Macromolecule non-specific clearance assay

Abstract

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

Description

[0001] This application is a divisional application of PCT application PCT / EP2021 / 058839, filed on April 6, 2021, entitled “Macromolecular non-specific clearance assay”, which entered the Chinese national phase on October 8, 2022, with the application number 202180027436.8.

[0002] A new method for estimating the clearance of therapeutic proteins in humans using a novel in vitro assay based on human primary cells is reported herein. This macromolecular non-specific clearance assay (LUCA) provides an in vitro-based approach to assess and predict key PK properties for therapeutic proteins. BACKGROUND

[0003] G class 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.

[0004] 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 of 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, protecting IgG from lysosomal degradation (see, e.g., Rouseenian, 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 critical for IgG recycling, and any deviation in the binding properties at different pH values can strongly impact the circulating half-life of IgG (see, e.g., Vaccaro, C., et al., Nat. Biotechnol. 23 (2005) 1283-1288).

[0005] Eigenmann, M.J. et al. summarize that cellular uptake of antibodies is thought to occur primarily 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 the physiological pH of ~7.4 in plasma. Thus, antibodies bound to FcRn in endosomes are released into the plasma at neutral pH, allowing for antibody recycling rather than lysosomal degradation (MABS 9 (2017) 1007-1015).

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

[0007] 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).

[0008] 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

[0009] A method for determining the level of non-specific clearance of a therapeutic protein, especially an antibody, by pinocytosis and lysosomal degradation is reported herein.

[0010] The present invention is based at least in part on the finding that the uptake of an antibody into primary human endothelial cells in vitro can be used as a surrogate for assessing the non-specific clearance of said antibody in vivo, in particular in mice, cynomolgus monkeys and humans.

[0011] The present invention is based at least in part on the finding that only primary human endothelial cells can be used to determine in vivo clearance rates 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.

[0012] The present invention is based at least in part on the finding that the contribution of pinocytosis of an antibody 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.

[0013] 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: a) incubating an antibody conjugated to a pH-sensitive fluorescent dye with primary human endothelial cells (for a defined time), and b) determining the (intracellular) fluorescence intensity of the primary human endothelial cells obtained in step a) (after the defined incubation time), wherein the increase of 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) determines the non-specific clearance of the antibody (i.e. is indicative for the non-specific clearance of the antibody).

[0014] In certain embodiments, the method further comprises the steps of: - determining the (intracellular) fluorescence intensity of the primary human endothelial cells before incubation with / without the antibody, and wherein 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 determines the non-specific clearance of the antibody (i.e. is indicative for the non-specific clearance of the antibody).

[0015] 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, the method comprising the steps of: 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; 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, thereby selecting one or more antibodies with a low relative non-specific (non-target mediated) clearance (rate).

[0016] Further, the present application comprises a method for ranking a multitude of antibodies based on their non-specific (non-target mediated) clearance (rate), the method comprising the steps of: 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; b) ranking the antibodies based on the (intracellular) fluorescence intensity (change) from low to high or from high to low, thereby ranking the antibodies based on their non-specific (non-target mediated) clearance (rate).

[0017] 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, the method comprising the steps of: a) incubating an antibody conjugated to a pH sensitive fluorescent dye with primary human endothelial cells for a defined time, and thereafter determining the (intracellular) fluorescence intensity (change) of the primary human endothelial cells; 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, 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 with the ratio of the fluorescence intensity (change) determined in a) to the (intracellular) fluorescence intensity (change) determined in b).

[0018] In certain embodiments, step b) is

[0019] b) i) incubating each member of a multitude of reference antibodies (i.e. at least two) with 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, ii) thereafter determining the (intracellular) fluorescence intensity (change) of the primary human endothelial cells for each of the reference antibodies, and iii) for the values obtained in ii) calculating the best fit 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).

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

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

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

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

[0024] (Formula I).

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

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

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

[0028] 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 sulfo DBCO-PEG4-amine of Formula II.

[0029] (Formula II)

[0030] The conjugation to the antibody is at the free amino group of Formula II.

[0031] 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.

