Methods for determining therapeutic antibody amounts in the brain

Abstract

A method for determining the concentration of a therapeutic antibody in a tissue of an experimental animal to which a therapeutic antibody has been administered is reported herein, wherein the interference of residual blood in a tissue sample from the experimental animal for determining the concentration of the therapeutic antibody in said tissue is reduced, wherein the concentration of the therapeutic antibody in the tissue of the experimental animal is calculated with the formula: C tmAb,组织,det. = obtained by determining the concentration of the therapeutic antibody in a tissue sample of the experimental animal, C tmAb,血浆,det. = obtained by determining the concentration of the therapeutic antibody in a blood sample of the experimental animal directly before taking the tissue sample, C refmAb,组织,det. = obtained by determining the concentration of an inert reference antibody in a tissue sample of the experimental animal, C refmAb,血浆,det. = obtained by determining the concentration of an inert reference antibody in a blood sample of the experimental animal directly before taking the tissue sample, C 组织,样品 = obtained by determining the tissue concentration in the tissue sample, wherein the inert reference antibody does not penetrate into said tissue, wherein the inert reference antibody is administered 2 to 10 minutes before obtaining the tissue and blood samples.

Description

[0001] The present invention belongs to the field of immunoassays. More specifically, a method for determining the amount of a therapeutic antibody in brain tissue is reported herein, more specifically, the amount of a therapeutic antibody transported from the blood across the blood brain barrier into the brain is determined. BACKGROUND

[0002] For the analysis of therapeutic monoclonal antibodies (tmAb) in samples of extracorporeal or in vivo origin, a corresponding assay is necessary.

[0003] The determination of the amount of a therapeutic antibody is usually carried out by determining the amount of said therapeutic antibody in the sample. Thus, immunoassays such as ELISA, RIA, Western blot assay and the like can be used.

[0004] Katsinelos et al. (Front. Immunol. 10 (2019) A1139) review the protective role of antibodies and their receptors in preventing neurodegeneration on ordered protein assembly. They outline that IgG levels in human serum are maintained at about 10 mg / ml. The brain is isolated from serum by the blood brain barrier (BBB), which is impermeable to macromolecules including IgG, and is bathed in cerebrospinal fluid (CSF), which is produced after filtration and ion transport through the choroid plexus from blood. Thus, the IgG concentration in the produced CSF is about 500 to 1,000 times lower than in serum.

[0005] Hanzatian et al. (mAbs 10 (2018) 765-777) report that therapeutic monoclonal antibodies and endogenous IgG antibodies show limited uptake into the central nervous system (CNS) due to the blood brain barrier (BBB) regulation and control of selective and specific transport of both exogenous and endogenous substances into the brain. There have been studies in rodents and monkeys using natural transport mechanisms, such as receptor-mediated transcytosis (RMT), to deliver antibody therapeutics into the brain. Hanzatian et al. used two independent methods in parallel to observe specific uptake in the brain: a sensitive quantitative assay based on electrochemiluminescence and a semi-quantitative immunohistochemical technique for brain concentration determination and biodistribution / localization in the brain, respectively, after systemic administration of each DVD-Ig. Regardless of the chosen CNS target or systemic route of administration, a significant enhancement of brain uptake and retention of all TfR1 DVD-Ig proteins was observed. To prepare brain samples for analysis, C57BL / 6N mice used were transcardially perfused with cold heparinized Dulbecco’s PBS at a rate of 2 ml / min for 10 minutes using a programmable peristaltic pump.

[0006] Zuchero et al. (Neuron 89 (2016) 70-82; wild-type mice, which were collected for whole blood and PBS perfusion after intravenous injection of the target antibody) and Janowicz et al. (Nature Sci. Rep. 9 (2019) 9255; pR5 mice transgenic for P301 L tau, which had been administered Alexa-647 labeled IgG, Fab or scFv by retro-orbital injection, were perfused after treatment to remove the antibody from their vasculature) have used similar approaches.

[0007] In WO 2018 / 152359, mice overexpressing human tau from the PS 19 line were used to assess target engagement of the chimeric IgG anti-tau antibody clones 1C7 and 1A1. Thus, mice were injected intravenously (35 mg / kg) or intraperitoneally (50 mg / kg) with control IgG, chimeric IgG clone 1C7 or chimeric IgG clone 1A1. Two or seven days after injection, cerebrospinal fluid (CSF) was collected via the cisterna magna and visually inspected for potential blood contamination, and after cardiac perfusion with ice-cold PBS, brain tissue was removed and snap-frozen.

[0008] Ayabe, M. et al. report on intravenous administration of an anti-human interleukin 6 receptor (hIL-6R) antibody or a control antibody to tumor-bearing hIL-6R transgenic mice and intravenous administration of bovine serum albumin (BSA) as a marker for residual blood volume in tissues. Lysate samples were treated with immunoprecipitation using anti-BSA antibody and protein A magnetic beads, followed by trypsin digestion. Each surrogate peptide was analyzed simultaneously by LC / ESI-MS / MS. Corrected tissue concentrations were calculated.

[0009] Vedeler et al. report on immunoglobulins in serum and cerebrospinal fluid of patients with acute Guillain-Barre syndrome (Acta Neurol. Scand. 73 (1986) 388-393).

[0010] Shah et al. report on the biodistribution coefficients of antibodies, especially the extrapolation of tissue concentrations of monoclonal antibodies from plasma concentrations in several preclinical species and humans (MABS, 5 (2013) 297-305).

[0011] Lavezzi et al. report on MPBPK-TMDD models for monoclonal antibodies (mAbs), especially surrogate models, comparisons and identifiability issues (J. Pharmacokin. Pharmcodyn. 45 (2018) 787-802). SUMMARY

[0012] A method for determining the amount of a therapeutic antibody that has been transported across the blood brain barrier from the blood into the brain of an experimental animal is reported herein. The amount is preferably determined in a brain lysate sample. The gist of the invention is the additional administration of an inert antibody that is not transported across the blood brain barrier, the amount of the therapeutic antibody transported across the blood brain barrier in the brain sample is determined before the brain sample is obtained. By administering the inert antibody a corrected value of the therapeutic antibody present in the brain sample that is present in the residual blood can be obtained. This amount originating from the residual blood is used to correct the determined amount of antibody that is not located in the brain. Without correction the determination will determine the total amount of therapeutic antibody in the sample, i.e. the amount transported across the blood brain barrier into the brain and the amount in the residual blood of the sample. The amount of therapeutic antibody in the residual blood cannot be neglected because only about 0.1 % of the antibody in the blood will pass the blood brain barrier. Therefore the concentration of therapeutic antibody in the blood is at least two and up to three orders of magnitude higher than the concentration of therapeutic antibody in the brain. The result obtained will thus be too high if not corrected in the method as described in the invention.

[0013] The invention is at least partly based on the finding that in order to reliably and correctly determine the amount of therapeutic antibody in a brain lysate that has been transported across the blood brain barrier into the brain, the amount of therapeutic antibody in the residual blood of the brain lysate sample has to be corrected for (i.e. subtracted).

[0014] The invention is at least partly based on the finding that the amount of residual blood in a brain lysate can be determined by administering a correction antibody before the brain sample is taken. It has been found that it is particularly advantageous to use as a reference antibody an antibody that does not bind to any target in the experimental animal from which the brain sample is obtained, most preferably a human germline antibody.

[0015] One aspect of the invention is a method / assay for determining the concentration of a therapeutic antibody in a tissue of an experimental animal, wherein the tissue has a barrier to the blood circulation of said animal, and wherein the therapeutic antibody has been administered to said experimental animal, wherein the interference of residual blood in a tissue sample from an experimental animal for determining the concentration of the therapeutic antibody in said tissue is reduced, the method comprising the following steps:

[0016] i) determining the concentration of the therapeutic antibody in a blood sample of the experimental animal,

[0017] ii) determining the concentration of the therapeutic antibody in a tissue sample of the experimental animal,

[0018] iii) determining the concentration of an inert reference antibody in a blood sample of the experimental animal,

[0019] iv) determining the concentration of the inert reference antibody in a tissue sample of the experimental animal,

[0020] v) the tissue concentration in the tissue sample,

[0021] and determining the concentration of the therapeutic antibody in the tissue of the experimental animal with the following formula:

[0022]

[0023] wherein,

[0024] C tmAb,血浆,det. = the concentration of the therapeutic antibody of i)

[0025] C tmAb,组织,det. = the concentration of the therapeutic antibody of ii)

[0026] C refmAb,组织,det. = the concentration of the therapeutic antibody of iii)

[0027] C refmAb,血浆,det. = the concentration of the therapeutic antibody of iv)

[0028] C 组织,样品 = the tissue concentration of v)

[0029] - wherein the inert reference antibody does not cross the said barrier between the tissue and the blood circulation,

[0030] - wherein the inert reference antibody has been i) administered together with the therapeutic antibody, if the sample is to be taken within 5 minutes after administration of the therapeutic antibody, or ii) administered 2 to 10 minutes before taking the tissue sample.

[0031] The same aspect in alternative expression is a method for determining the concentration of a therapeutic antibody in a tissue of an experimental animal to which the therapeutic antibody has been administered, wherein the interference of residual blood in a tissue sample from the experimental animal for determining the concentration of the therapeutic antibody in the tissue is reduced,

[0032] wherein the concentration of the therapeutic antibody in the tissue of the experimental animal is calculated with the following formula:

[0033]

[0034] wherein,

[0035] C tmAb,组织,det. = is obtained by determining the concentration of the therapeutic antibody in a tissue sample of the experimental animal,

[0036] C tmAb,血浆,det. = is obtained by determining the concentration of the therapeutic antibody in a blood sample of the experimental animal,

[0037] C refmAb,组织,det. = is obtained by determining the concentration of the inert reference antibody in a tissue sample of the experimental animal,

[0038] C refmAb,血浆,det. = obtained by determining the concentration of the inert reference antibody in a blood sample of the experimental animal,

[0039] C 组织,样品 = obtained by determining the tissue concentration in the tissue sample,

[0040] - wherein the inert reference antibody does not penetrate into the tissue,

[0041] - wherein the inert reference antibody is administered 2 to 10 minutes before the tissue sample is obtained.

[0042] The following are all individual embodiments of each and any aspect of the invention. Thus, with respect to any individual aspect of the invention, all and any possible permutations of the disclosed embodiments are disclosed,

[0043] In one embodiment, the blood sample is taken up to 5 minutes before the tissue sample. In one embodiment, the blood sample is taken before the tissue sample. In one embodiment, the blood sample is taken together with or simultaneously with the tissue sample.

[0044] In one embodiment, the tissue is brain tissue and the therapeutic antibody can cross the blood brain barrier, or eye tissue and the therapeutic antibody can cross the blood eye barrier.

[0045] One aspect of the invention is a method / assay for determining the concentration of a therapeutic antibody in brain tissue or a brain tissue sample of an experimental animal, wherein the brain tissue has a barrier to the blood circulation of said animal, and wherein a therapeutic antibody has been administered to the experimental animal, wherein the interference from residual blood in the brain tissue sample from the experimental animal for determining the concentration of the therapeutic antibody in the brain tissue is reduced, the method comprising the steps of:

[0046] i) determining the concentration of the therapeutic antibody in a blood sample of the experimental animal,

[0047] ii) determining the concentration of the therapeutic antibody in a brain tissue sample of the experimental animal,

[0048] iii) determining the concentration of the inert reference antibody in a blood sample of the experimental animal,

[0049] iv) determining the concentration of the inert reference antibody in a brain tissue sample of the experimental animal,

[0050] v) determining the brain tissue concentration in the tissue sample,

[0051] and determining the concentration of the therapeutic antibody in the brain tissue or brain tissue sample of the experimental animal with the following formula:

[0052]

[0053] wherein,

[0054] C tmAb,血浆,det. = concentration of the therapeutic antibody of i)

[0055] C tmAb,组织,det. = concentration of the therapeutic antibody of ii)

[0056] C refmAb,组织,det. = concentration of the therapeutic antibody of iii)

[0057] C refmAb,血浆,det. = concentration of the therapeutic antibody of iv)

[0058] C 组织,样品 = concentration of the tissue of v)

[0059] - wherein the inert reference antibody does not cross the blood brain barrier between the brain tissue and the blood circulation,

[0060] - wherein the inert reference antibody has been i) administered together with the therapeutic antibody, if the brain tissue sample is to be taken within 5 minutes after administration of the therapeutic antibody, or ii) administered 2 to 10 minutes before taking the brain tissue sample.

[0061] The same aspect in alternative wording is a method for determining the concentration of a therapeutic antibody in brain tissue or a brain tissue sample of an experimental animal to which the therapeutic antibody has been administered, wherein the interference of residual blood in the brain tissue sample from the experimental animal for determining the concentration of the therapeutic antibody in the brain tissue is reduced,

[0062] wherein the concentration of the therapeutic antibody in the brain tissue of the experimental animal is calculated with the formula:

[0063]

[0064] wherein,

[0065] C tmAb,组织,det. = is obtained by determining the concentration of the therapeutic antibody in a brain tissue sample of the experimental animal,

[0066] C tmAb,血浆,det. = is obtained by determining the concentration of the therapeutic antibody in a blood sample of the experimental animal,

[0067] C refmAb,组织,det. = is obtained by determining the concentration of the inert reference antibody in a brain tissue sample of the experimental animal,

[0068] C refmAb,血浆,det. = is obtained by determining the concentration of the inert reference antibody in a blood sample of the experimental animal,

[0069] C 组织,样品= Obtained by measuring the concentration of brain tissue in brain tissue samples.

[0070] -The inert reference antibody will not penetrate into the brain tissue.

[0071] -The inert reference antibody is administered 2 to 10 minutes before obtaining the brain tissue sample.

[0072] In one embodiment, the therapeutic antibody is a bispecific antibody.

[0073] In one embodiment, the therapeutic antibody specifically binds to human transferrin receptors and brain targets.

[0074] In one embodiment, the brain target is human CD20, human Aβ, human α-synuclein, human τ, human glucocerebrosidase, human lingo-1, or human huntingtin.

[0075] In one embodiment, the experimental animals are selected from mice, rats, rabbits, dogs, sheep, apes, and monkeys.

[0076] In one embodiment, the experimental animal is a non-human experimental animal weighing more than 100g and less than 15kg.

[0077] In one embodiment, the experimental animal was a cynomolgus monkey.

[0078] In one embodiment, the inert reference antibody is a human lineage antibody.

[0079] In one embodiment, the inert reference antibody is DP47GS. In one embodiment, the inert reference antibody comprises the heavy chain variable domain of SEQ ID NO:67 and the light chain variable domain of SEQ ID NO:68. In one embodiment, the inert reference antibody comprises the heavy chain of SEQ ID NO:69 and the light chain of SEQ ID NO:70.

[0080] In one embodiment, the inert reference antibody does not cross the barrier in a detectable amount within 15 minutes of its administration.

[0081] In one embodiment, the inert reference antibody does not cross the barrier in a detectable amount within 10 minutes of its administration.

