present application, the true scope of which is set out in the appended claims. It should be understood that modifications can be made to the procedures set forth without departing from the spirit of the application.

[0187] Example

[0188] I Materials and Methods

[0189] Antibodies

[0190] The reference antibodies used in the experiments were an anti-pTau antibody with the heavy chain amino acid sequence of SEQ ID NO: 01 and the light chain amino acid sequence of SEQ ID NO: 02 and an anti-Her 3 antibody with the heavy chain amino acid sequence of SEQ ID NO: 03 and the light chain amino acid sequence of SEQ ID NO: 04.

[0191] Synthetic genes were produced at Geneart (Life Technologies GmbH, Carlsbad, CA, USA).

[0192] The monoclonal antibodies used herein were transiently expressed in HEK293 cells (see below) and purified by protein A chromatography using standard procedures (see below).

[0193] Biochemical characterization included size exclusion chromatography (Waters BioSuite TM 250 7.8 x 300 mm, eluent: 200 mM KH2PO4, 250 mM KC1, pH 7.0) and analysis of the molecular weight distribution using a BioAnalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA).

[0194] Expression plasmids

[0195] For the expression of the above antibodies, variants of expression plasmids for transient expression of cells (e.g. in HEK293-F) were applied based on cDNA organization with or without CMV-intron A promoter or based on genomic organization with CMV promoter.

[0196] In addition to the antibody expression cassette, the plasmid comprises:

[0197] - an origin of replication allowing replication of the plasmid in E. coli,

[0198] - a beta-lactamase gene conferring ampicillin resistance in E. coli, and

[0199] - a dihydrofolate reductase gene from Mus musculus as selection marker in eukaryotic cells.

[0200] The transcriptional unit of the antibody gene consists of:

[0201] - a unique restriction site at the 5’ end,

[0202] - an immediate early enhancer and promoter from human cytomegalovirus,

[0203] - in case of cDNA organization, followed by an intron A sequence,

[0204] - a 5’ untranslated region of the human antibody gene,

[0205] - an immunoglobulin heavy chain signal sequence,

[0206] - a human antibody chain either as cDNA or as genomic organization with immunoglobulin exon-intron organization,

[0207] - a 3’ untranslated region with a polyadenylation signal sequence, and

[0208] - unique restriction site at the 3' end.

[0209] Fusion genes comprising antibody chains are generated by PCR and / or gene synthesis and assembled by known recombination methods and techniques by ligating the respective nucleic acid segments, e.g. using unique restriction sites in the respective plasmids. Subcloned nucleic acid sequences are verified by DNA sequencing. For transient transfection, larger amounts of plasmids are prepared from transformed E. coli cultures by plasmid preparation (Nucleobond AX, Macherey-Nagel).

[0210] Cell culture techniques

[0211] Standard cell culture techniques as described in Current Protocols in Cell Biology (2000), Bonifacino, J.S., Dasso, M., Harford, J.B., Lippincott-Schwartz, J. and Yamada, K.M. (eds.), John Wiley & Sons, Inc are used.

[0212] Transient transfection in the HEK293-F system

[0213] Antibodies are generated by transient transfection of the respective plasmids (e.g. encoding heavy chain as well as the respective light chain) using the HEK293-F system (Invitrogen) according to the manufacturer's instructions. Briefly, HEK293-F cells (Invitrogen) grown in suspension in serum-free FreeStyle 293 Expression Medium (Invitrogen) in shake flasks or stirred fermenter tubes are transfected with a mixture of the respective expression plasmids and 293fectin or fectin (Invitrogen). For 2 L shake flasks (Corning), HEK293-F cells are seeded at a density of 1 * 10 TM cells / mL in 600 mL and incubated at 120 rpm, 8% C02. Cells are transfected at a density of about 1.5 * 10 TM cells / mL in 600 mL and incubated at 120 rpm, 8% C02. Cells are transfected at a density of about 1.5 * 10 6 cells / mL in 600 mL and incubated at 120 rpm, 8% C02. Cells are transfected at a density of about 1.5 * 10 6Cell densities of 1 cell / mL were transfected with about 42 mL of the following mixture in an equimolar ratio encoding the heavy chain and the corresponding light chain the day after: A) 20 mL Opti-MEM (Invitrogen) with 600 μg total plasmid DNA (1 μg / mL) and B) 20 ml Opti-MEM + 1.2 mL 293fectin or fectin (2 μL / mL). Glucose solution was added during the fermentation process according to the glucose consumption. The supernatant containing the secreted antibody was harvested after 5-10 days and the antibody was purified directly from the supernatant or the supernatant was frozen and stored. Some of the antibodies that have been produced:

[0214]

[0215]

[0216] Purification

[0217] The antibodies were purified from the cell culture supernatant by affinity chromatography using MabSelect Sure - Sepharose TM (GE Healthcare, Sweden), hydrophobic interaction chromatography using Butyl Sepharose (GE Healthcare, Sweden) and Superdex 200 size exclusion (GE Healthcare, Sweden) chromatography.

[0218] Briefly, sterile filtered cell culture supernatant was captured on MabSelect SuRe 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, pH 3.0. Eluted antibody fractions were pooled and neutralized with 2 M Tris, pH 9.0. The antibody pool was prepared for hydrophobic interaction chromatography by addition of a 1.6 M ammonium sulphate solution to a final concentration of 0.8 M ammonium sulphate and adjusting the pH to pH 5.0 using acetic acid. After equilibration of the butyl sepharose resin with 35 mM sodium acetate, 0.8 M ammonium sulphate, 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, pH 5.0. Antibody containing fractions were pooled and further purified by size exclusion chromatography using a Superdex 200 26 / 60 GL (GE Healthcare, Sweden) column equilibrated with 20 mM histidine, 140 mM NaCl, pH 6.0. Antibody containing fractions were pooled, concentrated to the required concentration using a Vivaspin ultrafiltration device (Sartorius Stedim Biotech S.A., France) and stored at -80 °C.

[0219] Following each purification step, purity and antibody integrity were analysed by CE-SDS using microfluidic Labchip technology (Caliper Life Science, USA). 5 μl of protein solution was prepared for CE-SDS analysis using the HT Protein Express kit according to the manufacturer's instructions and analysis was performed on a LabChip GXII system using the HT Protein Express chip. Data was analysed using LabChip GX software.

[0220] Mice

[0221] B6.Cg-Fcgrt tm1DcrTg(FCGRT)276Dcr mice lack the mouse FcRnα-chain gene, but a hemizygous transgene targeting the human FcRnα-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, weighing 17–22 g at administration). All animal experiments were approved by the Upper Bavarian State Government (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.

[0222] Pharmacokinetic studies

[0223] A single dose of antibody was administered intravenously via the lateral tail vein at a dose level of 5 mg / kg. Mice were randomly divided into three groups of six mice each, covering nine serum collection time points (0.08, 2, 8, 24, 48, 168, 336, 504, and 672 hours post-administration). Each mouse underwent two retroorbital blood samplings, administered via isoflurane. TM (CP-Pharma GmbH, Burgdorf, Germany) The procedure was performed under mild anesthesia; a third blood sample was collected at the time of euthanasia. Blood was collected into serum tubes (Microvette 500Z-Gel, Sarstedt, Nümbrecht, Germany). After incubation for 2 hours, the sample was centrifuged at 9.300g for 3 minutes to obtain serum. After centrifugation, the serum sample was frozen at -20°C until analysis.