[0032] (Formula III)

[0033] In one embodiment of all aspects and embodiments, the dye is conjugated to the antibody by chemical cross-linking.

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

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

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

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

[0038] In one embodiment of all aspects and embodiments, 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 of 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 4 to 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.

[0039] In certain embodiments, the determination is made directly after the incubation.

[0040] In one embodiment of all aspects and embodiments, the antibody has a human-derived Fc region. 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.

[0041] 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.

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

[0043] In one embodiment of all aspects and embodiments, the first reference antibody is moxidomycin with mutations M252Y / S254T / T256E, and / or a bispecific antibody in the format TCB. BRIEF DESCRIPTION OF DRAWINGS

[0044] Figure 1 Time course of fluorescence intensity of different antibodies that have been 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 = avelumab; 6 = anti-human alpha-synuclein antibody; 7 = anti-CD20 antibody (2).

[0045] Figure 2 Time course of fluorescence intensity of different antibodies that have been 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).

[0046] Figure 3 Protocol of fluorescently labeled antibodies used in the method according to the application; pHAb dye conjugated to antibodies via Sulfo DBCO-PEG4-Amine linker.

[0047] Figure 4 Protocol of the method according to the application.

[0048] Figure 5 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: 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.

[0049] Figure 6 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: 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.

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

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

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

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

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

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

[0056] The method according to the present application can be used to estimate the pharmacokinetic (PK) properties of different antibody molecules (different in form, valency and specificity).

[0057] Thus, the method according to the present application can be used to

[0058] - support the selection of a suitable clinical lead molecule with respect to PK (pharmacokinetic) properties (clearance and half-life, respectively); - deselect antibodies from a library with PK properties that are not suitable for therapeutic applications, i.e. with high clearance or short in vivo half-life, respectively; - rank members of a set of antibodies according to their PK properties (clearance and half-life, respectively); - 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; - guide the engineering of antibodies with respect to their 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); - determine the need for PK engineering and evaluate the results of PK engineering.

[0059] Thus, the assay according to the present application can reduce or even replace animal PK studies.

[0060] I. Definitions

[0061] As used herein, the amino acid positions of all constant regions and domains of 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 "according to Kabat numbering". Specifically, the Kabat numbering system (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991), pages 647-660) is used for the light chain constant domains 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).

[0062] The knob-into-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).

[0063] 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).

[0064] 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, CHSL 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).

[0065] Using recombinant DNA technology it is possible to generate nucleic acid derivatives. 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).

[0066] 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" and "at least one" are used interchangeably herein. It is further noted that the terms "comprise", "comprising", "include", "including" and "have" are used interchangeably.

[0067] 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.

[0068] As used herein, the term "determining" also includes the terms measuring and analyzing.

[0069] The term "comprising" also includes the term "consisting of".

[0070] 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.

[0071] "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')2 bispecific 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).

[0072] 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".

[0073] The term "buffering substance" denotes a substance which, when in solution, can adjust changes in the pH value of the solution, e.g., due to the addition or release of acidic or basic substances.

[0074] The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and 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, , , and µ, respectively.

[0075] 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 desired targeting and / or protein A and / or FcRn binding activity.

[0076] The term "human Fc region" denotes the C-terminal region of a human immunoglobulin heavy chain, which contains at least a portion 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 is composed of two heavy chain Fc region polypeptides, which can be covalently linked to one another via a hinge cysteine residue, forming an interchain disulfide bond.

[0077] 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 1 :2 stoichiometry, 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 neutral cell surface (pH is 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 endosomes, enabling recycling of IgG to the cell surface, whereupon the FcRn-IgG complex is released into the bloodstream upon exposure to the extracellular neutral pH environment.

[0078] 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 are altered according to EU numbering by Kabat: 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).

[0079] 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.

[0080] 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. Also, 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.

[0081] 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.

[0082] 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.

[0083] 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). For a review of methods for assessment of antibody purity, see Flatman, S. et al., J. Chrom. B 848 (2007) 79-87.

[0084] 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 at which it naturally occurs.

[0085] 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 generally being 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, as described herein and in examples of such methods and other exemplary methods for making monoclonal antibodies.