[0082] In one embodiment, the inert antibody is administered 5 to 10 minutes before tissue sample collection.

[0083] In one embodiment, the tissue is perfused directly with an aqueous solution after blood sample collection and before tissue sample collection.

[0084] In one embodiment, the concentration is determined by bridging ELISA. Attached Figure Description

[0085] Figure 1 An exemplary calculation for determining the residual plasma-corrected brain lysate concentration of tmAb.

[0086] Figure 2 In an example of the method described in this invention, the structure of an exemplary brain shuttle construct is used.

[0087] Figure 3 The detection methods for therapeutic antibodies and reference antibodies used in the examples.

[0088] Figure 4 Overlay of calibration curves for the detection assay as described in Example 1, with 1% cynomolgus and mouse brain lysates present respectively.

[0089] Figure 5 The ratio of the corrected to the uncorrected concentration of antibody 2 in mouse brain lysate.

[0090] Figure 6 ELISA for determining antibody 2.

[0091] Figure 7 Calibration curve of the complement factor H Elecsys assay.

[0092] Figure 8 An ELISA for quantifying α2-macroglobulin in cCSF. The capture antibody is a murine anti-human α2-macroglobulin antibody; the detection antibody is a biotinylated goat anti-human α2-macroglobulin antibody.

[0093] Figure 9 Figure Z shows the calibration curve for ELISA.

[0094] Figure 10 Calibration curve of complement component 5a ELISA assay.

[0095] Figure 11 Magnevist time course in plasma and brain tissue in PK studies.

[0096] Figure 12 The ELISA capture mAb used to quantify cynomolgus IgG in cCSF is an anti-cynomolgus IgG antibody; the detection mAb is an anti-cynomolgus IgG antibody that binds to a non-interfering antigenic determinant relative to the primary antibody.

[0097] Figure 13 Figure X The calibration curve of ELISA. Detailed Implementation

[0098] A method for determining the amount of a therapeutic antibody that has been transported across the blood brain barrier from the blood into the brain of an experimental animal is reported herein. The amount is preferably determined in a brain lysate sample. The gist of the invention is the additional administration of an inert antibody that is not transported across the blood brain barrier, the amount of the therapeutic antibody transported across the blood brain barrier in the brain sample is determined before the brain sample is obtained. By administering the inert antibody a corrected value of the therapeutic antibody present in the brain sample that is present in the residual blood can be obtained. This amount originating from the residual blood is used to correct the determined amount of antibody that is not located in the brain. The determination without correction would determine the total amount of therapeutic antibody in the sample, i.e. the amount transported across the blood brain barrier into the brain and the amount in the residual blood of the sample. The amount of therapeutic antibody in the residual blood cannot be neglected because only about 0.1 % of the antibody in the blood will pass the blood brain barrier. Therefore the concentration of therapeutic antibody in the blood is at least two and up to three orders of magnitude higher than the concentration of therapeutic antibody in the brain. The result obtained would thus be too high if not corrected in the method as described in the invention.

[0099] The invention is at least partly based on the finding that in order to reliably and correctly determine the amount of therapeutic antibody in a brain lysate that has been transported across the blood brain barrier into the brain, the amount of therapeutic antibody in the residual blood of the brain lysate sample has to be corrected for (i.e. subtracted).

[0100] The invention is at least partly based on the finding that the amount of residual blood in a brain lysate can be determined by administering a correction antibody before the brain sample is taken. It has been found that it is particularly advantageous to use as a reference antibody an antibody that does not bind to any target in the experimental animal from which the brain sample is obtained, most preferably a human germline antibody.

[0101] One aspect of the invention is a method / assay for determining the concentration of a therapeutic antibody in a tissue of an experimental animal, wherein the tissue has a barrier to the blood circulation of said animal, and wherein the therapeutic antibody has been administered to said experimental animal, wherein the interference of residual blood in a tissue sample from an experimental animal for determining the concentration of the therapeutic antibody in said tissue is reduced, the method comprising the steps of:

[0102] i) determining the concentration of the therapeutic antibody in a blood serum sample of the experimental animal,

[0103] ii) determining the concentration of the therapeutic antibody in a tissue sample of the experimental animal,

[0104] iii) determining the concentration of an inert reference antibody in a blood serum sample of the experimental animal,

[0105] iv) determining the concentration of the inert reference antibody in a tissue sample of the experimental animal,

[0106] v) the concentration of the tissue in the tissue sample,

[0107] and determining the concentration of the therapeutic antibody in the tissue of the experimental animal with the following formula:

[0108]

[0109] wherein,

[0110] C tmAb,血浆,det. = the concentration of the therapeutic antibody of i)

[0111] C tmAb,组织,det. = the concentration of the therapeutic antibody of ii)

[0112] C refmAb,组织,det. = the concentration of the therapeutic antibody of iii)

[0113] C refmAb,血浆,det. = the concentration of the therapeutic antibody of iv)

[0114] C 组织,样品 = the concentration of the tissue of v)

[0115] - wherein the inert reference antibody does not cross the said barrier between the tissue and the blood circulation,

[0116] - wherein the inert reference antibody has been i) administered together with the therapeutic antibody, if the sample will be taken within 5 minutes after administration of the therapeutic antibody, or ii) administered 2 to 10 minutes before taking the tissue sample,

[0117] - wherein the blood sample is taken together with the tissue sample / directly before the tissue sample / simultaneously.

[0118] Definitions

[0119] Knobs into holes dimerization modules and their use in antibody engineering have been described in Carter P.; Ridgway J.B.B.; Presta L.G.: Immunotechnology, Vol. 2, No. 1, February 1996, pages 73-73(1).

[0120] For general information on human immunoglobulin light and heavy chain nucleotide sequences, see: Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0121] The amino acid positions of all constant regions and domains of the heavy and light chains used herein are based on the Kabat numbering system described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991), and are referred to herein as “according to Kabat numbering”. Specifically, the Kabat numbering system (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991), pp. 647–660) is used for the constant domain CL of the κ and λ isoforms of the light chain, and the Kabat EU index numbering system (see pp. 661–723) is used for the constant domains of the heavy chain (CH1, hinge, CH2, and CH3, which in this case are further clarified herein by reference to “according to Kabat EU indexing”).

[0122] The term "about" indicates a range of + / - 20% for the following value. In one embodiment, the term "about" indicates a range of + / - 10% for the following value. In another embodiment, the term "about" indicates a range of + / - 5% for the following value.

[0123] The term "antibody-dependent cytotoxicity (ADCC)" refers to a function mediated by Fc receptor binding and specifically to the lysis of target cells by the antibody reported herein in the presence of effector cells. In one embodiment, ADCC is measured in the presence of effector cells, such as freshly isolated PBMCs (peripheral blood mononuclear cells) or effector cells purified from the erythrocyte sedimentation rate buffy coat, such as monocytes or NK (natural killer) cells, by treating a CD19-expressing erythroid cell formulation (e.g., K562 cells expressing recombinant human CD19) with the antibody reported herein. Target cells are labeled with 51Cr and then cultured with the antibody. The labeled cells are co-cultured with effector cells, and the release of 51Cr in the supernatant is analyzed. Controls consist of culturing target endothelial cells with effector cells but not with the antibody. The ability of the antibody to induce the initial step of ADCC is investigated by measuring the binding of the antibody to cells expressing the Fcγ receptor (e.g., recombinant cells expressing FcγRI and / or FcγRIIA or NK cells (which essentially express FcγRIIIA)). In a preferred embodiment, the binding to FcγR on NK cells is measured.

[0124] The term "amplifier" denotes an entity or process that enhances a signal in an assay method, e.g. in an ELISA (e.g. an enzyme amplifier used in an ELISA).

[0125] The terms "anti-human A-beta antibody" and "an antibody specifically binding to human A-beta" refer to an antibody that is capable of binding to an A-beta peptide with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in targeting A-beta peptide.

[0126] It is noted that human A-beta has several naturally occurring forms, of which the human forms are referred to as Aβ39, Aβ40, Aβ41, Aβ42and Aβ43. The most important form, Aβ42, has the amino acid sequence of SEQ ID NO: 01. In Aβ41, Aβ40, Aβ39, respectively, the C-terminal amino acid A, IA and VIA is missing. In the Aβ43 form, the C-terminus of SEQ ID NO: 01 (33106) comprises an additional threonine residue.

[0127] The term thus also encompasses antibodies that bind to a truncated fragment of a human A-beta polypeptide.

[0128] The term "antibody" herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, or multispecific antibodies (e.g., bispecific antibodies).

[0129] An antibody typically comprises two so-called light chain polypeptides (light chains) and two so-called heavy chain polypeptides (heavy chains). Each heavy and light chain polypeptide comprises a variable domain (variable region), typically the amino terminal portion of the polypeptide chain, which comprises the binding site capable of interacting with an antigen. Each heavy and light chain polypeptide comprises a constant region, typically the carboxyl terminal portion. The constant region of the heavy chain mediates the binding of the antibody to i) cells bearing Fc gamma receptors (FcγR), such as phagocytic cells, or ii) cells bearing the neonatal Fc receptor (FcRn) (also known as the Brambell receptor). It also mediates binding to certain factors, including factors of the classical complement system, e.g. component (C1q). The constant domain of the heavy chain of an antibody comprises a CH1-domain, a CH2-domain and a CH3-domain, while the light chain comprises only one constant domain CL, which can be of the kappa or lambda isotype.

[0130] The variable domain of an immunoglobulin light or heavy chain comprises in order three regions of hypervariability, i.e. the Complementarity Determining Regions (CDRs), and four more conserved framework regions (FRs).

[0131] An "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv and scFab), single-domain antibodies (dAbs), and multispecific antibodies formed from antibody fragments. For a review of certain antibody fragments, see Holliger and Hudson, Nature Biotechnology 23:1126-1136 (2005).

[0132] "Blood brain barrier" or "BBB" refers to the physiological barrier between the peripheral circulation and the brain and spinal cord that is formed by tight junctions in the endothelial cell membranes of brain capillaries, creating a tight barrier that limits the transport of molecules into the brain, even very small molecules like urea (60 daltons). The BBB within the brain, the blood spinal cord barrier within the spinal cord, and the blood-retinal barrier within the retina are contiguous capillary barriers within the CNS and are collectively referred to herein as the blood brain barrier or BBB. The BBB also includes the blood CSF barrier (choroid plexus), where the barrier is composed of ependymal cells, not capillary endothelial cells.

[0133] A "blood brain barrier receptor" (abbreviated herein as "BBBR") is an extracellular membrane-bound receptor protein expressed on brain endothelial cells that is capable of transporting molecules across the BBB or for use in transporting exogenously administered molecules. Examples of BBBRs herein include: transferrin receptor (TfR), insulin receptor, insulin-like growth factor receptor (IGF-R), low density lipoprotein receptors (including but not limited to low density lipoprotein receptor-related protein 1 (LRP1) and low density lipoprotein receptor-related protein 8 (LRP8)), and heparin-binding epidermal growth factor-like growth factor (HB-EGF). One preferred BBBR is the transferrin receptor (TfR).

[0134] The term "brain effector entity" denotes a molecule that is transported across the BBB to the brain. The effector entity generally has a characteristic therapeutic activity that is desired to be delivered to the brain. Effector entities include neurological disorder drugs and cytotoxic agents, such as polypeptides and antibodies, particularly monoclonal antibodies or fragments thereof directed to brain targets.

[0135] The term "capture antibody" denotes an antibody used in a sandwich ELISA format to bind (i.e., capture) a target material present in a sample for detection. A second antibody (i.e., detection antibody) then binds to the captured target, and allows detection of the antibody-target-antibody complex (forming a "sandwich" consisting of antibody-target-antibody).

[0136] The "central nervous system" or "CNS" refers to the complex of nervous tissue that controls body functions and includes the brain and spinal cord.

[0137] The terms "CNS antigen" and "brain target" refer to antigens and / or molecules expressed in the CNS, including the brain, that can be targeted by an antibody or small molecule. Examples of such antigens and / or molecules include, but are not limited to, beta-secretase 1 (BACE1), amyloid beta (Αβ), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau protein, apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), glucocerebrosidase, and caspase 6.

[0138] A "conjugate" is a fusion protein of the present application conjugated to one or more heterologous molecules, including but not limited to a label, a neurological disorder drug, or a cytotoxic agent.

[0139] The term "detection antibody" refers to an antibody that carries a means for visualization or quantitation. Such means are typically enzymes (which can catalyze a colorimetric or fluorescent reaction product upon addition of appropriate substrates), such as horseradish peroxidase, urease, alkaline phosphatase, glucoamylase, and beta-galactosidase. In some embodiments, the detection antibody is directed against the antigen of interest. In some embodiments, the detection antibody is an anti-species antibody. In some embodiments, the detection antibody is conjugated to a detectable label, such as biotin, a fluorescent marker, or a radioisotope, and detection and / or quantitation is performed using the label.

[0140] The term "detection reagent" refers to a reagent that allows for detection and / or quantitation of an antibody that binds to an antigen. In some embodiments, the detection reagent is a colorimetric substrate for an enzyme that has been conjugated to an antibody. Addition of the appropriate substrate to the antibody-enzyme conjugate results in a colorimetric or fluorescent signal (e.g., upon binding of the conjugated antibody to the antigen of interest). This definition also encompasses the use of biotin and avidin-based compounds (e.g., including but not limited to neutravidin and streptavidin) as part of a detection system.

[0141] As used herein, the term "immediately thereafter" refers to the time period between taking a first sample and a second sample, which encompasses only the change in sampling device and the actual time of taking the sample. In one embodiment, the term immediately thereafter refers to a time period of 5 minutes or less, in another embodiment, a time period of 3 minutes or less, and in a preferred embodiment, a time period of 2 minutes or less.

[0142] "Effector function" refers to those biological activities attributable to the Fc region of an antibody, which vary with the antibody class. Examples of antibody effector functions include Clq binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor); and B cell activation.

[0143] Fc receptor binding-dependent effector functions can be mediated by the interaction of an antibody's Fc region with Fc receptors (FcRs), which are specialized cell surface receptors of hematopoietic cells. Fc receptors belong to the immunoglobulin superfamily, and have been shown to mediate the removal of antibody-coated pathogens by phagocytosis of immune complexes, as well as the lysis of erythrocytes and various other cellular targets (e.g., tumor cells) coated with the appropriate antibodies via antibody-dependent cell-mediated cytotoxicity (ADCC) (see, e.g., Van de Winkel, J.G. and Anderson, C.L., J. Leukoc. Biol. 49 (1991) 511-524). FcRs are defined by their specificity for immunoglobulin isotypes: Fc receptors for IgG antibodies are referred to as FcyRs. Fc receptor binding is described in, e.g., Ravetch, J.V. and Kinet, J.P., Immunol. 9 (1991) 457-492; Capel, P.J. et al., Immunomethods 4 (1994) 25-34; de Haas, M. et al., J. Lab. Clin. Med. 126 (1995) 330-341; and Gessner, J.E. et al. Ann. Hematol. 76 (1998) 231-248.