[0224] Determination of human antibody serum concentrations

[0225] The concentration of antibodies in murine serum was determined by a specific enzyme-linked immunoassay. A biotinylated capture reagent specific for each antibody and a digoxigenin-labeled anti-human Fc mouse monoclonal antibody (Roche Diagnostics, Penzberg, Germany) were used for capture and detection, respectively. Streptavidin-coated microtiter plates (Roche Diagnostics, Penzberg, Germany) were coated with the biotinylated capture reagent diluted in assay buffer (Roche Diagnostics, Penzberg, Germany) for 1 h. After washing, serum samples of different dilutions were added and incubated for another 1 h. After repeated washing, the bound antibodies were detected by subsequent incubation with the detection antibody followed by an anti-digoxigenin antibody conjugated to horseradish peroxidase (HRP; Roche Diagnostics, Penzberg, Germany). ABTS (2,2'Azino-di[3-ethylbenzthiazoline sulfonate]; Roche Diagnostics, Germany) was used as HRP substrate to form a colored reaction product. The absorbance of the resulting reaction product was read at 405 nm with a Tecan Sunrise plate reader (Tecan, Austria) with a reference wavelength of 490 nm. Switzerland) in 405 nm with a reference wavelength of 490 nm.

[0226] All serum samples, positive and negative control samples were analyzed in duplicate and calibrated against reference standards.

[0227] PK analysis

[0228] Pharmacokinetic parameters were calculated by non-compartmental analysis using WinNonlin TM 1.1.1 (Pharsight, CA, USA).

[0229] In brief, the area under the curve (AUC 0-inf ) values were calculated by log-trapezoidal method and extrapolated to infinity using the apparent terminal rate constant λz, extrapolated from the concentration observed at the last time point.

[0230] The plasma clearance was calculated as the dose rate (D) divided by the AUC 0-inf . The apparent terminal half-life (T1 / 2) was derived from the equation T1 / 2 = ln2 / λz.

[0231] Example 1

[0232] Cynomolgus monkey SDPK study

[0233] Pharmacokinetics of test compounds were determined after single intravenous administration at dose levels ranging from 0.3 mg / kg to 150 mg / kg in cynomolgus monkeys. Serial blood samples were collected from the monkeys over several weeks and serum / plasma was prepared from the collected blood samples. Serum / plasma levels of test compounds were determined by ELISA. In the case of linear pharmacokinetics, pharmacokinetic parameters were determined by standard non-compartmental methods. The clearance was calculated according to the following formula:

[0234] Clearance = Area under the concentration-time curve

[0235] In the case of non-linear pharmacokinetics, the linear fraction of clearance was determined via the following alternative methods: Either the clearance value was estimated after IV administration at high dose levels at which additional non-linear clearance pathways are practically saturated. Or, a PK model was established that includes both linear and non-linear, saturable clearance terms. In these cases, the linear clearance fraction determined by the model was used for correlation.

[0236] Example 2

[0237] Preparation of FcRn affinity column

[0238] Expression of FcRn in HEK293 cells

[0239] FcRn was transiently expressed by transfection of HEK293 cells with two plasmids containing the coding sequences for FcRn and beta-2-microglobulin. Transfected cells were cultivated in a 14l stainless steel bioreactor at 36.5°C, 120 rpm (shaker amplitude 5 cm), 80% humidity and 7% CO2. Cells were diluted every 2-3 days to a density of 3 to 4*10 5 cells / ml.

[0240] For transient expression, a 14l stainless steel bioreactor was started at 36.5°C, pH 7.0 ± 0.2, pO2 35% (aeration with N2 and air, total gas flow 200 ml min -1 -1), a culture volume of 8.1 and a stirrer speed of 100-400 rpm. When the cell density reached 20*10 5For transfection of 1.5 x 106cells / ml, 10 mg of plasmid DNA (equimolar amounts of both plasmids) was diluted in 400 ml Opti-MEM (Invitrogen). 20 ml 293fectin (Invitrogen) was added to the mixture, which was then incubated for 15 min at room temperature before being transferred to the fermenter. From the next day, the cells were provided with nutrients in continuous mode: a feed solution was added at a rate of 500 ml per day, and glucose was added as needed to maintain a level above 2 g / l. After 7 days of transfection, the supernatant was collected using a swing-bucket centrifuge with a 1 1 bucket at 4000 rpm for 90 min. The supernatant (13 L) was cleared by a Sartobran P filter (0.45 μm + 0.2 μm, Sartorius) and the FcRn β-2-microglobulin complex was purified therefrom.