[0086] “Native antibodies” refer to naturally occurring immunoglobulin molecules with varying structures. For example, native 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 (CHI, 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.

[0087] The term "pharmaceutical formulation" refers to a preparation which is in a form which can be used for administration to a subject and which is suitable for combined administration of the active ingredients contained therein.

[0088] A "pharmaceutically acceptable carrier" refers to a non-toxic component of a pharmaceutical formulation which is not biologically or otherwise undesirable, e.g., a buffer, excipient, stabilizer, or preservative.

[0089] The term "recombinant antibody" as used herein means all antibodies which are prepared, expressed, created or isolated by recombinant means (chimeric antibodies, humanized antibodies and human antibodies). This includes antibodies isolated from a host cell such as a NS0, HEK, BHK or CHO cell or from a transgenic animal (e.g., a mouse) of human immunoglobulin genes, or 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 which, although derived from and related to human germline VH and VL sequences, can not naturally exist within the human antibody germline repertoire in vivo.

[0090] The term "TCB" as used herein means 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.

[0091] In a preferred embodiment, the TCB comprises: a) a first Fab fragment and a second Fab fragment, each binding a first antigen, b) one domain exchanged Fab fragment specifically binding a second antigen, in which the CH1 domain and the CL domain are exchanged with each other, c) one Fc region comprising a first heavy chain Fc region polypeptide and a second heavy chain Fc region polypeptide, 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-swapped Fab fragment is connected to the N-terminus of the other heavy chain Fc-region polypeptide, and 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 VL domain of the domain-swapped Fab fragment, and wherein the first antigen or the second antigen is human CD3.

[0092] In another equally preferred embodiment, the TCB comprises: a) a first Fab fragment and a second Fab fragment, each binding to a first antigen, b) one domain-swapped Fab fragment specifically binding to a second antigen, in which the VH domain and the VL domain are swapped with each other, c) one Fc region comprising a first heavy chain Fc-region polypeptide and a second heavy chain Fc-region polypeptide, 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-swapped Fab fragment is connected to the N-terminus of the other heavy chain Fc-region polypeptide, and 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 VL domain of the domain-swapped Fab fragment, and wherein the first antigen or the second antigen is human CD3.

[0093] 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".

[0094] 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 and light chains of an antibody (VH and VL, respectively) 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), pp. 91). A single VH or VL domain can be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen can be isolated using a VH or VL domain from an antibody that binds the antigen for screening a library of complementary VL or VH domains, respectively. See, e.g., Portolano, S. et al., J. Immunol . 150 (1993) 880-887; Clackson, T. et al., Nature 352 (1991) 624-628).

[0095] The terms "variant," "modified antibody," and "modified fusion polypeptide" refer to molecules having 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.

[0096] A "primary human endothelial cell" is a human cell that is 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 referred to as anchorage-dependent cells. Adherent cells are usually derived from organ tissues. Suspension cells do not require attachment to grow and are referred to as anchorage-independent cells. Most suspension cells are isolated from blood.

[0097] The term "pH sensitive fluorescent dye" denotes a dye that has different fluorescence intensities or emission wavelengths at physiological pH of about pH 7.4 and at lysosomal pH of about pH 4.5.

[0098] II. In vivo antibodies

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

[0100] In case the antigen is a membrane-bound antigen, the administered therapeutic antibody binds to the membrane-bound antigen on the cell surface. Subsequently, the antibody is taken up into an endosome within the cell by internalization of the membrane-bound antigen bound to the antibody. 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 referred to as antigen-dependent elimination. This has been reported for different antibody molecules (see, e.g., Drug Discov. Today, 11 (2006) 81-88). Since a single IgG antibody molecule, when bivalently bound to an antigen, binds to two antigen molecules and is then internalized and directly degraded by the lysosome, a single ordinary IgG antibody cannot neutralize two or more antigen molecules.

[0101] The reason for the long retention (slow elimination) of IgG molecules in the plasma is the FcRn, called the IgG molecule salvage receptor (see, e.g., Nat. Rev. Immunol. 7 (2007) 715-725). IgG molecules that have been taken up into endosomes by endocytosis bind to the FcRn expressed in the endosome under the acidic conditions in the endosome. IgG molecules bound to the FcRn move to the cell surface, where they dissociate from the FcRn under the neutral conditions in the plasma. IgG molecules that cannot bind to the FcRn enter the lysosome, where they are degraded.