[0144] Receptor cross-linking of the Fc region for IgG antibodies (FcyRs) can trigger a wide variety of effector functions, including phagocytosis, antibody-dependent cellular cytotoxicity, and the release of inflammatory mediators as well as clearance of immune complexes and modulation of antibody production. In humans, three classes of FcyRs have been identified, which are:

[0145] - FcγRI (CD64) binds monomeric IgG with high affinity and is expressed on macrophages, monocytes, neutrophils and eosinophils. Modifications of at least one of the amino acid residues E233-G236, P238, D265, N297, A327 and P329 in the Fc region of IgG (numbering according to EU index of Kabat) reduce binding to FcγRI. The IgG2 residues at positions 233-236 are replaced by IgG1 and IgG4, binding to FcγRI is reduced by 10 3 fold and the reaction of human monocytes to antibody sensitized erythrocytes is abolished (Armour, K. L. et al., Eur. J. Immunol. 29 (1999) 2613-2624).

[0146] - FcγRII (CD32) binds complexed IgG with medium to low affinity and is widely expressed. This receptor can be divided into two subtypes, FcγRIIA and FcγRIIB. FcγRIIA is found on many cells involved in killing, such as macrophages, monocytes, neutrophils and appears to be able to activate the killing process. FcγRIIB appears to play a role in inhibition and is found on B-cells, macrophages and on mast cells and eosinophils. On B-cells it appears to have the function of inhibiting further immunoglobulin production and isotype switching to, for example, the IgE class. On macrophages, FcγRIIB can inhibit phagocytosis mediated through FcγRIIA. On eosinophils and mast cells, the B-type can contribute to the inhibition of activation of these cells through IgE binding to its separate receptors. For example, antibodies comprising an IgG Fc region with at least one of the amino acid residues E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, R292 and K414 (numbering according to EU index of Kabat) mutated have been found to bind less to FcγRIIA.

[0147] - FcγRIII (CD16) binds IgG with medium to low affinity and exists in two types. FcγRIIIA is found on NK cells, macrophages, eosinophils and some monocytes and T-cells and mediates ADCC. FcγRIIIB is highly expressed on neutrophils. For example, antibodies comprising an IgG Fc region with at least one of the amino acid residues E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, S239, E269, E293, Y296, V303, A327, K338 and D376 (numbering according to EU index of Kabat) mutated have been found to bind less to FcγRIIIA.

[0148] The above mutation sites and methods for measuring binding to FcyRI and FcyRIIA are described in Shields, R.L. et al., J. Biol. Chem. 276 (2001) 6591-6604, which locates the binding site on human IgGl for Fc receptors.

[0149] An "effective amount" of an agent, e.g., a pharmaceutical formulation, is an amount effective for eliciting the desired therapeutic or prophylactic effect at a reasonable benefit / risk ratio applicable to any medical treatment.

[0150] The term "ELISA" denotes enzyme-linked immunosorbent assay. Different ELISA formats and applications are known in the art (see, e.g., Crowther, "Enzyme-Linked Immunosorbent Assay (ELISA)" in Molecular Biomethods Handbook, Rapley et al., eds., pp. 595-617, Humana Press, Inc., Totowa, NJ (1998); Harlow and Lane, eds., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1988); Ausubel et al., eds., Current Protocols in Molecular Biology, Chapter 11, John Wiley & Sons, Inc., New York (1994)).

[0151] One particular ELISA format is the so-called "direct ELISA". In this ELISA format, the target (e.g., a polypeptide) is present in the sample being tested. In a direct ELISA, the sample containing the target is contacted with a solid phase, e.g., an immobile or immobilized support (e.g., a microtiter plate well). If the target is present in the sample, it will become immobilized on the solid phase, and then detected directly using an enzyme-conjugated detection molecule. If the target is an antigen, the detection molecule is an antibody specific for the antigen, or if the target is an antibody specific for an antigen, the detection molecule is an enzyme-conjugated antibody specific for the antigen.

[0152] Another particular ELISA format is the so-called "indirect ELISA". In this ELISA format, an antigen (or antibody) is immobilized on a solid phase (e.g., a microtiter plate well). Thereafter, an antigen-specific antibody (or antigen) is added, followed by a detection antibody specific for the antibody specifically bound to the antigen. The detection antibody can be a "species-specific" antibody (e.g., a goat anti-rabbit antibody).

[0153] Another particular ELISA format is the so-called "sandwich ELISA". In this format, the antigen is captured by an antibody (i.e. the capture antibody) that specifically binds to the antigen, which is immobilized on a solid phase (e.g. a microtiter plate well), either covalently or via specific binding partners. Typically, a sample comprising the antigen is added to the solid phase and then washed. If the target antigen is present in the sample, it will bind to the solid phase via the capture antibody.

[0154] The above particular ELISA formats can be used in combination. The sandwich ELISA can be a "direct sandwich ELISA", in which the captured antigen is detected directly by using an enzyme-conjugated antibody against the antigen. The sandwich ELISA can be an "indirect sandwich ELISA", in which the captured antigen is detected indirectly by using an antibody against the antigen, which is then detected by another enzyme-conjugated antibody that binds to the specific antibody against the antigen, either directly or via an attached label. The third antibody is detected with a reporter reagent.

[0155] The term "Fc receptor" as used herein refers to activating receptors characterized by the presence of cytoplasmic ITAM sequences associated with the receptor (see e.g. Ravetch, JV and Bolland, S., Annu. Rev. Immunol. 19 (2001) 275-290). Such receptors are FcyRI, FcyRIIA and FcyRIIIA. The term "does not bind FcyR" means that the binding of the antibody to NK cells at an antibody concentration of 10 μg / ml, as reported herein, is 10% or less of the binding of the anti-OX40L antibody LC.001 as reported in WO 2006 / 029879.

[0156] While IgG4 shows reduced FcR binding, antibodies of other IgG subclasses show strong binding. However, Pro238, Asp265, Asp270, Asn297 (loss of Fc carbohydrate), Pro329 and 234, 235, 236 and 237, Ile253, Ser254, Lys288, Thr307, Gln311, Asn434 and His435 are residues which, if altered, also provide reduced FcR binding (Shields, R.L. et al., J. Biol. Chem. 276 (2001) 6591-6604; Lund, J. et al., FASEB J. 9 (1995) 115-119; Morgan, A. et al., Immunology 86 (1995) 319-324; and EP 0 307 434). In one embodiment, the antibody reported herein is of the IgGl or IgG2 subclass and comprises the mutations PVA236, GLPSS331 and / or L234A / L235A. In one embodiment, the antibody reported herein is of the IgG4 subclass and comprises the mutation L235E. In one embodiment, the antibody further comprises the mutation S228P.

[0157] The term "Fc region", as used herein, defines the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxy-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region can or can not be present. Unless otherwise indicated, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system (also referred to as the EU index), as described in Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242.

[0158] In one embodiment, the antibody reported herein comprises as Fc region an Fc region derived from human origin. In one embodiment, the Fc region comprises all parts of a human constant region. The Fc region of an antibody is directly involved in complement activation, Clq binding, C3 activation and Fc receptor binding. While the influence of an antibody on the complement system depends on certain conditions, the binding to Clq is caused by a binding site defined in the Fc region. This binding site is known in the prior art and described, for example, in Lukas, T.J. et al., J. Immunol. 127 (1981) 2555-2560; Brunhouse, R. and Cebra, J.J., Mol. Immunol. 16 (1979) 907-917; Burton, D.R. et al., Nature 288 (1980) 338-344; Thommesen, J.E. et al., Mol. Immunol. 37 (2000) 995-1004; Idusogie, E.E. et al., J. Immunol. 164 (2000) 4178-4184; Hezareh, M. et al., J. Virol. 75 (2001) 12161-12168; Morgan, A. et al., Immunology 86 (1995) 319-324; and EP 0 307 434. For example, this binding site is L234, L235, D270, N297, E318, K320, K322, P331 and P329 (numbering according to the EU index of Kabat; unless otherwise indicated herein, the numbering of amino acid residues in the Fc region or constant region is in accordance with the EU numbering system (also referred to as the EU index), as described in Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242). Antibodies of subclass IgGl, IgG2 and IgG3 generally show complement activation, Clq binding and C3 activation, while IgG4 does not activate the complement system, does not bind Clq and does not activate C3. The "Fc region of an antibody" is a term well known to the skilled person and defined on the basis of papain cleavage of an antibody. In one embodiment, the Fc region is a human Fc region. In one embodiment, the Fc region is a Fc region of the human IgG4 subclass, which comprises the mutations S228P and / or L235E (numbering according to the EU index of Kabat). In one embodiment, the Fc region is a Fc region of the human IgGl subclass, which comprises the mutations L234A and L235A (numbering according to the EU index of Kabat).

[0159] The terms "full-length antibody," "intact antibody," and "whole antibody" are used herein interchangeably to refer to an antibody with a structure substantially similar to a native antibody structure, i.e., comprising two light chains and two heavy chains.

[0160] A "human antibody" is one having an amino acid sequence corresponding to an antibody produced by a human or a human cell, or derived from a non-human source using human antibody repertoire or other human antibody encoding sequences. This definition specifically excludes a humanized antibody comprising non-human antigen binding residues.

[0161] The term "in vitro" denotes such an artificial environment, or a process or reaction carried out within such an artificial environment.

[0162] The term "in vivo" denotes the natural environment of a compound (e.g., an animal or a cell), or a process or reaction carried out within its natural environment.

[0163] The term "immunoassay" denotes any technique that utilizes specific binding molecules (e.g., antibodies) to capture and / or detect a particular target for qualitative or quantitative analysis. Typically, immunoassays feature the following steps: 1) immobilization or capture of the analyte, and 2) detection and measurement of the analyte. The analyte can be captured (i.e., bound) to any solid surface, such as a membrane, plastic plate, or other solid surface.

[0164] The term "linker" denotes a chemical linker or linker of a single chain peptide that covalently connects different entities of a blood brain barrier shuttling module and / or fusion polypeptide and / or conjugate as reported herein. For example, a linker connects a brain effector entity to a monovalent binding entity. For example, if the monovalent binding entity comprises a CH2-CH3 Ig entity and a scFab against a blood brain barrier receptor, the linker is conjugated to the C-terminus of the CH3-CH2 Ig entity of the scFab. The linker that conjugates the brain effector entity to the monovalent binding entity (first linker), and the linker that connects the scFab to the C-terminus of the CH2-CH3 Ig domain (second linker), can be the same or different.

[0165] Linkers of single chain peptides can be used, which consist of 1 to 20 amino acid residues linked by peptide bonds. In certain embodiments, the amino acids are selected from the twenty naturally occurring amino acids. In certain other embodiments, one or more of the amino acids are selected from glycine, alanine, proline, asparagine, glutamine and lysine. In other embodiments, the linker is a chemical linker. In certain embodiments, the linker is a linker of a single chain peptide having an amino acid sequence of at least 25 amino acid residues in length, in a preferred embodiment, 32 to 50 amino acid residues in length. In one embodiment, the linker of the peptide is a (GxS)n linker, where G = glycine, S = serine, (x = 3, n = 8, 9 or 10) or (x = 4 and n = 6, 7 or 8), in one embodiment, x = 4, n = 6 or 7, in a preferred embodiment, x = 4, n = 7. In one embodiment, the linker is (G4S)4 (SEQ ID NO: 02). In one embodiment, the linker is (G4S)6G2 (SEQ ID NO: 03).

[0166] Conjugation can be performed using a variety of chemical linkers. For example, the monovalent binding entity or fusion polypeptide and the brain effector entity can be conjugated using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), 4-(N-maleimidomethyl) cyclohexane-l-carboxylate succinimidyl ester (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis-(p-azidophenyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as l,5-difluoro-2,4-dinitrobenzene). The linker can be a "cleavable linker" that facilitates release of the effector entity when delivered to the brain. For example, acid-labile linkers, linkers sensitive to peptidases, photo-labile linkers, dimethyl linkers, or disulfide-containing linkers can be used (Chari et al., Cancer Res. 52 (1992) 127-131; US 5,208,020).

[0167] Covalent conjugation can be direct or via a linker. In certain embodiments, direct conjugation is by construction of a polypeptide fusion (i.e., by genetic fusion of two genes encoding the monovalent binding entity for the BBBR and the effector entity, and expression as a single polypeptide (chain)). In certain embodiments, direct conjugation is by formation of a covalent bond between a reactive group on one of the two parts of the monovalent binding entity for the BBBR and a corresponding group or acceptor on the brain effector entity. In certain embodiments, direct conjugation is by modification (i.e., genetic modification) of one of the two molecules to be conjugated to include a reactive group (mercapto or carboxyl as non-limiting examples) that forms a covalent linkage with the other molecule to be conjugated under appropriate conditions. As a non-limiting example, a molecule (i.e., amino acid) having a desired reactive group (i.e., a cysteine residue) can be introduced into the monovalent binding entity for the BBBR antibody, for example, to form a disulfide bond with a neurotherapeutic antibody. Methods of covalently conjugating nucleic acids to proteins are also known in the art (i.e., photocrosslinking, see, e.g., Zatsepin et al., Russ. Chem. Rev. 74 (2005) 77-95). Conjugation can also be performed with a variety of linkers. For example, the monovalent binding entity and the effector entity can be conjugated using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), 4-(N-maleimidomethyl) cyclohexane-l-carboxylate succinimidyl ester (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), azino compounds (such as bis-(p-azidophenyl) hexanediamine), bis-diazonium

[0168] 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 except for possible naturally occurring mutations that can be 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 methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, including those described herein and in other exemplary methods for making monoclonal antibodies.

[0169] The term "monovalent binding entity" denotes a molecule capable of specifically binding to a BBBR in a monovalent binding mode. The blood brain shuttle modules and / or conjugates reported herein are characterized by the presence of a single unit of a monovalent binding entity, i.e. the blood brain shuttle modules and / or conjugates of the present application comprise exactly one unit of a monovalent binding entity. The monovalent binding entity includes, but is not limited to, polypeptides, full length antibodies, antibody fragments containing Fab, Fab', Fv fragments, single chain antibody molecules (e.g. single chain Fab, scFv). The monovalent binding entity can for example be a scaffold protein engineered using state-of-the-art techniques such as phage display or immunization. The monovalent binding entity can also be a polypeptide. In certain embodiments, the monovalent binding entity comprises a CH2-CH3 Ig domain and a single chain Fab (scFab) directed against a blood brain barrier receptor. The scFab is coupled to the C-terminal end of the CH2-CH3 Ig domain via a linker. In certain embodiments, the scFab is directed against the transferrin receptor.

[0170] The term "monovalent binding mode" denotes a specific binding to a BBBR, wherein the interaction between the monovalent binding entity and the BBBR occurs via one single antigenic epitope. The monovalent binding mode prevents any dimerization / polymerization of the BBBR due to the single antigenic epitope interaction point. The monovalent binding mode prevents the intracellular sorting of the BBBR from being altered.

[0171] A "naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The naked antibody can be present in a pharmaceutical composition.