[0241] Biotinylation of the neonatal Fc receptor

[0242] Three mg of FcRn β-2-microglobulin complex was dissolved / diluted in 5.3 mL 20 mM sodium phosphate monobasic buffer containing 150 mM sodium chloride and added to 250 μΐ^PBS and 1 tablet complete protease inhibitor (Complete ULTRA tablets, Roche Diagnostics GmbH). Biotinylation of FcRn was performed using a biotinylation kit from Avidity according to the manufacturer's instructions (Bulk BIRA, Avidity LLC). The biotinylation reaction was completed overnight at room temperature.

[0243] The biotinylated FcRn was dialyzed against 20 mM MES buffer (containing 140 mM NaCl, pH 5.5) (Buffer A) overnight at 4°C to remove excess biotin.

[0244] Coupling to streptavidin sepharose

[0245] For coupling to streptavidin sepharose, 1 mL of streptavidin sepharose (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 sepharose was packed into a 4.6 mm x 50 mm chromatography 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).

[0246] Example 3

[0247] Chromatography using FcRn affinity column and pH gradient

[0248] Conditions:

[0249] Column dimensions: 50 mm x 4.6 mm

[0250] Load: 30 pg sample

[0251] Buffer A: 20 mM MES with 140 mM NaCI, adjusted to pH 5.5

[0252] Buffer B: 20 mM Tris / HCI with 140 mM NaCI, adjusted to pH 8.8

[0253] Apply 30 pg sample to an FcRn affinity column equilibrated with buffer A. After a 10 minute wash step at 20% buffer B at a flow rate of 0.5 mL / min, elute over 70 minutes with a linear gradient from 20% to 70% buffer B. Detection is performed using UV absorption at a wavelength of 280 nm. Regenerate the column after each run for 10 minutes using 20% buffer B.

[0254] To calculate the relative retention time, run according to Bertoletti-Ciarlet, A. et al. (Mol. Immunol. 46 (2009) 1878-1882) with a standard sample (anti-Her3 antibody (SEQ ID NO: 03 and 04)) oxidized with 0.02% hydrogen peroxide for 18 hours at the beginning of the sequence and after every 10 sample injections.

[0255] Briefly, mix antibody (9 mg / mL) in 10 mM sodium phosphate pH 7.0 with H2O2 to a final concentration of 0.02% and incubate for 18 hours at room temperature. To quench the reaction, dialyze the sample extensively into pre-chilled 10 mM sodium acetate buffer pH 5.0.

[0256] Example 4

[0257] Chromatography using heparin affinity column and pH gradient

[0258] Conditions:

[0259] Column dimensions: 50 mm x 5.0 mm

[0260] Load: 20-50 pg sample

[0261] Buffer A: 50 mM TRIS, pH 7.4

[0262] Buffer B: 50 mM TRIS, pH 7.4, 1000 mM NaCI

[0263] A protein sample of 20 to 50 pg in low salt buffer (≤ 25 mM ionic strength) was applied to a 5.0 x 50 mm TSKgel Heparin-5PW glass column (Tosoh Bioscience, Tokyo / Japan) pre-equilibrated with buffer A at room temperature. Elution was performed with a linear gradient of 0-100% buffer B over 32 min at a flow rate of 0.8 mg / mL. Detection was performed using UV absorption at a wavelength of 280 nm.

[0264] Example 5

[0265] Examination of antibody internalization

[0266] This method is based on a previously reported method that uses pH-activated probes for uniform fluorescence imaging to detect internalizing antibodies, which enables the maximum fluorescence signal of the antibody under intracellular acidic conditions without detecting any fluorescence signal in the extracellular environment (Li, Z., et al., Int. Immunopharm. 62 (2018) 299-308).