[0102] If, when an IgG antibody is taken up into an endosome in a cell by internalization, the IgG antibody dissociates from the antigen under the acidic conditions in the endosome, the dissociated antibody can bind to the FcRn also present in the endosome. Thus, IgG molecules that dissociate from the antigen and are bound by the FcRn are transferred to the cell surface and released from the FcRn into the plasma under the neutral pH conditions. The antibodies are thereby recirculated into the plasma. IgG molecules that return to the plasma are able to bind to new antigens again. The repetition of this process allows a single IgG molecule to repeatedly bind to antigens, thereby enabling a single IgG molecule to neutralize multiple antigens.

[0103] In the case of soluble antigens, the therapeutic antibodies administered bind to the antigens in the plasma and are retained in the plasma in the form of antigen-antibody complexes. As in the case of IgG molecules that do not bind to antigens, IgG molecules that bind to antigens in the plasma are taken up into endosomes by endocytosis. In the endosomes, they can bind to the FcRn expressed in the endosome under the acidic conditions in the endosome. IgG molecules bound to the FcRn move to the cell surface and then dissociate from the FcRn under the neutral conditions in the plasma. If the IgG molecules can dissociate from the antigens under the acidic conditions in the endosome, the dissociated antigens cannot bind to the FcRn and can thus be degraded by the lysosome. As the IgG molecules that have returned to the plasma have dissociated from the antigens in the endosome, they are able to bind to new antigens in the plasma again. The 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.

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

[0105] More specifically, a single IgG molecule binds strongly to the antibody at the cell surface pH of 7.4, and weakly to the antigen at the pH of 5.5 to 6.0 in the endosome, and can be able to neutralize a variety of antigens, thereby improving the pharmacokinetics (it has been reported that the pH in the endosome is generally pH 5.5 to 6.0 (see, for example, Nat. Rev. Mol. Cell. Biol. 5 (2004) 121-132)).

[0106] Generally, 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 apparent binding constant (avidity). The binding strength of pH-dependent binding varies between neutral conditions (pH 7.4) and acidic conditions (pH 5.5 to 6.0), which is present in naturally occurring protein-protein interactions. 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 a histidine residue present in IgG (see, for example, Mol. Cell. 7 (2001) 867-877).

[0107] III. Methods according to the application

[0108] A new method for estimating therapeutic antibody clearance 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 method to assess and predict the PK properties of therapeutic antibodies.

[0109] 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.

[0110] The present invention is based at least in part on the finding that only primary human endothelial cells can be used to predict in vivo clearance rates from in vitro experiments, as non-primary endothelial cells do not show the same correlation and are therefore 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 depicts a scheme of the method according to the present invention.

[0111] 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: a) incubating an antibody conjugated to a pH sensitive fluorescent dye with primary human endothelial cells, and b) determining the (intracellular) fluorescence intensity of the primary human endothelial cells of step a) (after a defined incubation time), wherein an increase of the (intracellular) fluorescence intensity of the primary human endothelial cells determined in step b) relative to the background level (i.e. the (intracellular) fluorescence of the primary human endothelial cells not incubated with the antibody) is indicative for a non-specific clearance of the antibody.

[0112] The present invention is at least partially based on the finding that the sum of the antibody taken up and recycled into primary human endothelial cells in vitro can be used as a surrogate to estimate the non-specific clearance of said antibody in vivo.

[0113] The present invention is at least partially based on the finding that only primary human endothelial cells can be used to predict the clearance 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, no differentiation between different antibodies can be achieved (see Figures 1 and 2).

[0114] The present invention is at least partially based on the finding that the contribution to the fluorescence of primary endothelial cells is greatest by pinocytosis of the antibody and its transport to the lysosomal compartment without recycling by the FcRn of the primary endothelial cells.