[0172] A "native antibody" refers to naturally occurring immunoglobulin molecules having different structures. For example, a native IgG antibody is a heterotetrameric glycoprotein of about 150,000 Daltons, composed of two identical light chains and two identical heavy chains that are linked by disulfide bonds. From N- to C-terminus, each heavy chain has a variable domain (VH), also called a variable heavy domain or a heavy chain variable region, followed by three heavy chain constant domains (CHI, CH2, and CH3). Similarly, from N- to C-terminus, each light chain has a variable domain (VL), also called a variable light domain or a light chain variable region, followed by a light chain constant (CL) domain.

[0173] The term "pharmaceutical composition" refers to a preparation which is in a form suitable for administration into a subject and which is in further admixture, if desired, with suitable additional carriers, excipients, stabilizers, or preservatives.

[0174] A "pharmaceutically acceptable carrier" refers to a component of a pharmaceutical composition other than the active ingredient which is not toxic to the subject to which the composition will be administered. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0175] The term "sample" includes, but is not limited to, any material content from a biological or originally biological. These biologicals include mice, monkeys, rats, rabbits, and other animals. In one embodiment, the sample is taken from a monkey, particularly a cynomolgus monkey, or a rabbit, or a mouse, or a rat.

[0176] The term "signal" as used herein encompasses any detectable physical change that can be used to indicate that a reaction has occurred, such as the binding of an antibody to its antigen. Signals in the form of fluorescent or colorimetric products / reagents are particular forms of signals and can be used in the methods as described in the present application. In some embodiments of the present application, the signal is evaluated quantitatively.

[0177] The term "solid phase" denotes a non-fluid substance and includes particles (including microparticles and beads) made of materials such as polymers, metals (paramagnetic, ferromagnetic particles), glass and ceramics; gel materials, e.g. silica, alumina and polymeric gels; capillaries, which can be made of polymers, metals, glass and / or ceramics; zeolites and other porous materials; electrodes; microtiter plate wells; solid phase test strips and cuvettes, test tubes or other spectroscopic sample containers. The solid phase component is distinguished from inert solid surfaces in that the "solid phase" comprises at least one moiety on its surface which is intended to interact with a substance in the sample. The solid phase can be a fixed component such as a test tube, test strip, cuvette or microtiter plate well, or a non-fixed component such as a bead or microparticle. A variety of microparticles can be used which allow non-covalent or covalent attachment of proteins and other substances. Such particles include polymeric particles such as polystyrene and poly(methyl methacrylate); gold particles such as gold nanoparticles and gold colloids; and ceramic particles such as silica, glass and metal oxide particles. See, e.g., Martin, C. R. et al., Analytical Chemistry - News & Features, 70 (1998) 322A-327A, or Butler, J. E., Methods 22 (2000) 4-23.

[0178] The terms "therapeutic (monoclonal) antibody" and "drug" are used interchangeably herein. These terms are used in the broadest sense and encompass various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, and antibody fragments so long as they exhibit the desired antigen-binding activity.

[0179] The "transferrin receptor" ("TfR") is a transmembrane glycoprotein (molecular weight of about 180,000 Da) consisting of two disulfide-bonded subunits (each with an apparent molecular weight of about 90,000 Da) involved in iron uptake in vertebrates. In one embodiment, the TfR referred to herein is the human TfR, e.g. comprising the amino acid sequence in Schneider et al. (Nature 311 (1984) 675-678).

[0180] Multispecific antibodies

[0181] In certain embodiments, the therapeutic antibody is a bispecific antibody. In one embodiment, the therapeutic antibody is a bispecific, trivalent antibody. In a preferred embodiment, the therapeutic antibody is a monoclonal, bispecific, trivalent antibody.

[0182] In some embodiments, the therapeutic antibody is a multispecific antibody, such as a bispecific antibody. A multispecific antibody is a monoclonal antibody that has binding specificity to at least two different antigens. In some embodiments, one of the binding specificities is against a first antigen, while the others are against different second antigens. Bispecific antibodies can be formulated as full-length antibodies or antibody fragments. In one embodiment, the antibody is a bispecific antibody that specifically binds to both the first and second antigens. In one embodiment, the bispecific antibody has: i) a first binding specificity that specifically binds to the first antigen, and ii) a second binding specificity that specifically binds to the second antigen. In one embodiment, the antibody is a bispecific, trivalent antibody. In a preferred embodiment, the antibody is a monoclonal, bispecific, trivalent antibody.

[0183] In one embodiment, a specific binding site binds to BBBR.

[0184] Techniques for preparing multispecific antibodies include, but are not limited to, recombinant co-expression of heavy-light chain pairs of two immunoglobulins with different specificities (see Milstein and Cuello, Nature 305(1983)537-540, WO 93 / 08829 and Traunecker, A. et al., EMBO J.10(1991)3655-3659), and “protrusion-in-pore” engineering (see, for example, US 5,731,168). Multispecific antibodies can also be prepared by: engineered electrostatic redirection effects for preparing antibody Fc-heterodimer molecules (WO 2009 / 089004); crosslinking two or more antibodies or fragments (see, for example, US Patent 4,676,980; and Brennan et al., Science, 229(1985) 81-83); generating bispecific antibodies using leucine zippers (see, for example, Kostelny, SA et al., J. Immunol., 148(1992) 1547-1553); and preparing double-chain antibody fragments using "dual antibody" technology (see, for example, Holliger, P. et al., Proc. Natl. Acad. Sci. USA). 90 (1993) 6444-6448); and the use of single-chain Fv (scFv) dimers (see, for example, Gruber, M. et al., J. Immunol. 152 (1994) 5368-5374); and the preparation of trispecific antibodies according to, for example, the methods described by Tutt, A. et al. (J. Immunol. 147 (1991) 60-69).

[0185] Multispecific antibodies are described in WO 2009 / 080251, WO 2009 / 080252, WO 2009 / 080253, WO 2009 / 080254, WO 2010 / 1 1293, WO 2010 / 1 15589, WO 2010 / 136172, WO 2010 / 145792 or WO 2010 / 145793.

[0186] Different bispecific antibody formats are known.

[0187] Exemplary bispecific antibody formats that can be used using the methods reported herein

[0188] - CrossMab format (= CrossMab): a multispecific IgG antibody comprising a first Fab fragment and a second Fab fragment, wherein in the first Fab fragment

[0189] a) only the CH1 and CL domains are replaced by each other (i.e. the light chain of the first Fab fragment comprises a VL and a CH1 domain, whereas the heavy chain of the first Fab fragment comprises a VH and a CL domain);

[0190] b) only the VH and VL domains are replaced by each other (i.e. the light chain of the first Fab fragment comprises a VH and a CL domain, whereas the heavy chain of the first Fab fragment comprises a VL and a CH1 domain); or

[0191] c) the CH1 and CL domains are replaced by each other and the VH and VL domains are replaced by each other (i.e. the light chain of the first Fab fragment comprises a VH and a CH1 domain, whereas the heavy chain of the first Fab fragment comprises a VL and a CL domain); and

[0192] wherein the second Fab fragment comprises a light chain comprising a VL and a CL domain, and a heavy chain comprising a VH and a CH1 domain;

[0193] The CrossMab can comprise a first heavy chain comprising a CH3 domain and a second heavy chain comprising a CH3 domain, wherein both CH3 domains are engineered in a complementary fashion by respective amino acid substitutions to support heterodimerization of the first heavy chain and the modified second heavy chain, e.g. as disclosed in WO 96 / 27011, WO 98 / 050431, EP 1870459, WO 2007 / 1 10205, WO 2007 / 147901, WO 2009 / 089004, WO 2010 / 129304, WO 201 1 / 90754, WO 201 1 / 143545, WO 2012 / 058768, WO 2013 / 157954 or WO 2013 / 096291 (incorporated herein by reference);

[0194] - single arm single chain format (= single arm single chain antibody): antibody comprising a first binding site that specifically binds to a first antigen, and a second binding site that specifically binds to a second antigen, wherein the chains are as follows:

[0195] - light chain (variable light domain + light chain kappa constant domain)

[0196] - light / heavy chain combination (variable light domain with knob mutation + light chain constant domain + linker of peptide + variable heavy domain + CH1 + hinge + CH2 + CH3)

[0197] - heavy chain (variable heavy domain with hole mutation + CH1 + hinge + CH2 + CH3);

[0198] - double arm single chain format (= double arm single chain antibody): antibody comprising a first binding site that specifically binds to a first antigen, and a second binding site that specifically binds to a second antigen, wherein the chains are as follows:

[0199] - light / heavy chain 1 combination (variable light domain with hole mutation + light chain constant domain + linker of peptide + variable heavy domain + CH1 + hinge + CH2 + CH3)

[0200] - light / heavy chain 2 combination (variable light domain with knob mutation + light chain constant domain + linker of peptide + variable heavy domain + CH1 + hinge + CH2 + CH3);

[0201] - common light chain double specificity format (= common light chain bispecific antibody): antibody comprising a first binding site that specifically binds to a first antigen, and a second binding site that specifically binds to a second antigen, wherein the chains are as follows:

[0202] - light chain (variable light domain + light chain constant domain)

[0203] - heavy chain 1 (variable heavy domain with hole mutation + CH1 + hinge + CH2 + CH3)

[0204] - heavy chain 2 (variable heavy domain with knob mutation + CH1 + hinge + CH2 + CH3);

[0205] - bispecific Fab format: the Fab comprises two (non-overlapping) paratopes in the complementary pairing of the VH and VL domains, wherein the first paratope comprises (is composed of) amino acid residues from CDR1 and CDR3 of the VL domain and CDR2 of the VH domain, and the second paratope comprises (is composed of) residues from CDR1 and CDR3 of the VH domain and CDR2 of the VL domain; the term "non-overlapping" herein means that the amino acid residues comprised within the first paratope of the bispecific Fab, and the amino acid residues comprised within the second paratope of the bispecific Fab are not comprised in the first paratope;

[0206] - TCB format: the bispecific antibody comprises

[0207] - a first and a second Fab fragment, wherein each binding site of the first and the second Fab fragment specifically binds to a second antigen,

[0208] - a third Fab fragment, wherein the binding site of the third Fab fragment specifically binds to a first antigen, and wherein the third Fab fragment comprises a domain crossover, such that the variable light chain domain (VL) and the variable heavy chain domain (VH) are replaced by each other, and

[0209] - the Fc region comprises a first Fc region polypeptide and a second Fc region polypeptide,

[0210] wherein the first and the second Fab fragment each comprise a heavy chain fragment and a full length light chain,

[0211] wherein the C-terminus of the heavy chain fragment of the first Fab fragment is fused to the N-terminus of the first Fc region polypeptide,

[0212] wherein the C-terminus of the heavy chain fragment of the second Fab fragment is fused to the N-terminus of the variable light chain domain of the third Fab fragment, and the C-terminus of the heavy chain constant domain 1 of the third Fab fragment is fused to the N-terminus of the second Fc region polypeptide.

[0213] - brain shuttle format (BS): the bispecific antibody comprises

[0214] a) one (full length) antibody comprising two pairs of (full length) antibody light chains and (full length) antibody heavy chains each, wherein each pair of (full length) heavy chain and (full length) light chain forms a binding site that specifically binds to a first antigen, and

[0215] b) one additional Fab fragment, wherein the additional Fab fragment is fused to the C-terminus of either one of the heavy chains of the (full length) antibody, wherein the binding site of the additional Fab fragment specifically binds to a second antigen,

[0216] wherein the additional Fab fragment that specifically binds to the second antigen comprises a domain crossover such that the constant light chain domain (CL) and the constant heavy chain domain 1 (CH1 ) are replaced by each other, and

[0217] wherein the first antigen is a brain target and the second antigen is human transferrin receptor.

[0218] In one embodiment, the bispecific antibody is a CrossMab.

[0219] In one embodiment, the bispecific antibody is a single-arm single-chain antibody.

[0220] In one embodiment, the bispecific antibody is a two-arm single-chain antibody.

[0221] In one embodiment, the bispecific antibody is a common light chain bispecific antibody.

[0222] In one embodiment, the bispecific antibody is a bispecific Fab.

[0223] In one embodiment, the bispecific antibody is a TCB.

[0224] In one embodiment, the bispecific antibody is a BS.

[0225] Multivalent, multispecific antibodies specifically bind to different targets, each target most likely with different affinity and complex stability. Only fully active multivalent, multispecific antibodies can bind all targets and show intact biological activity in the respective assays.

[0226] A. Exemplary bispecific antibody: Anti-human A-beta / human transferrin receptor antibody

[0227] In certain embodiments, the therapeutic antibody to be determined in a method as described in the present application is an antibody that binds to human A-beta and human transferrin receptor. The antibody is a bispecific antibody composed of a full-length core antibody and a fusion Fab fragment in which certain domains are cross-exchanged. The resulting bispecific antibody is thus asymmetric. The bispecific antibody is generated using a technique called knob-into-hole heterodimerization, which uses a first heavy chain with a so-called knob mutation (Hcknob) and a second heavy chain with a so-called hole mutation (Hchole).

[0228] Exemplary antibody 0012 is composed of four polypeptides with the amino acid sequences of SEQ ID NOs: 04 to 07.

[0229] Exemplary antibody 0015 is composed of four polypeptides with the amino acid sequences of SEQ ID NOs: 08 to 11.

[0230] Exemplary antibody 0020 consists of three polypeptides having the amino acid sequences of SEQ ID NOs: 12 to 14.

[0231] Exemplary antibody 0024 consists of four polypeptides having the amino acid sequences of SEQ ID NOs: 15 to 18.

[0232] In one aspect, the therapeutic antibody is a bispecific antibody comprising

[0233] a) one full length antibody comprising two pairs of a full length antibody light chain and a full length antibody heavy chain, wherein the binding site formed by each pair of full length heavy chain and full length light chain specifically binds to a first antigen, and

[0234] b) one additional Fab fragment, wherein the additional Fab fragment is fused to the C-terminus of one of the heavy chains of the full length antibody, wherein the binding site of the additional Fab fragment specifically binds to a second antigen,

[0235] wherein each full length antibody light chain comprises an amino acid residue arginine (instead of the wild type glutamic acid residue; E123R mutation) at position 123 in the constant light chain domain (CL) and an amino acid residue lysine (instead of the wild type glutamine residue; Q124K mutation) at position 124 (according to Kabat numbering),

[0236] wherein each full length antibody heavy chain comprises a glutamic acid residue (instead of the wild type lysine residue; K147E mutation) at position 147 in the first constant heavy chain domain (CH1) and a glutamic acid residue (instead of the wild type lysine amino acid residue; K213E mutation) at position 213 (according to Kabat EU index numbering),

[0237] wherein the additional Fab fragment specifically binding to the second antigen comprises a domain crossover such that the constant light chain domain (CL) and the constant heavy chain domain 1 (CH1) are replaced by each other, and

[0238] wherein the first antigen is human A-beta protein and the second antigen is human transferrin receptor.