[0267] Briefly, the respective antibody was conjugated to the pHAb amine-reactive dye and then diluted with cell culture medium. At the same time, cells were seeded into 6-well plates (1 x 10 5 cells per well) and 100 pL of culture medium containing the pHAb amine-reactive dye conjugated antibody (final concentration of 10 pg / mL) was added per well. After incubation at 37°C, the internalization of the antibody 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).

[0268] Example 6

[0269] Antibody labeling

[0270] According to the manufacturer’s instructions, antibodies were labeled using the SiteClick TM Antibody Azido Modification Kit (Thermo Fisher Scientific). Briefly, the N-linked galactose residue of the Fc region was removed by b-galactosidase and replaced by azide-containing galactose (GalNaz) via b-1,4-galactosyltransferase (GalT). This azide modification enables copper-free conjugation of the sDIBO modified dye. The pH-sensitive amine-reactive dye (523 nm) was purchased from Promega and coupled to the sulfo-DBCO PEG4 amine. Antibodies were labeled with a 2-fold molar excess of dye. A MWCO of 50 kDa (EMD Millipore, #UFC200324) spin filter was used to remove unbound dye. Excess dye was removed using an Ultra-2 centrifugal filter, and the antibody was reburied in 20 mM histidine buffer (pH 5.5). The antibody was then analyzed using a Nanodrop spectrometer at 280 nm (A). 280nm ) and 532nm (A 532nm The concentration of labeled antibody[1] and the dye-to-antibody ratio (DAR)[2] were determined.

[0271] CAB = [A 280nm -[A 280nm *CF Dye ]] / ε mAb [1]

[0272] DAR = [A 532nm *MW mAb ] / [c mAb *ε Dye [2]

[0273] ε Dye =47225

[0274] CF Dye =0.36

[0275] Example 7

[0276] Cell maintenance and preparation

[0277] Cryopreserved human liver-derived endothelial cells (HLEC-P2) were purchased from Lonza (Lonza, #HLECP2). Cells were maintained in an environment supplemented with EGM. TM -2 MV microvascular endothelial cell growth medium SingleQuots TM EBM of (Lonza, #CC-4176) TM -2 Endothelial cell growth basal medium-2 (Lonza, #CC-3156). Five days before antibody treatment, cells were seeded into 100 mm culture dishes coated with collagen I. BioCoat TM Cells were cultured on collagen I-coated 96-well plates two days before treatment (#354450) and passaged. BioCoat TM In (#354407), the cell density is 4 x 10⁻⁶. 4 Cells per well were spaced to allow adhesion for 48 hours. The culture medium was changed after 24 hours, and the cells were maintained at 37°C and 5% CO2.

[0278] On the day of the experiment, cells were washed twice with 200 mI pre-warmed medium, followed by incubation with 400 nM of labeled antibody or 20 mM histidine buffer (pH 5.5) as a negative control in medium. After 2 and 4 hours, the antibody solution was removed and cells were washed once with 200 mI ice-cold DPBS (without Mg and Ca) and detached by applying 100 mI trypsin (with EDTA) for 2.5 min at 37°C. The trypsin was inactivated by adding 100 mI FACS buffer (20% FCS, 1 mM EDTA in DPBS).

[0279] Example 8

[0280] Flow cytometry and pharmacokinetic analysis

[0281] Use The mean fluorescence intensity (MFI, more specifically the geo-mean) of internalized antibodies was acquired using a MACSQuant® Analyzer 10 (Miltenyi Biotec) equipped with a laser exciting at 488 nm and filters for collecting emitted light at 585 nm / 540 nm. The exact same conditions, gain and gates were used for both time points (2 hours and 4 hours). Data extraction was performed using FloJo_V10 software. The value of the negative control was subtracted from all geo-means and then normalized to DAR. The normalized geo-means from each antibody were plotted as a linear regression curve using GraphPad Prism to extract the slope (geo-mean MFI / minute for 120 and 240 minutes). Two standard antibodies were chosen to normalize the slope: Mavrilimumab-YTE was set to 0 and TCB was set to 1. The final slope was plotted against published in vivo human, cynomolgus and hFcRn Tg32+ / + mouse clearance values using TIBCO Spotfire software.