[0115] In Figures 1 and 2, the time course of the fluorescence intensity of different antibodies 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) is compared, which different antibodies have been labeled with the same pH sensitive fluorescent dye. As can be seen from Figure 1, for five of the seven antibodies, no differentiation is possible when using simple endothelial cells. In contrast, when using primary endothelial cells, all seven antibodies can be differentiated (see Figure 2).

[0116] The labeled antibodies have been analyzed by heparin and FcRn chromatography. Exemplary retention times of 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 reliable within the assay according to the present invention, a difference to the geometric mean below 15% is expected.

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

[0118]

[0119] 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 times, preferably about 25 times and most preferably about 50 times between a physiological pH of about 7 and an acidic pH in the range of pH 4 to 5.

[0120] An exemplary suitable dye is the pHAb dye sold by Promega. These dyes are pH sensor dyes which have very low fluorescence at pH > 7 and the fluorescence increases sharply as the pH of the solution becomes acidic. The pHAb dye has an excitation maximum (Ex) at 532 nm and an emission maximum (Em) at 560 nm. The pHAb dye comes in two reactive forms suitable for conjugation to antibodies: pHAb amine-reactive dye and 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 by using a reducing agent such as DTT or TCEP. The pHAb dye retains its fluorescence response to a decrease in pH after conjugation to the antibody.

[0121] An unsuitable dye is the Click-iT™ pHrodo™ iFL Red sDIBO alkyne of Invitrogen. This dye has only a small change in fluorescence intensity when the pH value is changed by only 2 to 3 times.

[0122] A suitable linker is the Sulfo DBCO-PEG4-Amine sold by ClickChemistryTools. Sulfo DBCO-PEG4-Amine is a water-soluble reagent for derivatization of carboxyl-containing molecules or activated esters (e.g. NHS esters) with a DBCO moiety via stable amide bonds. The hydrophilic sulfonated spacer improves the water solubility of the DBCO-derivatized molecule, allowing it to be completely soluble in aqueous media in many cases. The PEG spacer provides a long and flexible linkage. Conjugation is achieved by azide activation of the antibody and reaction with the DBCO moiety using a click chemistry reaction.

[0123] In the following, conjugation using the pHAb dye and using a sulfo-DBCO-PEG4-amine linker is used to exemplify the present application. Any other dye showing the above-mentioned characteristics of the conjugation chemistry or linker or not interfering with the binding properties of the antibody as well as the pH-dependent fluorescence properties of the dye can equally be used. This is presented as an example of the present application only and should not be interpreted as limiting. The true scope is set forth in the appended claims.

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

[0125] The mean fluorescence intensity (MFI, more specifically the geometric mean fluorescence intensity) of the internalized antibodies was acquired using FACS with an excitation wavelength of 488 nm and a detection wavelength of 585 / 540 nm. The exact same conditions, gain and gates were used for all time points (i.e. 2 and 4 hours). Data extraction was performed using FloJo_V10 software. The values of the negative controls 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 a linear regression curve using GraphPad Prism to extract the slope (geometric mean MFI / minute for 120 and 240 minutes and including the origin, i.e. 0 / 0). Two antibodies were used for the normalization of the slope: Movlitumab with mutations M252Y / S254T / T256E was set to 0 and TCB was set to 1. These antibodies were chosen because they span a sufficient range of rates. The final slope was plotted against the respective in vivo human, cynomolgus and hFcRn Tg32 + / + mouse clearance values using TIBCO Spotfire software. The respective plots of the human, cynomolgus and human FcRn transgenic mice with different antibodies including Table 1 of the antibodies are shown in Figures 5 to 7.

[0126] Figure 8 shows that the method according to the present 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 properly captured in the method according to the present application.

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

[0128] In certain embodiments, the method according to the present application is a method for estimating or determining the in vivo clearance rate of an antibody in a human or cynomolgus or mouse, the method comprising the steps of: a) incubating an 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, 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, c) determining the relative normalized intracellular fluorescence intensity ratio by: i) subtracting the geometric mean intracellular fluorescence intensity of the primary human endothelial cells determined at the same time point from each of the geometric mean intracellular fluorescence intensities determined in a) for the antibody in question and the reference antibodies to obtain corrected geometric mean intracellular fluorescence intensities, 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, iii) determining the slope of the best fit straight 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) 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; iv) normalizing the slope of the best fit straight line of the antibody in question as follows: Normalized slope (antibody in question) = slope of the best fit straight line of the antibody in question / slope of the best fit straight line of the first reference antibody

[0129] wherein 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 intracellular fluorescence intensity ratio.