[0239] In another embodiment, the therapeutic antibody is a bispecific antibody comprising

[0240] a) one full length antibody comprising two pairs of a full length antibody light chain and a full length antibody heavy chain, wherein the binding site formed by each pair of full length heavy chain and full length light chain specifically binds to a first antigen, and

[0241] b) one additional Fab fragment, wherein the additional Fab fragment is fused to the C-terminus of one of the heavy chains of the full length antibody, wherein the binding site of the additional Fab fragment specifically binds to a second antigen,

[0242] wherein each full-length antibody light chain comprises an amino acid residue arginine (instead of the wild-type glutamic acid residue; E123R mutation) at position 123 in the constant light chain domain (CL), and an amino acid residue lysine (instead of the wild-type glutamine residue; Q124K mutation) at position 124 (according to Kabat numbering) in the constant light chain domain (CL),

[0243] wherein each full-length antibody heavy chain comprises a glutamic acid residue (instead of the wild-type lysine residue; K147E mutation) at position 147 in the first constant heavy chain domain (CH1), and a glutamic acid residue (instead of the wild-type lysine amino acid residue; K213E mutation) at position 213 (according to Kabat EU index numbering) in the constant heavy chain domain 1 (CH1),

[0244] wherein the further Fab fragment that specifically binds to the second antigen comprises a domain crossover, such that the constant light chain domain (CL) and the constant heavy chain domain 1 (CH1) are replaced by each other,

[0245] wherein the first antigen is human A-beta protein, and the second antigen is human transferrin receptor,

[0246] wherein the human A-beta binding site comprises a heavy chain variable domain (VH) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO: 19, and a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO: 20, and

[0247] wherein the human transferrin receptor binding site comprises a heavy chain variable domain (VH) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO: 21, and a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO: 22.

[0248] In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, includes substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but retains the ability to bind to its antigen. In certain embodiments, no more than 1, 2, 3, 4, or 5 amino acids in SEQ ID NO: 19 or 21 are substituted, inserted and / or deleted. In certain embodiments, substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs).

[0249] In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, includes substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but retains the ability to bind to its antigen. In certain embodiments, no more than 1, 2, 3, 4, or 5 amino acids in SEQ ID NO: 20 or 22 are substituted, inserted and / or deleted. In certain embodiments, substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs).

[0250] In one embodiment, the human A-beta binding site comprises a VH sequence as in SEQ ID NO: 19 (including post-translational modifications of that sequence) and a VL sequence as in SEQ ID NO: 20.

[0251] In one embodiment, the human transferrin receptor binding site comprises a VH sequence as in SEQ ID NO: 21 (including post-translational modifications of that sequence) and a VL sequence as in SEQ ID NO: 22.

[0252] In one embodiment, the bispecific antibody comprises

[0253] i) a light chain having at least 70%, at least 80%, at least 90%, or 95% or more sequence identity to SEQ ID NO: 23,

[0254] ii) a heavy chain having at least 70%, at least 80%, at least 90%, or 95% or more sequence identity to SEQ ID NO: 24,

[0255] iii) a light chain having at least 70%, at least 80%, at least 90%, or 95% or more sequence identity to SEQ ID NO: 25, and

[0256] iv) a heavy chain Fab fragment having at least 70%, at least 80%, at least 90%, or 95% or more sequence identity to SEQ ID NO: 26,

[0257] wherein,

[0258] SEQ ID NO: 23 has the amino acid sequence DIVLTQSPATLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGVPARFSGSGSGTDFTLTISSLEPEDFATYYCLQIYNMPITFGQGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC,

[0259] SEQ ID NO: 24 has the amino acid sequence QVELVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAINASGTRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGKGNTHKPYGYVRYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG,

[0260] SEQ ID NO:25 has the amino acid sequence AIQLTQSPSSLSASVGDRVTITCRASQSISSYLAWYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNYASSNVDNTFGGGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC, and SEQ ID NO:26 has the amino acid sequence QSMQESGPGLVKPSQTLSLTCTVSGFSLSSYAMSWIRQHPGKGLEWIGYIWSGGSTDYASWAKSRVTISKTSTTVSLKLSSVTAADTAVYYCARRYGTSYPDYGDASGFDPWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0261] In another embodiment, the therapeutic antibody is a bispecific antibody comprising

[0262] a) one full length antibody comprising two pairs of a full length antibody light chain and a full length antibody heavy chain, wherein the binding site formed by each pair of full length heavy chain and full length light chain specifically binds to a first antigen, and

[0263] b) one additional Fab fragment, wherein the additional Fab fragment is fused to the C-terminus of one heavy chain of the full length antibody, wherein the binding site of the additional Fab fragment specifically binds to a second antigen,

[0264] wherein each full length antibody light chain comprises an amino acid residue arginine (instead of the wild type glutamic acid residue; E123R mutation) at position 123 in the constant light chain domain (CL), and an amino acid residue lysine (instead of the wild type glutamine residue; Q124K mutation) at position 124 (according to Kabat numbering),

[0265] wherein each full length antibody heavy chain comprises a glutamic acid residue (instead of the wild type lysine residue; K147E mutation) at position 147 in the first constant heavy chain domain (CH1), and a glutamic acid residue (instead of the wild type lysine amino acid residue; K213E mutation) at position 213 (according to Kabat EU index numbering),

[0266] The additional Fab fragment that specifically binds to the second antigen contains domain crossovers, such that the constant light chain domain (CL) and the constant heavy chain domain 1 (CH1) are substituted for each other.

[0267] The first antigen is human A-β protein, and the second antigen is human transferrin receptor.

[0268] The human A-β binding site includes a heavy chain variable domain (VH) with the amino acid sequence of SEQ ID NO:19, and a light chain variable domain (VL) with the amino acid sequence of SEQ ID NO:20.

[0269] The human transferrin receptor binding site includes a heavy chain variable domain (VH) with the amino acid sequence of SEQ ID NO:21 and a light chain variable domain (VL) with the amino acid sequence of SEQ ID NO:22.

[0270] In another embodiment, the therapeutic antibody is a bispecific antibody containing...

[0271] a) A full-length antibody comprising two pairs of full-length antibody light chains and two pairs of full-length antibody heavy chains, wherein each pair of full-length heavy chains and full-length light chains forms a binding site that specifically binds to a first antigen, wherein the full-length antibody comprises an Fc region formed by Fc region polypeptides, each Fc region polypeptide comprising CH1, CH2, and CH3 domains of two full-length heavy chains, and

[0272] b) An additional Fab fragment, wherein the additional Fab fragment is fused to the C-terminus of a heavy chain of the full-length antibody, wherein the binding site of the additional Fab fragment specifically binds to a second antigen.

[0273] Each full-length antibody light chain contains the amino acid residue arginine (not the wild-type glutamate residue; E123R mutation) at position 123 in the constant light chain domain (CL), and the amino acid residue lysine (not the wild-type glutamine residue; Q124K mutation) at position 124 (according to Kabat numbering).

[0274] Each full-length antibody heavy chain contains a glutamate residue (not a wild-type lysine residue; K147E mutation) at position 147 in the first constant heavy chain domain (CH1), and a glutamate residue (not a wild-type lysine amino acid residue; K213E mutation) at position 213 (according to the Kabat EU index number).

[0275] The additional Fab fragment that specifically binds to the second antigen contains cross-domains, making...

[0276] the constant light chain domain (CL) and the constant heavy chain domain 1 (CH1) are replaced by each other, wherein the first antigen is the human A-beta protein and the second antigen is the human transferrin receptor, wherein the human A-beta binding site comprises a heavy chain variable domain (VH) having the amino acid sequence of SEQ ID NO: 19 and a light chain variable domain (VL) having the amino acid sequence of SEQ ID NO: 20,

[0277] wherein the human transferrin receptor binding site comprises a heavy chain variable domain (VH) having the amino acid sequence of SEQ ID NO: 21 and a light chain variable domain (VL) having the amino acid sequence of SEQ ID NO: 22, and

[0278] wherein the Fc region polypeptide is

[0279] a) human subclass IgGl,

[0280] b) human subclass IgG4,

[0281] c) human subclass IgGl with the mutations L234A, L235A and P329G

[0282] d) human subclass IgG4 with the mutations S228P, L235E and P329G

[0283] e) human subclass IgGl with the mutations L234A, L235A and P329G in both Fc region polypeptides and with the mutations T366W and S354C in one Fc region polypeptide and T366S, L368A, Y407V and Y349C in the respective other Fc region polypeptide,

[0284] f) human subclass IgG4 with the mutations S228P and P329G in both Fc region polypeptides and with the mutations T366W and S354C in one Fc region polypeptide and T366S, L368A, Y407V and Y349C in the respective other Fc region polypeptide,

[0285] g) human subclass IgGl with the mutations L234A, L235A, P329G, I253A, H310A and H435A in both Fc region polypeptides and with the mutations T366W and S354C in one Fc region polypeptide and T366S, L368A, Y407V and Y349C in the respective other Fc region polypeptide, or

[0286] h) human subclass IgGl with mutations L234A, L235A, P329G, M252Y, S254T, and T256E in both Fc region polypeptides, and mutations T366W and S354C in one Fc region polypeptide and mutations T366S, L368A, Y407V, and Y349C in the respective other Fc region polypeptide.

[0287] B. Exemplary Anti-transferrin Receptor Antibodies

[0288] The anti-transferrin receptor binding site of the therapeutic antibody to be determined in the method as described in the present application has a dissociation rate in a range that binds to human transferrin receptor to ensure proper BBB shuttling. One end of the range is determined by the dissociation rate of the murine anti-transferrin receptor antibody 128.1 (variable domain amino acid sequences given in SEQ ID NOs: 27 and 28) as determined by surface plasmon resonance to cynomolgus monkey transferrin receptor, and the other end is determined by 5% of the dissociation rate (i.e. 20-fold slower dissociation). The dissociation rate of the human transferrin receptor should be between 0.1 1 / s and 0.005 1 / s, inclusive.

[0289] One aspect reported herein is an anti-transferrin receptor antibody that specifically binds to human transferrin receptor and cynomolgus monkey transferrin receptor, comprising

[0290] i) a humanized heavy chain variable domain derived from the heavy chain variable domain of SEQ ID NO: 29, and

[0291] ii) a humanized light chain variable domain derived from the light chain variable domain of SEQ ID NO: 30,

[0292] wherein the dissociation rate of the antibody to human transferrin receptor is equal to or slower than (i.e. at most) the dissociation rate of the anti-transferrin receptor antibody 128.1 to cynomolgus monkey transferrin receptor,

[0293] wherein the dissociation rate is determined by surface plasmon resonance, and

[0294] wherein the anti-transferrin receptor antibody 128.1 has a heavy chain variable domain of SEQ ID NO: 27 and a light chain variable domain of SEQ ID NO: 28.

[0295] In one embodiment, the dissociation rate of the human transferrin receptor is between 0.1 1 / s and 0.005 1 / s, inclusive.

[0296] In one embodiment, the antibody has a proline amino acid residue (P) at position 80 of the light chain variable domain (numbering in accordance with Kabat).

[0297] In one embodiment, the antibody has an asparagine amino acid residue (N) at position 91 of the light chain variable domain (numbering in accordance with Kabat).

[0298] In one embodiment, the antibody has an alanine amino acid residue (A) at position 93 of the light chain variable domain (numbering in accordance with Kabat).

[0299] In one embodiment, the antibody has a serine amino acid residue (S) at position 100g of the heavy chain variable domain (numbering in accordance with Kabat).

[0300] In one embodiment, the antibody has a glutamine amino acid residue (Q) at position 100g of the heavy chain variable domain (numbering in accordance with Kabat).

[0301] In one embodiment, the antibody has a serine amino acid residue (S) at position 65 of the heavy chain variable domain (numbering in accordance with Kabat).

[0302] In one embodiment, the antibody has a glutamine amino acid residue (Q) at position 105 of the heavy chain variable domain (numbering in accordance with Kabat).

[0303] In one embodiment, the antibody has a proline amino acid residue (P) at position 80 of the light chain variable domain, an asparagine amino acid residue (N) at position 91 of the light chain variable domain, an alanine amino acid residue (A) at position 93 of the light chain variable domain, a serine amino acid residue (S) at position 100g of the heavy chain variable domain, a serine amino acid residue (S) at position 65 of the heavy chain variable domain, and a glutamine amino acid residue (Q) at position 105 of the heavy chain variable domain (numbering in accordance with Kabat).

[0304] In one embodiment, the antibody has a proline amino acid residue (P) at position 80 of the light chain variable domain, an asparagine amino acid residue (N) at position 91 of the light chain variable domain, an alanine amino acid residue (A) at position 93 of the light chain variable domain, a glutamine amino acid residue (Q) at position 100g of the heavy chain variable domain, a serine amino acid residue (S) at position 65 of the heavy chain variable domain, and a glutamine amino acid residue (Q) at position 105 of the heavy chain variable domain (numbering in accordance with Kabat).

[0305] Such anti-transferrin receptor bispecific antibodies can be used as blood brain barrier shuttle modules to deliver a brain effector entity across the blood brain barrier into the brain. A blood brain barrier shuttle module is a monovalent binding entity that specifically binds to the human transferrin receptor. In use as a blood brain barrier shuttle module, the anti-transferrin receptor bispecific antibody is useful, for example, in the diagnosis or treatment of neurological disorders, such as Alzheimer's disease, Parkinson's disease, and Alzheimer's disease and Parkinson's disease co-morbidity.

[0306] In one embodiment, the anti-transferrin receptor binding site of the therapeutic antibody comprises a heavy chain variable domain of SEQ ID NO: 31 and a light chain variable domain of SEQ ID NO: 32, which reflects the binding properties of murine antibody 128.1 relative to cynomolgus monkey transferrin receptor with respect to off-rate, relative to human transferrin receptor.

[0307] In one embodiment, the anti-transferrin receptor binding site of the therapeutic antibody specifically binds to human transferrin receptor (huTfR) and cynomolgus monkey transferrin receptor (cyTfR), and comprises i) a humanized heavy chain variable domain derived from the heavy chain variable domain of SEQ ID NO: 29, and ii) a humanized light chain variable domain derived from the light chain variable domain of SEQ ID NO: 30, wherein the light chain variable domain has a proline amino acid residue (P) at position 80, an asparagine amino acid residue (N) at position 91, and an alanine amino acid residue (A) at position 93 (according to Kabat numbering).

[0308] In one embodiment, the anti-transferrin receptor binding site of the therapeutic antibody further has a serine amino acid residue (S) at position 100g of the heavy chain variable domain (according to Kabat numbering).

[0309] In one embodiment, the anti-transferrin receptor binding site of the therapeutic antibody further has a serine amino acid residue (S) at position 65 of the heavy chain variable domain (according to Kabat numbering).

[0310] In one embodiment, the anti-transferrin receptor binding site of the therapeutic antibody further has a glutamine amino acid residue (Q) at position 105 of the heavy chain variable domain (according to Kabat numbering).