[0282] Example 9

[0283] Quality control

[0284] Biophysical binding properties are key determinants influencing the clearance mechanism. Therefore, it is important to assess whether the binding affinity of the antibody changes during the labeling process. Heparin chromatography and neonatal Fc receptor binding have previously been shown to be predictive for in vitro antibody clearance (Kraft, T.E., et. al., MABS 12 (2020) e1683432). Here, this method was used to illustrate potential aberrant binding properties introduced by the click tag. Details of the method are provided in Examples 3 and 4.

[0285] To confirm the absence of unbound dye and to verify the concentration measured on the spectrometer, the labeled antibodies were subjected to size exclusion chromatography. The samples were separated using a BioSuite Diol (OH) column (Waters, 186002165) with potassium dihydrogen phosphate buffer (pH 6.2) as mobile phase at a flow rate of 0.5 ml / min. The labeled antibodies were quantified and analyzed using detectors at 280 nm and 532 nm. The area under the curve (AUC) at 280 nm and 532 nm was extracted to calculate the concentration. The geometric mean of the AUCs of all antibodies was calculated and the deviation of each antibody from this geometric mean was determined. For antibodies that are reliable within the assay according to the application, the deviation from the geometric mean is expected to be below 15%. SEQUENCE LISTING <110> F. Hoffmann-La Roche AG <120> Macromolecular non-specific clearance assay <130> P35993 <150> EP20168671.4 <151> 2020-04-08 <160> 5 <170> PatentIn Version 3.5 <210> 1 <211> 441 <212> PRT <213> Artificial Sequence <220> <223> Anti-pTau antibody HC <400> 1 Glu Val Gin Val Leu Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Ser Leu Ser Ser Asn 20 25 30 Ala lie Asn Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Tyr lie Ala Val Ser Gly Asn Thr Tyr Tyr Ala Ser Trp Ala Lys 50 55 60 Gly Arg Phe Thr lie Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Gly 85 90 95 Lys Ser Asn lie Trp Gly Gin Gly Thr Leu Val Thr Val Ser Ser Ala 100 105 110 Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser 115 120 125 Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe 130 135 140 Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly 145 150 155 160 Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu 165 170 175 Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gin Thr Tyr 180 185 190 Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys 195 200 205 Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 210 215 220 Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 225 230 235 240 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 245 250 255 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 260 265 270 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 275 280 285 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 290 295 300 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 305 310 315 320 Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 325 330 335 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu 340 345 350 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 355 360 365 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 370 375 380 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 385 390 395 400 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 405 410 415 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 420 425 430 Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 <210> 2 <211> 218 <212> PRT <213> Artificial Sequence <220> <223> Anti-pTau antibody LC <400> 2 Ala Gln Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Gln Ser Val Arg Thr Asn 20 25 30 Arg Leu Ala Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro Lys Arg Leu 35 40 45 Ile Tyr Ser Ala Ser Thr Leu Asp Tyr Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gly Tyr Phe Asp Ser Ser 85 90 95 Ala Asp Ile Val Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 3 <211> 449 <212> PRT <213> artificial sequence <220> <223> Anti-Her3 antibody HC <400> 3 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Arg Ser Ser 20 25 30 Tyr Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Tyr Ala Gly Thr Gly Ser Pro Ser Tyr Asn Gln Lys Leu 50 55 60 Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg His Arg Asp Tyr Tyr Ser Asn Ser Leu Thr Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly <210> 4 <211> 220 <212> PRT <213> Artificial Sequence <220> <223> Anti-Her3 antibody LC <400> 4 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Ser 85 90 95 Asp Tyr Ser Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 115 120 125 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 130 135 140 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 145 150 155 160 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 165 170 175 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 180 185 190 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 195 200 205 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 220 <210> 5 <211> 227 <212> PRT <213> Homo sapiens <400> 5 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225

Claims

1. A method for determining non-specific 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 of step a), wherein the dye is conjugated to a linker, and the linker is conjugated to the antibody and has the structure of Formula III: wherein the primary human endothelial cells are primary human liver endothelial cells; wherein the antibody has an Fc region of human IgGl or IgG4 subclass; wherein non-specific 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 determined in the absence of the antibody.