[0130] It has been found that by using the relative normalized ratio (intracellular) fluorescence intensity (ratio), assay-to-assay and intra-assay bias can be minimized.

[0131] ​By using the correlation between the relative normalized intracellular fluorescence intensity ratio 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.

[0132] 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 intracellular fluorescence intensity ratio as the x-value in the in vitro-in vivo correlation according to the present application.

[0133]

[0134] 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.

[0135] Examples

[0136] I Materials and methods

[0137] Antibodies

[0138] 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.

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

[0140] The monoclonal antibodies used herein were transiently expressed in HEK293 cells (see below) and purified by protein A chromatography using standard procedures (see below).

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

[0142] Expression plasmids

[0143] For the expression of the above-mentioned antibodies, variants of expression plasmids for transient expression of cells, e.g. in HEK293-F, based on cDNA organization with or without CMV-intron A promoter or based on genomic organization with CMV promoter were applied.

[0144] In addition to the antibody expression cassette, the plasmid comprises: - an origin of replication, which allows replication of the plasmid in Escherichia coli - an ampicillin resistance gene, which confers ampicillin resistance in Escherichia coli - a dihydrofolate reductase gene from E. coli as a selection marker in eukaryotic cells. Mus musculus

[0145] The transcriptional unit of the antibody gene consists of the following elements: - a unique restriction site at the 5' end, - an immediate early enhancer and promoter from human cytomegalovirus, - in case of a cDNA organization, followed by an intron A sequence, - a 5' untranslated region of the human antibody gene, - an immunoglobulin heavy chain signal sequence, - the human antibody chain as cDNA or as genomic organization with immunoglobulin exon-intron organization, - a 3' untranslated region with a polyadenylation signal sequence, and - a unique restriction site at the 3' end.

[0146] The fusion genes comprising the antibody chains were 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 the unique restriction sites in the respective plasmids. The subcloned nucleic acid sequences were verified by DNA sequencing. For transient transfection, larger amounts of plasmids were prepared from transformed Escherichia coli cultures by plasmid preparation (Nucleobond AX, Macherey-Nagel).

[0147] Cell culture techniques

[0148] ​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.

[0149] Transient transfection in the HEK293-F system

[0150] Antibodies are generated using the HEK293-F system (Invitrogen) by transient transfection of the respective plasmids (e.g. encoding heavy chain as well as the respective light chain) 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 6 x 106cells / mL in 600 mL and incubated at 120 rpm, 8% C02. Cells are transfected at a cell density of about 1.5 10 6 x 106cells / mL with about 42 mL of the following mixture the day after the second: A) 20 mL Opti-MEM (Invitrogen) with 600 pg total plasmid DNA (1 pg / mL) and B) 20 ml Opti-MEM + 1.2 mL 293 fectin or fectin (2 pL / mL) in equimolar ratios encoding heavy chain and the respective light chain, respectively. Glucose solution is added during the fermentation depending on glucose consumption. Supernatant containing secreted antibodies is harvested after 5-10 days and antibodies are purified directly from the supernatant or the supernatant is frozen and stored. Some of the antibodies that have been produced are:

[0151] Purification

[0152] by using MabSelect Sure-Sepharose TMAntibodies were purified from cell culture supernatant by affinity chromatography using MabSelect SuRe resin (GE Healthcare, Sweden), hydrophobic interaction chromatography using butyl sepharose (GE Healthcare, Sweden) and size exclusion chromatography on Superdex 200 (GE Healthcare, Sweden).

[0153] 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. 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 desired concentration using Vivaspin ultrafiltration devices (Sartorius Stedim Biotech S.A., France) and stored at -80°C.