[0311] In one embodiment, the anti-transferrin receptor binding site of the therapeutic antibody specifically binds to both human transferrin receptor (huTfR) and cynomolgus transferrin receptor (cyTfR) and comprises i) a humanized heavy chain variable domain derived from the heavy chain variable domain of SEQ ID NO: 29, and ii) a humanized light chain variable domain derived from the light chain variable domain of SEQ ID NO: 30, wherein the off-rate of the therapeutic antibody for human transferrin receptor is equal to or less than (i.e., at most) the off-rate of the anti-transferrin receptor antibody 128.1 for cynomolgus transferrin receptor in units of 1 / s, wherein the off-rate is determined by surface plasmon resonance, and wherein the anti-transferrin receptor antibody 128.1 has a heavy chain variable domain of SEQ ID NO: 27 and a light chain variable domain of SEQ ID NO: 28.

[0312] In one embodiment, the anti-transferrin receptor binding site of the therapeutic antibody has an off-rate for human transferrin receptor in units of 1 / s that is i) equal to or less than (i.e., at most) the off-rate of the anti-transferrin receptor antibody 128.1 for cynomolgus transferrin receptor in units of 1 / s, and ii) equal to or greater than (i.e., at least) 5% of the off-rate of the anti-transferrin receptor antibody 128.1 for cynomolgus transferrin receptor in units of 1 / s.

[0313] In one embodiment, the anti-transferrin receptor binding site of the therapeutic antibody comprises (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 33; (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 34; (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 35, 36, or 37, in a preferred embodiment, SEQ ID NO: 36; (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 38; (e) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 39; and (f) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 40.

[0314] In any of the above embodiments, the anti-transferrin receptor binding site is humanized. In one embodiment, the anti-transferrin receptor binding site comprises the HVRs as described in any of the above embodiments, and further comprises a recipient human framework, e.g., a human immunoglobulin framework or a human consensus framework.

[0315] In one embodiment, the antibody is a bispecific antibody comprising at least one pair of a heavy chain variable domain of SEQ ID NO: 31 and a light chain variable domain of SEQ ID NO: 32, which form a binding site for transferrin receptor, and at least one pair of a heavy chain variable domain of SEQ ID NO: 41 and a light chain variable domain of SEQ ID NO: 42 for human CD20. In one embodiment, the heavy chain variable region comprises a substitution of the amino acid residue at Kabat position 11 with any amino acid other than leucine. In one embodiment, the substitution comprises a substitution of the amino acid residue at Kabat position 11 with a non-polar amino acid. In a preferred embodiment, the substitution comprises a substitution of the amino acid residue at Kabat position 11 in the heavy chain variable domain of SEQ ID NO: 41 with an amino acid residue selected from the group consisting of valine, leucine, isoleucine, serine, and phenylalanine.

[0316] In one embodiment, the antibody is a bispecific antibody comprising at least one pair of a heavy chain variable domain of SEQ ID NO: 31 and a light chain variable domain of SEQ ID NO: 32, which form a binding site for transferrin receptor, and at least one pair of a heavy chain variable domain of SEQ ID NO: 43 and a light chain variable domain of SEQ ID NO: 44 for human alpha-synuclein.

[0317] In one embodiment, the antibody is a bispecific antibody comprising at least one pair of a heavy chain variable domain of SEQ ID NO: 31 and a light chain variable domain of SEQ ID NO: 32, which form a binding site for transferrin receptor, and at least one pair of a heavy chain variable domain of SEQ ID NO: 43 and a light chain variable domain of SEQ ID NO: 44 for human alpha-synuclein.

[0318] In one embodiment, the antibody is a bispecific antibody comprising at least one pair of a heavy chain variable domain of SEQ ID NO: 31 and a light chain variable domain of SEQ ID NO: 32, which form a binding site for transferrin receptor, and at least one pair of a heavy chain variable domain of SEQ ID NO: 43 and a light chain variable domain of SEQ ID NO: 44 for human alpha-synuclein.

[0319] In one embodiment, the antibody is a bispecific antibody comprising at least one pair of a heavy chain variable domain of SEQ ID NO: 31 and a light chain variable domain of SEQ ID NO: 32, which form a binding site for transferrin receptor, and at least one pair of a humanized heavy chain variable domain derived from SEQ ID NO: 49 and a humanized light chain variable domain derived from SEQ ID NO: 50 for human alpha-synuclein.

[0320] In one embodiment, the antibody is a bispecific antibody comprising at least one pair of a heavy chain variable domain of SEQ ID NO: 31 and a light chain variable domain of SEQ ID NO: 32, which form a binding site for transferrin receptor, and at least one pair of a humanized heavy chain variable domain derived from SEQ ID NO: 51 and a humanized light chain variable domain derived from SEQ ID NO: 52 for human alpha-synuclein.

[0321] In one embodiment, the antibody is a bispecific antibody comprising at least one pair of a heavy chain variable domain of SEQ ID NO: 31 and a light chain variable domain of SEQ ID NO: 32, which form a binding site for transferrin receptor, and at least one pair of a humanized heavy chain variable domain derived from SEQ ID NO: 53 and a humanized light chain variable domain derived from SEQ ID NO: 54 for human alpha-synuclein.

[0322] In one embodiment, the antibody is a bispecific antibody comprising at least one pair of a heavy chain variable domain of SEQ ID NO: 31 and a light chain variable domain of SEQ ID NO: 32, which form a binding site for transferrin receptor, and at least one pair of a humanized heavy chain variable domain derived from SEQ ID NO: 49 and a humanized light chain variable domain derived from SEQ ID NO: 50 for human alpha-synuclein.

[0323] In another embodiment, the therapeutic antibody is a multispecific antibody. In one such embodiment, the multispecific antibody comprises a first antigen binding site that binds to TfR and a second antigen binding site that binds to a brain antigen. In one such aspect, the brain antigen is selected from the group consisting of beta-secretase 1 (BACE1), Abeta, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau protein, apolipoprotein E (ApoE), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), glucocerebrosidase, and caspase 6. In another embodiment, the multispecific antibody binds to both TfR and BACE1. In another embodiment, the multispecific antibody binds to both TfR and Abeta. In another embodiment, the multispecific antibody binds to both TfR and alpha-synuclein. In another embodiment, the multispecific antibody binds to both TfR and CD20. In another embodiment, the multispecific antibody binds to both TfR and glucocerebrosidase. In another embodiment, the therapeutic compound is a neurological disorder therapeutic antibody.

[0324] In one embodiment, the effector function is reduced or eliminated by at least one modification of the Fc region. In one embodiment, the reduction or elimination of effector function or complement activation function is by deletion of all or part of the Fc region, or by engineering the antibody to not include an Fc region or a non-Fc region that is capable of exerting effector function or complement activation function. In one embodiment, the at least one modification of the Fc region is selected from the group consisting of: a point mutation in the Fc region that impairs binding of one or more Fc receptors selected from the group consisting of positions 238, 239, 248, 249, 252, 254, 265, 268, 269, 270, 272, 278, 289, 292, 293, 294, 295, 296, 297, 298, 301, 303, 322, 324, 327, 329, 333, 30 335, 338, 340, 373, 376, 382, 388, 389, 414, 416, 419, 434, 435, 437, 438, and 439; and a point mutation in the Fc region that impairs binding to the Clq receptor selected from the group consisting of positions 270, 322, 329, and 321; deletion of part or all of the Fc region, and a point mutation at position 132 of the CHI domain. In one embodiment, the modification is a point mutation in the Fc region that impairs binding to the Clq receptor selected from the group consisting of positions 270, 322, 329, and 321. In another embodiment, the modification is deletion of part or all of the Fc region. In another embodiment, the reduction or elimination of complement triggering function is by deletion of all or part of the Fc region, or by engineering the antibody to not include an Fc region that binds to the complement pathway. In another embodiment, the antibody is selected from the group consisting of a Fab or a single chain antibody. In another embodiment, the non-Fc region of the antibody is modified to reduce or eliminate activation of the complement pathway by the antibody. In one embodiment, the modification is a point mutation in the CHI region to impair binding to C3. In one embodiment, the point mutation is at position 132 (see, e.g., Vidarte et al., J. Biol. Chem. 276 (2001) 38217-38223).

[0325] In one aspect of the above embodiments, the antibody has reduced affinity for TfR relative to a wild-type antibody of the same isotype that does not have reduced affinity for TfR. In one such aspect, the antibody has a K D or IC 50 of about 1 pM to about 100 μM.

[0326] In one embodiment, the antibody reported herein is effector function silent. In one embodiment, the antibody has no effector function. In one embodiment, the antibody is of the human IgGl subclass and has the mutations L234A, L235A, and P329G (numbering according to the Kabat EU index) in both heavy chains.

[0327] In one embodiment, the antibody is

[0328] a) a full-length antibody of human subclass IgGl, or

[0329] b) a full-length antibody of human subclass IgG4, or

[0330] c) a full-length antibody of human subclass IgGl with mutations L234A, L235A and P329G,

[0331] d) a full-length antibody of human subclass IgG4 with mutations S228P, L235E and optionally P329G,

[0332] e) a full-length antibody of human subclass IgGl with mutations L234A, L235A and P329G in both heavy chains and mutations T366W and S354C in one heavy chain and T366S, L368A, Y407V and Y349C in the respective other heavy chain, or

[0333] f) a full-length antibody of human subclass IgG4 with mutations S228P and optionally P329G in both heavy chains and mutations T366W and S354C in one heavy chain and T366S, L368A, Y407V and Y349C in the respective other heavy chain.

[0334] In one embodiment, the bispecific therapeutic antibody comprises

[0335] i) a homodimeric Fc region of human IgGl subclass, optionally with mutations P329G, L234A and L235A, or

[0336] ii) a homodimeric Fc region of human IgG4 subclass, optionally with mutations P329G, S228P and L235E, or

[0337] iii) a heterodimeric Fc region, which

[0338] a) one Fc region polypeptide comprises mutation T366W and the other Fc region polypeptide comprises mutations T366S, L368A and Y407V, or

[0339] b) one Fc region polypeptide comprises mutations T366W and Y349C and the other Fc region polypeptide comprises mutations T366S, L368A, Y407V and S354C, or

[0340] c) one Fc region polypeptide comprises mutations T366W and S354C and the other Fc region polypeptide comprises mutations T366S, L368A, Y407V and Y349C,

[0341] or

[0342] iv) a heterodimeric Fc region of the human IgG4 subclass, wherein both Fc region polypeptides comprise the mutations P329G, L234A and L235A, and

[0343] a) one Fc region polypeptide comprises the mutation T366W and the other Fc region polypeptide comprises the mutations T366S, L368A and Y407V, or

[0344] b) one Fc region polypeptide comprises the mutations T366W and Y349C and the other Fc region polypeptide comprises the mutations T366S, L368A, Y407V and S354C, or

[0345] c) one Fc region polypeptide comprises the mutations T366W and S354C and the other Fc region polypeptide comprises the mutations T366S, L368A, Y407V and Y349C.

[0346] or

[0347] v) a heterodimeric Fc region of the human IgG4 subclass, wherein both Fc region polypeptides comprise the mutations P329G, S228P and L235E, and

[0348] a) one Fc region polypeptide comprises the mutation T366W and the other Fc region polypeptide comprises the mutations T366S, L368A and Y407V, or

[0349] b) one Fc region polypeptide comprises the mutations T366W and Y349C and the other Fc region polypeptide comprises the mutations T366S, L368A, Y407V and S354C, or

[0350] c) one Fc region polypeptide comprises the mutations T366W and S354C and the other Fc region polypeptide comprises the mutations T366S, L368A, Y407V and Y349C.

[0351] Immunoassay

[0352] The principles of different immunoassays have been described in the art. For example, Hage, D.S. (Anal. Chem. 71 (1999) 294R-304R). Lu, B. et al. (Analyst 121 (1996) 29R-32R) report the directed immobilization of antibodies for immunoassays. For example, avidin-biotin mediated immunoassays have been reported in Wilchek, M. and Bayer, E.A., Methods Enzymol. 184 (1990) 467-469.

[0353] Monoclonal antibodies and their constant domains contain a number of reactive amino acid side chains for conjugation to binding partners, such as polypeptides / proteins, polymers (e.g., PEG, cellulose, or polystyrene), or enzymes. For example, chemically reactive groups of amino acids are amino groups (lysine, alpha-amino), thiol groups (cystine, cysteine, and methionine), carboxylic acid groups (aspartic acid, glutamic acid), and sugar alcohol groups. For example, such methods are described in Aslam M. and Dent, A., "Bioconjugation", MacMillan Ref. Ltd. 1999, pages 50-100.

[0354] One of the most common reactive groups of antibodies is the aliphatic epsilon-amine of the amino acid lysine. Generally, almost all antibodies contain a rich supply of lysine. Lysine amines are quite good nucleophiles above pH 8.0 (pKa = 9.18) and thus readily react with a variety of reagents to form stable linkages. Amine reactive reagents react primarily with lysine and the alpha-amino groups of proteins. The most commonly used amine modification reagents are reactive esters, particularly N-hydroxysuccinimide (NHS) esters. The optimal pH for reaction in aqueous environments is 8.0 to 9.0. Isothiocyanates are amine modification reagents that form thiourea linkages with proteins. They react with protein amines in aqueous solution (optimal pH 9.0 to 9.5). Aldehydes react with aliphatic and aromatic amines, hydrazines, and hydrazides under mild aqueous conditions to form imine intermediates (Schiff bases). The Schiff bases can be selectively reduced with mild or strong reducing agents (e.g., sodium borohydride or sodium cyanoborohydride) to generate stable alkyl amine linkages. Other reagents that have been used to modify amines are acid anhydrides. For example, diethylenetriamine pentaacetic acid anhydride (DTPA) is a bifunctional chelating agent that contains two amine reactive acid anhydride groups. It can react with the N-terminus and epsilon-amino groups of amino acids to form amide linkages. The acid anhydride ring opens to produce a polyvalent metal chelating arm that is capable of tightly binding metals in coordination complexes.

[0355] Another common reactive group in antibodies is the thiol residue from the sulfur-containing amino acid cystine, and its reduced product cysteine (or half of cystine). Cysteine contains a free thiol group that is more nucleophilic than amines and is generally the most reactive functional group in proteins. Thiols are generally reactive at neutral pH and thus can be selectively coupled to other molecules in the presence of amines. Because of the relative reactivity of the free thiol group, proteins bearing these groups are usually found in oxidized form as disulfide groups or disulfide bonds. In such proteins, the disulfide bonds need to be reduced with a reagent such as dithiothreitol (DTT) to generate the reactive free thiol. Thiol reactive reagents are those that will couple to the thiol group on a polypeptide to form a thioether coupling product. These reagents react rapidly at weakly acidic to neutral pH and thus can react selectively in the presence of amine groups. Several thiolating cross-linkers have been reported in the literature, such as Traut's reagent (2-iminothiolane), succinimidyl (acetylthio)acetate (SATA), and sulfosuccinimidyl 6-[3-(2-pyridyldithio)propionamido]hexanoate (sulfo-LC-SPDP), providing an efficient means of introducing multiple sulfhydryl groups via reactive amines. Haloacetyl derivatives, such as iodoacetamide, form thioether linkages and are reagents for thiol group modification. Further useful reagents are maleimides. The reaction of maleimides with thiols is essentially the same as that of iodoacetamide. Maleimides react rapidly at weakly acidic to neutral pH.