2. The method according to claim 1, the method 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 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 2, wherein the dye has a change in fluorescence intensity of about 10-fold between physiological pH of about 7 and acidic pH in the range of pH 4 to 5 determined at the same concentration of the dye and using the same excitation wavelength.

5. The method according to claim 3, wherein the dye has a change in fluorescence intensity of about 10-fold between physiological pH of about 7 and acidic pH in the range of pH 4 to 5 determined at the same concentration of the dye and using the same excitation wavelength.

6. The method according to any one of claims 1 to 2 and 5, wherein the dye is conjugated to the antibody at residue 297 according to Kabat numbering.

7. The method according to claim 3, wherein the dye is conjugated to the antibody at residue 297 according to Kabat numbering.

8. The method according to claim 4, wherein the dye is conjugated to the antibody at residue 297 according to Kabat numbering.

9. The method according to any one of claims 1 to 2, 5, 7 to 8, wherein the fluorescence intensity is determined by FACS to determine the shift of the fluorescence maximum.

10. The method according to claim 3, wherein the fluorescence intensity is determined by FACS to determine the shift of the fluorescence maximum.

11. The method according to claim 4, wherein the fluorescence intensity is determined by FACS to determine the shift of the fluorescence maximum.

12. The method according to claim 6, wherein the fluorescence intensity is determined by FACS to determine the shift of the fluorescence maximum.

13. The method according to any one of claims 1 to 2, 5, 7 to 8, 10 to 12, wherein the fluorescence intensity is the geometric mean fluorescence intensity determined by FACS.

14. The method of claim 3, wherein the fluorescence intensity is geometric mean fluorescence intensity determined by FACS.

15. The method of claim 4, wherein the fluorescence intensity is geometric mean fluorescence intensity determined by FACS.

16. The method of claim 6, wherein the fluorescence intensity is geometric mean fluorescence intensity determined by FACS.

17. The method of claim 9, wherein the fluorescence intensity is geometric mean fluorescence intensity determined by FACS.

18. The method of any one of claims 1-2, 5, 7-8, 10-12, 14-17, wherein the incubating lasts up to 4 hours.

19. The method of claim 3, wherein the incubating lasts up to 4 hours.

20. The method of claim 4, wherein the incubating lasts up to 4 hours.

21. The method of claim 6, wherein the incubating lasts up to 4 hours.

22. The method of claim 9, wherein the incubating lasts up to 4 hours.

23. The method of claim 13, wherein the incubating lasts up to 4 hours.

24. The method of any one of claims 1-2, 5, 7-8, 10-12, 14-17, 19-23, wherein the incubating lasts at least 0.5 hours.

25. The method of claim 3, wherein the incubating lasts at least 0.5 hours.

26. The method of claim 4, wherein the incubating lasts at least 0.5 hours.

27. The method of claim 6, wherein the incubating lasts at least 0.5 hours.

28. The method of claim 9, wherein the incubating lasts at least 0.5 hours.

29. The method of claim 13, wherein the incubating lasts at least 0.5 hours.

30. The method of claim 18, wherein the incubating lasts at least 0.5 hours.

31. The method of any one of claims 1-2, 5, 7-8, 10-12, 14-17, 19-23, 25-30, wherein the antibody is a bispecific antibody.

32. The method of claim 3, wherein the antibody is a bispecific antibody.

33. The method of claim 4, wherein the antibody is a bispecific antibody.

34. The method of claim 6, wherein the antibody is a bispecific antibody.

35. The method of claim 9, wherein the antibody is a bispecific antibody.

36. The method of claim 13, wherein the antibody is a bispecific antibody.

37. The method of claim 18, wherein the antibody is a bispecific antibody.

38. The method of claim 24, wherein the antibody is a bispecific antibody.

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