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

[0155] Mice

[0156] B6.Cg- Fcgrt tm1Dcr Tg(FCGRT)276Dcr mice lack the mouse FcRn a-chain gene, but harbor a hemizygous transgene for the human FcRn a-chain gene (muFcRn- / - huFcRn tg + / -, line 276) 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 of age, body weight 17-22 g at the time of dosing). All animal experiments were approved by the Government of Upper Bavaria, Germany (license number 55.2-1-54-2532.2-28-10) and performed in accordance with the European Union guidelines for the care and use of laboratory animals in AAALAC-accredited animal facilities. Animals were housed in standard cages and had free access to food and water throughout the study period.

[0157] Pharmacokinetic studies

[0158] A single dose of antibody was injected i.v. via the lateral tail vein at a dose level of 5 mg / kg. Mice were divided into 3 groups of 6 mice each covering 9 serum collection time points (0.08, 2, 8, 24, 48, 168, 336, 504 and 672 hours post-dose). Each mouse received two retro-orbital bleeds under light isoflurane™ (CP-Pharma GmbH, Burgdorf, Germany) anesthesia; a third blood sample was taken at the time of euthanasia. Blood was collected into serum tubes (Microvette 500 Z-Gel, Sarstedt, Nümbrecht, Germany). After 2 hours of incubation, samples were centrifuged at 9.300 g for 3 minutes to obtain serum. After centrifugation, serum samples were stored frozen at -20°C until analysis.

[0159] Determination of human antibody serum concentrations

[0160] 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, 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 out at 405 nm with a Tecan sunrise plate reader (Männedorf, Switzerland) with a reference wavelength of 490 nm.

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

[0162] PK analysis

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

[0164] 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.

[0165] 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.

[0166] Example 1

[0167] Cynomolgus monkey SDPK study

[0168] Pharmacokinetics of test compounds were determined following single IV 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. Clearance was calculated according to the following formula: Clearance = Dose / AUC In the case of non-linear pharmacokinetics, the linear fraction of clearance was determined via the following surrogate methods: either the clearance value was estimated following IV administration at high dose levels at which additional non-linear clearance pathways are practically saturated. Or, a PK model was established that included both linear and non-linear, saturable clearance terms. In these cases, the linear clearance fraction determined from the model was used for correlation.

[0169] Example 2

[0170] Preparation of FcRn affinity column

[0171] Expression of FcRn in HEK293 cells

[0172] FcRn was transiently expressed by transfecting HEK293 cells with two plasmids containing the coding sequences for FcRn and beta-2-microglobulin. Transfected cells were cultured in shake flasks at 36.5°C, 120 rpm (shaker amplitude 5 cm), 80% humidity and 7% C02. Cells were diluted every 2-3 days to a density of 3 to 4 10 5 cells / ml.

[0173] For transient expression, a 14 I stainless steel bioreactor was started at 36.5°C, pH 7.0 ± 0.2, p02 35% (aeration with N2 and air, total gas flow 200 ml min -1 -400 rpm, culture volume 8.1. When the cell density reached 20 10 5When the cell density reached 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 at room temperature for 15 minutes 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-bowl centrifuge with 1 I buckets at 4000 rpm for 90 minutes. The supernatant (13 L) was cleared by Sartobran P filters (0.45 pm + 0.2 pm, Sartorius) and the FcRn b-2-microglobulin complex was purified therefrom.

[0174] Biotinylation of the neonatal Fc receptor

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

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

[0177] Coupling to streptavidin sepharose

[0178] For coupling to streptavidin agarose, 1 mL of streptavidin agarose (GE Healthcare, United Kingdom) was added to the biotinylated and dialysed FcRn β-2-microglobulin complex and incubated overnight at 4°C. The derivatized FcRn β-2-microglobulin complex agarose was packed into a 4.6 mm x 50 mm 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).

[0179] Example 3

[0180] Chromatography using FcRn affinity column and pH gradient

[0181] Conditions: Column dimensions: 50 mm x 4.6 mm Load: 30 pg sample Buffer A: 20 mM MES with 140 mM NaCl, adjusted to pH 5.5 Buffer B: 20 mM Tris / HCl with 140 mM NaCl, adjusted to pH 8.8 30 pg sample was applied to the 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, elution was performed over 70 minutes with a linear gradient from 20% to 70% buffer B. Detection was performed using ultraviolet light absorption at a wavelength of 280 nm. The column was regenerated after each run using 20% buffer B for 10 minutes.