[0356] Another common reactive group in antibodies is the carboxylic acid. Antibodies contain carboxylic acid groups at the C-terminal position as well as within the side chains of aspartic acid and glutamic acid. The relatively low reactivity of carboxylic acids in water generally makes it difficult to use these groups to selectively modify polypeptides and antibodies. When this is done, the carboxylic acid group is usually converted to a reactive ester using a water-soluble carbodiimide and reacted with nucleophiles such as amines, hydrazides, or hydrazines. The amine-containing reagent should be weakly basic so as to selectively react with the activated carboxylic acid in the presence of the epsilon-amine of the basic lysine. Protein cross-linking can occur when the pH is raised above 8.0.

[0357] Sodium periodate can be used to oxidize the alcohol portion of the carbohydrate moieties of antibodies to aldehydes. As described for carboxylic acids, each aldehyde group can be reacted with an amine, hydrazide, or hydrazine. Because the carbohydrate moieties are primarily present in the fragment crystallizable region (Fc region) of antibodies, conjugation can be achieved by site-specific modification of the carbohydrates, far from the antigen-binding site. A Schiff base intermediate is formed, which is reduced by a water-soluble reducing agent, either sodium cyanoborohydride (modest and selective) or sodium borohydride (strong), which reduces the Schiff base intermediate to an alkylamine.

[0358] The conjugation of the tracer and / or capture and / or detection antibody to its conjugation partner can be performed by different methods, e.g. chemical binding or via binding of a binding pair. As used herein, the term "conjugation partner" denotes e.g. a solid support, a polypeptide, a detectable label, a specifically binding pair member. In one embodiment, the conjugation of the capture and / or tracer and / or detection antibody to its conjugation partner is performed by chemical binding via N-terminus and / or epsilon-amino groups (lysine), epsilon-amino groups of different lysines, carboxyl groups of the amino acid backbone of the antibody, thiol groups, hydroxyl groups and / or phenolic functions, and / or sugar alcohol groups of the carbohydrate structure of the antibody. In one embodiment, the capture antibody is conjugated to its conjugation partner via binding of a binding pair. In a preferred embodiment, the capture antibody is conjugated to biotin and immobilization of the capture antibody to the solid support is performed via avidin or streptavidin immobilized on the solid support. In one embodiment, the capture antibody is conjugated to its conjugation partner via binding of a binding pair. In a preferred embodiment, the tracer antibody is conjugated to digoxygenin as detectable label via a covalent bond.

[0359] Examples of "detectable labels" are chromogens (fluorescent or luminescent groups and dyes), enzymes, NMR-active groups or metal particles, incomplete antigens, e.g. digoxygenin. The detectable label can also be a photoactivatable cross-linking group, e.g. an azido or aziridine group. Metal chelates which can be detected by electrochemiluminescence science are also preferred signal emitting groups, particularly preferred are ruthenium chelates, e.g. ruthenium (bipyridyl)32+chelates. Suitable ruthenium label groups are described, e.g. in EP 0 580 979, WO 90 / 05301, WO 90 / 11511 and WO 92 / 14138. For direct detection, the label group can be selected from any known detectable label group, e.g. a dye, a luminescent label group, e.g. a chemiluminescent group, e.g. an acridinium ester or dioxetane, or a fluorescent dye, e.g. fluorescein, coumarin, rose Bengal, oxazine, resorufin, cyanine and derivatives thereof. Other examples of label groups are luminescent metal complexes, e.g. ruthenium or europium complexes, enzymes, e.g. for ELISA or CEDIA (clone enzyme donor immunoassay, e.g. EP-A-0 061 888) and radioisotopes.

[0360] For example, the indirect detection system comprises a detection reagent, such as a detection antibody labeled with a first partner of a binding pair. Examples of suitable binding pairs are antigen / antibody, biotin or biotin analogues, such as aminobiotin, iminobiotin or desthiobiotin / avidin or streptavidin, saccharide / lectin, nucleic acid or nucleic acid analogue / complementary nucleic acid, and receptor / ligand, such as steroid hormone receptor / steroid hormone. In a preferred embodiment, the first binding pair member comprises an incomplete antigen, an antigen and a hormone. In a preferred embodiment, the incomplete antigen is selected from the group consisting of digoxin, digitoxin and biotin and analogues thereof. The second partner of such binding pair (e.g. antibody, streptavidin etc.) is usually labeled to allow direct detection, e.g. by a label as described above.

[0361] Immunoassays can generally be performed in three different formats. One is direct detection, one is indirect detection or by sandwich assay. Direct detection immunoassays use a detection (or tracer) antibody that can be measured directly. Enzymes or other molecules can be used to generate a signal that will produce a color, fluorescence or luminescence, so that the signal can be seen or measured (radioactive isotopes can also be used, although they are not currently common). In indirect assays, the primary antibody that binds to the analyte is used to provide a clear target for a secondary antibody (tracer antibody) that specifically binds to the target provided by the primary antibody (called detection or tracer antibody). The secondary antibody generates a measurable signal. Sandwich assays use two antibodies, a capture antibody and a tracer (detection) antibody. The capture antibody is used to bind (immobilize) the analyte in solution or to bind to it in solution. This allows the analyte to be specifically removed from the sample. The tracer (detection) antibody is used in a second step to generate a signal (directly or indirectly as described above). The sandwich format requires two antibodies, each with a different epitope on the target molecule. In addition, they cannot interfere with each other, because both antibodies must bind to the target at the same time.

[0362] Different principles for determining bispecific antibodies in immunoassays are known to the person skilled in the art:

[0363] 1) Capture uses:

[0364] - one of the antigens;

[0365] - an anti-idiotypic antibody against one of the binding sites;

[0366] 2) Detection uses:

[0367] - the other corresponding antigen;

[0368] - an anti-idiotypic antibody directed to a respective other binding site;

[0369] These can be combined independently of each other.

[0370] a blood brain barrier penetrating antibody of the method as described herein

[0371] In one aspect, the application relates to determining the concentration of a bispecific antibody in a patient for treating a disease of the brain tissue,

[0372] wherein the bispecific therapeutic antibody comprises

[0373] i) an Fc region (capable of performing effector functions),

[0374] ii) two binding sites that specifically bind to a first (cell surface) target, and

[0375] iii) one binding site that specifically binds to a second (cell surface) target,

[0376] wherein the side effects of the treatment are reduced after administration,

[0377] wherein the side effects are one or more selected from the group consisting of vasodilation, bronchoconstriction, laryngeal edema, a drop in cardiac pressure, and hypothermia.

[0378] In one embodiment, the two binding sites that specifically bind to the first target and the binding site that specifically binds to the second target are arranged in opposite directions, i.e. one is conjugated to the N-terminus of the Fc region and the other is conjugated to the C-terminus of the Fc region.

[0379] In one embodiment, the first (cell surface) target and the second (cell surface) target are different.

[0380] In one embodiment, the binding site that specifically binds to the first (cell surface) target and the binding site that specifically binds to the second (cell surface) target are located at opposite ends, i.e. the binding sites that specifically bind to the first target are both / each at the N-terminus of the (full length) antibody heavy chain, while the binding site for the second target is at the C-terminus of one of the (full length) antibody heavy chains of the bispecific antibody.

[0381] In one embodiment, the binding site that specifically binds to the first (cell surface) target and the binding site that specifically binds to the second (cell surface) target are located at opposite ends of the bispecific antibody, i.e. one binding site that specifically binds to the first target is conjugated to the first N-terminus of the Fc region and the other is conjugated to the second N-terminus of the Fc region, while the binding site that specifically binds to the second target is conjugated to one of the C-termini of the Fc region.

[0382] In one embodiment, the binding site that specifically binds to the second (cell surface) target is linked via a peptide linker to one of the binding sites that specifically binds to the first (cell surface) target. In one embodiment, the peptide linker comprises the amino acid sequence of SEQ ID NO: 56 or 57.

[0383] In one embodiment, the binding site that specifically binds to the second (cell surface) target is in the Fc region, wherein at least one structural loop region of any of the CH2 domain, the CH3 domain or the CH4 domain, comprises at least one modification, such that the at least one modified loop region binds to the second (cell surface) target, wherein the unmodified immunoglobulin constant domain does not bind to said target.

[0384] In one embodiment, the binding site is a pair of an antibody heavy chain variable domain and an antibody light chain variable domain.

[0385] In one embodiment, the bispecific therapeutic antibody comprises

[0386] i) a pair of a first antibody light chain and a first antibody heavy chain,

[0387] ii) a pair of a second antibody light chain and a second antibody heavy chain, and

[0388] iii) a further antibody fragment selected from the group consisting of a scFv, a Fab, a scFab, a dAb fragment, a DutaFab and a CrossFab,

[0389] wherein the pair of antibody chains of i) and ii) comprises a binding site that specifically binds to the first (cell surface) target, and the further antibody fragment of iii) comprises a binding site that specifically binds to the second (cell surface) target.

[0390] In one embodiment, the further antibody fragment of iii) is conjugated directly or via a peptide linker to the first antibody heavy chain or the second antibody heavy chain. In one embodiment, the further antibody fragment of iii) is conjugated directly or via a peptide linker to the C-terminus of the antibody heavy chain of i) or ii). In one embodiment, the peptide linker comprises the amino acid sequence of SEQ ID NO: 56 or 57. In one embodiment, the first antibody light chain and the second antibody light chain have the same amino acid sequence, while the first antibody heavy chain and the second antibody heavy chain differ by mutations required for heterodimerization. In one embodiment, the mutations required for heterodimerization are knob-into-hole mutations. In one embodiment, the antibody heavy chain that is not conjugated to the further antibody fragment of iii) does not comprise i) a C-terminal lysine residue, or ii) a C-terminal glycine-lysine dipeptide.

[0391] In one embodiment, the first target is a brain target and the second target is human transferrin receptor. In one embodiment, the first target is a brain target and the second target is human transferrin receptor 1.

[0392] In one embodiment, the brain target is selected from the group consisting of beta- secretase 1 (BACE1), amyloid beta (Αβ), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), human tau, phosphorylated human tau, apolipoprotein E4 (ApoE4), human alpha-synuclein, human CD20, huntingtin, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), glucocerebrosidase and caspase 6. In a preferred embodiment, the brain target is selected from the group consisting of human CD20, human tau, phosphorylated human tau, human alpha-synuclein and human amyloid beta protein. In a preferred embodiment, the brain target is human amyloid beta protein. In one embodiment, the brain target is selected from SEQ ID NO: 58, 59, 60, 01, 61.

[0393] In one preferred embodiment, the bispecific therapeutic antibody comprises

[0394] i) a pair of a first antibody light chain and a first antibody heavy chain comprising a first light chain variable domain and a first heavy chain variable domain forming a first binding site that specifically binds to a brain target selected from the group consisting of human CD20, human tau, phosphorylated human tau, human alpha-synuclein and human amyloid beta protein,

[0395] ii) a pair of a second antibody light chain and a second antibody heavy chain comprising a second light chain variable domain and a second heavy chain variable domain forming a second binding site that specifically binds to the same brain target as the first binding site,

[0396] iii) a further antibody fragment selected from the group consisting of scFv, Fab, scFab, dAb fragment, DutaFab and CrossFab comprising a third light chain variable domain and a third heavy chain variable domain forming a third binding site that specifically binds to human transferrin receptor (transferrin receptor 1), and

[0397] iv) an (of human IgGl subclass) Fc region capable of performing (human) effector functions,

[0398] wherein the further antibody fragment of iii) is conjugated to the C-terminus of the antibody heavy chain of i) or ii) directly or via a peptide linker.

[0399] In one embodiment, the additional antibody fragment is a Fab fragment which specifically binds to a second antigen and which is fused via a linker of a peptide to the C-terminus of one of the heavy chains of i) or ii), wherein the constant domains CL and CH1 of the second light chain and the second heavy chain are replaced by each other, comprising a third light chain variable domain and a third heavy chain variable domain which form a third binding site which specifically binds to human transferrin receptor (transferrin receptor 1).

[0400] In one embodiment, the binding site which specifically binds to human transferrin receptor (transferrin receptor 1) comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 33 or 62; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 34 or 63 or 35; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 36, 37 or 64; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 38 or 65; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 39; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 66 or 40.

[0401] In one embodiment, the binding site which specifically binds to human transferrin receptor (transferrin receptor 1) comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 33; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 34; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 37; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 38; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 39; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 40.

[0402] In one embodiment, the therapeutic antibody comprises one pair of a heavy chain variable domain of SEQ ID NO: 31 and a light chain variable domain of SEQ ID NO: 32 which form a binding site to transferrin receptor (transferrin receptor 1) and at least one (i.e. one or two) pair of a heavy chain variable domain of SEQ ID NO: 19 and a light chain variable domain of SEQ ID NO: 20 which form a binding site to human amyloid beta protein (Aβ).

[0403] In one embodiment, the therapeutic antibody comprises a pair of heavy chain variable domains of SEQ ID NO: 31 and light chain variable domains of SEQ ID NO: 32, which form a binding site for human transferrin receptor (transferrin receptor 1), and two pairs of heavy chain variable domains of SEQ ID NO: 41 and light chain variable domains of SEQ ID NO: 42, which form binding sites for human CD20, respectively. In one embodiment, the heavy chain variable region comprises a substitution of the amino acid residue at Kabat position 11 with any amino acid other than leucine. In one embodiment, the substitution comprises a substitution of the amino acid residue at Kabat position 11 with a non-polar amino acid. In a preferred embodiment, the substitution comprises a substitution of the amino acid residue at Kabat position 11 in the heavy chain variable domain of SEQ ID NO: 41 with an amino acid residue selected from the group consisting of valine, leucine, isoleucine, serine, and phenylalanine.

[0404] In one embodiment, the therapeutic antibody comprises a pair of heavy chain variable domains of SEQ ID NO: 31 and light chain variable domains of SEQ ID NO: 32, which form a binding site for human transferrin receptor (transferrin receptor 1), and two pairs of heavy chain variable domains of SEQ ID NO: 43 and light chain variable domains of SEQ ID NO: 44, which form binding sites for human alpha-synuclein, respectively.

[0405] In one embodiment, the therapeutic antibody comprises a pair of heavy chain variable domains of SEQ ID NO: 31 and light chain variable domains of SEQ ID NO: 32, which form a binding site for human transferrin receptor (transferrin receptor 1), and two pairs of humanized heavy chain variable domains derived from SEQ ID NO: 45 and humanized light chain variable domains derived from SEQ ID NO: 46, which form binding sites for human alpha-synuclein, respectively.