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

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

[0184] Example 4

[0185] Chromatography using heparin affinity column and pH gradient

[0186] Conditions: Column dimensions: 50 mm x 5.0 mm Load: 20-50 pg sample Buffer A: 50 mM TRIS, pH 7.4 Buffer B: 50 mM TRIS, pH 7.4, 1000 mM NaCl Apply 20 to 50 pg of protein sample in low salt buffer (< 25 mM ionic strength) to a 5.0 x 50 mm TSKgel Heparin-5PW glass column (Tosoh Bioscience, Tokyo / Japan) pre-equilibrated with buffer A at room temperature. Elute with a linear gradient of 0-100% buffer B over 32 minutes at a flow rate of 0.8 mg / mL. Detect using UV absorption at a wavelength of 280 nm.

[0187] Example 5

[0188] Check antibody internalization

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

[0190] Briefly, the respective antibody was conjugated to a 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 5Cells (104cells per well) were incubated with 100 μL of medium containing pHAb amine-reactive dye-conjugated antibody (final concentration 10 μg / mL). 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).

[0191] Example 6

[0192] Antibody labeling

[0193] Antibodies were labeled according to the manufacturer’s instructions using the SiteClick™ Antibody Azido Modification Kit (Thermo Fisher Scientific). Briefly, N-linked galactose residues of the Fc region were removed by β-galactosidase and replaced by azide-containing galactose (GalNaz) via β-1,4-galactosyltransferase (GalT). This azide modification enables copper-free conjugation of sDIBO-modified dyes. pH-sensitive amine-reactive dyes (523 nm) were purchased from Promega and coupled to sulfo DBCO PEG4 amine. Antibodies were labeled with a 2-fold molar excess of dye. Excess dye was removed using Amicon® Ultra-2 centrifugal filters with a MWCO of 50 kDa (EMD Millipore, # UFC200324) and the antibody was rebuffered in 20 mM histidine buffer (pH 5.5). The concentration of labeled antibody [1] and the ratio of dye to antibody (DAR) [2] were determined with a Nanodrop spectrometer at 280 nm (A 280nm ) and 532 nm (A 532nm ).

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

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

[0196] ε Dye = 47225

[0197] CFDye = 0.36

[0198] Example 7

[0199] Cell maintenance and preparation

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

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

[0202] Example 8

[0203] Flow cytometry and pharmacokinetics analysis

[0204] The mean fluorescence intensity (MFI, more specifically the geo-mean) of internalized antibodies was acquired using a MACSQuant® Analyzer 10 (Miltenyi Biotec) equipped with a laser excitation at 488 nm and filters for collecting emitted light at 585 nm / 540 nm. The 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 linear regression curves using GraphPad Prism to extract the slope (geo-mean MFI / minute for 120 and 240 minutes). Two standard antibodies were selected to normalize the slope: Movlitumab-YTE set to 0 and TCB set to 1. The final slope was plotted against published in vivo human, cynomolgus and hFcRn Tg32 + / + mouse clearance values using TIBCO Spotfire software.

[0205] Example 9

[0206] Quality control

[0207] Biophysical binding properties are key determinants influencing clearance mechanisms. 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 of in vitro antibody clearance rates (Kraft, T.E., et. al., MABS 12 (2020) el683432). Here, this method was used to illustrate potential aberrant binding properties introduced by the click label. Details of the method are provided in Examples 3 and 4.

[0208] To confirm the absence of unbound dye and to verify the concentration measured on the spectrometer, the labeled antibodies were subjected to size exclusion chromatography. The samples were separated using a BioSuite Diol (OH) column (Waters, 186002165) with potassium phosphate monobasic 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 geo-mean of the AUCs of all antibodies was calculated and the deviation of each antibody from this geo-mean was determined. For antibodies that are reliable within the assay according to the present application, the deviation from the geo-mean is expected to be below 15%.

Claims

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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