[0406] In one embodiment, the therapeutic antibody comprises a pair of heavy chain variable domains of SEQ ID NO: 31 and light chain variable domains of SEQ ID NO: 32, which form a binding site for human transferrin receptor (transferrin receptor 1), and two pairs of humanized heavy chain variable domains derived from SEQ ID NO: 45 and humanized light chain variable domains derived from SEQ ID NO: 46, which form binding sites for human alpha-synuclein, respectively.

[0407] In one embodiment, the therapeutic antibody comprises a pair of heavy chain variable domains of SEQ ID NO: 31 and light chain variable domains of SEQ ID NO: 32, which form a binding site for human transferrin receptor (transferrin receptor 1), and two pairs of humanized heavy chain variable domains derived from SEQ ID NO: 49 and humanized light chain variable domains derived from SEQ ID NO: 50, which respectively form a binding site for human alpha-synuclein.

[0408] In one embodiment, the therapeutic antibody comprises a pair of heavy chain variable domains of SEQ ID NO: 31 and light chain variable domains of SEQ ID NO: 32, which form a binding site for human transferrin receptor (transferrin receptor 1), and two pairs of humanized heavy chain variable domains derived from SEQ ID NO: 51 and humanized light chain variable domains derived from SEQ ID NO: 52, which respectively form a binding site for human alpha-synuclein.

[0409] In one embodiment, the therapeutic antibody comprises a pair of heavy chain variable domains of SEQ ID NO: 31 and light chain variable domains of SEQ ID NO: 32, which form a binding site for human transferrin receptor (transferrin receptor 1), and two pairs of humanized heavy chain variable domains derived from SEQ ID NO: 53 and humanized light chain variable domains derived from SEQ ID NO: 54, which respectively form a binding site for human alpha-synuclein.

[0410] In one embodiment, the disease is a neurological condition. In one embodiment, the disease is a neurological disorder selected from the group consisting of neuropathy, amyloidosis, cancer, an eye disease or disorder, a viral or microbial infection, inflammation, ischemia, a neurodegenerative disease, a seizure disorder, a behavioral disorder, a lysosomal storage disease, Lewy body disease, post-polio syndrome, Shy-Draeger syndrome, olivopontocerebellar atrophy, Parkinson's disease, multiple system atrophy, striatonigral degeneration, tauopathy, Alzheimer's disease, supranuclear palsy, prion disease, bovine spongiform encephalopathy, scrapie, Creutzfeldt-Jakob syndrome, kuru, Gerstmann-Straussler-Scheinker disease, chronic wasting disease and fatal familial insomnia, bulbar palsy, motor neuron disease, a neurosystemic degenerative disorder, a spongiform leukodystrophy, Huntington's disease, neuronal ceroid lipofuscinosis, Alexander's disease, Tourette's syndrome, Menkes kinky hair syndrome, Cockayne syndrome, Hallervorden-Spatz syndrome, Lafora disease, Rett syndrome, hepatolenticular degeneration, Lesch-Nyhan syndrome, Unverricht-Lundborg syndrome, dementia, Pick's disease, spinocerebellar ataxia, cancer of the CNS and / or brain, including brain metastases from cancer in other parts of the body. In one embodiment, the disease is a neurological disorder selected from the group consisting of Alzheimer's disease, Parkinson's disease, cancer of the CNS and / or brain, including brain metastases from cancer in other parts of the body, and tauopathy. In one embodiment, the disease is a neurological disorder selected from the group consisting of Alzheimer's disease, Parkinson's disease, and tauopathy.

[0411] In one embodiment, the therapeutic antibody comprises an Fc region capable of performing an effector function. In one embodiment, the Fc region capable of performing an effector function is an Fc region that specifically binds to / can specifically bind to a human FcyR. In one embodiment, the Fc region capable of performing an effector function can elicit ADCC.

[0412] In one embodiment, the ADCC caused by the bispecific therapeutic antibody (upon injection / binding to the second (cell surface) target) is lower than the ADCC caused by a bivalent bispecific antibody having only one (i.e. exactly one) binding site that specifically binds to the first (cell surface) target, and (exactly) one binding site that specifically binds to the second (cell surface) target, i.e. one of the binding sites that specifically binds to the first (cell surface) target is deleted. In one embodiment, the ADCC is 10-fold or more lower.

[0413] In one embodiment, the administration is intravenous administration, subcutaneous administration, or intramuscular administration.

[0414] In one embodiment, the first antibody heavy chain of (i) and the second antibody heavy chain of (ii) form a heterodimer. In one embodiment, the first antibody heavy chain and the second antibody heavy chain comprise mutations that support heterodimer formation.

[0415] In one embodiment,

[0416] a) the antibody heavy chain is a full-length antibody heavy chain of the human IgGl subclass,

[0417] b) the antibody heavy chain is a full-length antibody heavy chain of the human IgG4 subclass,

[0418] c) one antibody heavy chain is a full-length antibody heavy chain of the human subclass IgGl with the mutations T366W and optionally S354C or Y349C, and the other antibody heavy chain is a full-length antibody heavy chain of the human subclass IgGl with the mutations T366S, L368A, Y407V and optionally Y349C or S354C,

[0419] d) both antibody heavy chains are full-length antibody heavy chains of the human subclass IgGl, wherein one antibody heavy chain has the mutations I253A, H310A and H435A, and the mutations T366W and optionally S354C or Y349C, and the respective other antibody heavy chain is a full-length antibody heavy chain of the human subclass IgGl with the mutations T366S, L368A, Y407V and optionally Y349C or S354C,

[0420] e) both antibody heavy chains are full-length antibody heavy chains of the human subclass IgGl, wherein one antibody heavy chain has the mutations M252Y, S254T and T256E, and the mutations T366W and optionally S354C or Y349C, and the respective other antibody heavy chain is a full-length antibody heavy chain of the human subclass IgGl with the mutations T366S, L368A, Y407V and optionally Y349C or S354C, or

[0421] f) both antibody heavy chains are antibody heavy chains of the human subclass IgGl, wherein one antibody heavy chain has the mutations T307H and N434H, and the mutations T366W and optionally S354C or Y349C, and the respective other antibody heavy chain is a full-length antibody heavy chain of the human subclass IgGl with the mutations T366S, L368A, Y407V and optionally Y349C or S354C.

[0422] In one embodiment,

[0423] a) the antibody heavy chain is an antibody heavy chain of the human subclass IgGl,

[0424] b) the antibody heavy chain is an antibody heavy chain of the human subclass IgG4,

[0425] c) one antibody heavy chain is an antibody heavy chain of human subclass IgG1 with the mutation T366W and optionally S354C or Y349C, and the other antibody heavy chain is an antibody heavy chain of human subclass IgG1 with the mutations T366S, L368A, Y407V and optionally Y349C or S354C,

[0426] d) both antibody heavy chains are antibody heavy chains of human subclass IgG1, wherein one antibody heavy chain has the mutations I253A, H310A and H435A, and the mutations T366W and optionally S354C or Y349C, and the respective other antibody heavy chain is an antibody heavy chain of human subclass IgG1 with the mutations T366S, L368A, Y407V and optionally Y349C or S354C,

[0427] e) both antibody heavy chains are antibody heavy chains of human subclass IgG1, wherein one antibody heavy chain has the mutations M252Y, S254T and T256E, and the mutations T366W and optionally S354C or Y349C, and the respective other antibody heavy chain is an antibody heavy chain of human subclass IgG1 with the mutations T366S, L368A, Y407V and optionally Y349C or S354C, or

[0428] f) both antibody heavy chains are antibody heavy chains of human subclass IgG1, wherein one antibody heavy chain has the mutations T307H and N434H, and the mutations T366W and optionally S354C or Y349C, and the respective other antibody heavy chain is an antibody heavy chain of human subclass IgG1 with the mutations T366S, L368A, Y407V and optionally Y349C or S354C,

[0429] wherein a C-terminal lysine or a glycine-lysine dipeptide is present or absent.

[0430] Embodiments of the method according to the present application

[0431] The relationship between CSF, blood-brain barrier and blood has been reviewed by Katsinelos, T. et al. (Front. Immunol. 10 (2019) 1139) as follows:

[0432] IgG levels are maintained in human serum at approximately 10 mg / ml. The brain is isolated from serum by the blood brain barrier (BBB), which is impermeable to large molecules including IgG (Neuwelt, E. A. et al. Nat. Rev. Neurosci. 12 (2011) 169-182). Instead, the brain is bathed in cerebrospinal fluid (CSF), which is produced after filtration and ion transport across the choroid plexus from blood. The concentration of IgG in the produced CSF is approximately 500 to 1,000-fold lower than in serum. At first glance, this low concentration of antibodies in the brain makes CNS antigens unattractive targets for passive immunotherapy, which is typically administered to the peripheral nervous system. This is further complicated by a poor understanding of the mechanisms that maintain steady-state levels of antibodies. CSF flows around the brain before exiting the CNS along the spinal and cranial nerves and via drainage to the lymphatic system (Louveau, A. et al. Nature 523 (2015) 337-341; Aspelund, A. J. Exp. Med. 212 (2015) 991-999). IgG administered intrathecally can be rapidly cleared from the brain, primarily by this extensive flow and the potential for selective transport out of the brain. The neonatal Fc receptor (FcRn) is abundantly expressed at the BBB (Schlachetzki, F. et al. J. Neurochem. 81 (2002) 203-206). Given the role of FcRn in transcytosis of antibodies across the placenta, it has been suggested that FcRn can perform retrotranscytosis to help maintain the low IgG environment of the CNS. There is evidence that the process of antibody clearance from the brain is mediated in part by the Fc domain of the antibody (Zhang, Y. and Pardridge, W. M. J. Neuroimmunol. 114 (2001) 168-172; Cooper, P. R. et al. Brain Res. 1534 (2013) 13-21), and that the efflux of anti-Ab monoclonal antibodies is reduced in FcRn-deficient mice (Deane, R. et al. J. Neurosci. 25 (2005) 11495-11503). However, brain concentrations of peripherally administered IgG do not differ significantly between wild-type and FcRn-deficient mice (Abuqayyas, L. and Balthasar, J. P. Mol. Pharma. 10 (2013) 1505-1513).

[0433] For small laboratory animals, blood is removed from the brain prior to harvesting by perfusion. For example, the brain can be harvested after 8 minutes of ice-cold PBS at 2 ml / min via cardiac perfusion.

[0434] For example, methods are currently being explored for using a multispecific antibody, bispecific antibody or trispecific antibody comprising one or more carrier molecules and one or more cargo molecules to transport a therapeutic antibody across the blood-brain barrier via a receptor-mediated endocytosis pathway. For example, transferrin receptor (TfR)-binding antibodies (and variants thereof) can be used as carriers, and when fused to cargo molecules, generate bispecific antibodies capable of crossing the blood-brain barrier (see, e.g., Zuchero, Y.J, et al., Neuron 89 (12016) 70-82; Bien-Ly, N, et al., J. Exp. Med. 211 (2014) 233-244; US 2018 / 8002433; CA 3,000,560; which are incorporated herein by reference). Alternatively, insulin-like growth factor 1 receptor (IGF-1R)-binding antibodies can be used as carriers and fused to cargo molecules to generate bispecific antibodies that cross the blood-brain barrier (see, e.g., WO 2015 / 131256; WO 2015 / 131257; WO 2015 / 131258; incorporated herein by reference).

[0435] To reliably and correctly determine the amount in the brain, the amount of lysate of the therapeutic antibody transported into the brain across the blood-brain barrier must be excluded, and the interference of residual blood in the sample must be avoided. As described above, the final concentration of IgG in the CSF is about 500 to 1,000 times lower than in the serum, and the brain is covered by an interwoven network of blood vessels. Therefore, the change of residual blood in the brain tissue sample is not negligible. Furthermore, even a small amount of residual blood can severely interfere with the quantitative determination of antibodies in the brain tissue.

[0436] Therefore, the amount of therapeutic antibody in the residual blood of the brain lysate sample must be corrected, i.e. reduced.

[0437] Therefore, the use of a quantitative blood correction marker that does not significantly diffuse to the brain during the perfusion phase is required. However, if these are determined to be in a steady state, a small constant concentration will exist behind the BBB.

[0438] The present invention is based at least in part on the finding that the amount of residual blood in the brain lysate can be determined by administering a correction antibody prior to the collection of the brain sample. It has been found that it is particularly advantageous to use an antibody that does not specifically bind to any target in the experimental animal from which the brain sample is obtained as a reference antibody, most preferably a human germline antibody.

[0439] Therefore, a method for determining the amount of therapeutic antibody that has been transported from the blood across the blood brain barrier into the brain of an experimental animal is reported herein. The amount is preferably determined in a brain lysate sample. The gist of the invention is the additional administration of an inert antibody that is not transported across the blood brain barrier, the amount of therapeutic antibody transported across the blood brain barrier in the brain sample is determined before the brain sample is obtained. By administering the inert antibody, a correction value for the amount of therapeutic antibody present in the brain sample that is due to residual blood can be obtained. This amount from residual blood is used to correct the determined amount of antibody that is not located in the brain. Without correction, the determination will determine the total amount of therapeutic antibody in the sample, i.e. the amount transported across the blood brain barrier into the brain and the amount in the sample that is due to residual blood. The amount of therapeutic antibody in residual blood cannot be neglected, because only about 0.1 % of the antibody in the blood will pass the blood brain barrier. Therefore, the concentration of therapeutic antibody in the blood is at least two and up to three orders of magnitude higher than the concentration of therapeutic antibody in the brain. Thus, if not corrected in a method as described in the invention, the result obtained will be too high.

[0440] This is particularly important for brain shuttle tools that resemble IgG or have a clearance close to IgG, because slowly cleared molecules remain in high concentration in the blood and a small amount of blood contamination can overshadow the determination of the brain concentration if the amount of clearance in the brain is relatively small.

[0441] The method as described in the invention can be applied to any brain tissue sample, independent of the method used to remove blood therefrom.

[0442] The method as described in the invention has the feasibility of cross-species analysis, sufficient determination stability, precision and accuracy, and a broad sensitivity range.

[0443] In short, the invention provides a method for determining residual blood in a brain sample of an experimental animal,

[0444] - wherein a second inert IgG is administered before perfusion, i.e. at the most 5 minutes before, a plasma sample is taken, perfusion is performed, and brain and plasma concentrations are measured and corrected

[0445] - wherein the advantage is that the amount that passes the blood brain barrier is limited; thus, the concentration of the second inert antibody only reflects the plasma volume

[0446] - wherein a first specificity assay for the therapeutic antibody and a second specificity assay for the second inert antibody are used

[0447] - wherein the problem of animals positive for anti-therapeutic antibody antibodies that can confound the measurement can be prevented.

[0448] Figure 1 Exemplary calculations for the determination of blood contamination in (brain) tissue samples using inter-antibody assays are provided.

[0449] For example, using a conventional ELISA, the concentration of a therapeutic monoclonal antibody (tmAb) and of an inert reference monoclonal antibody (refmAb) is determined in plasma as well as in homogenized brain tissue samples. The results of the ELISA are generally obtained in mass concentration in SI units [g / L]. In a first step, each of the determined mass concentrations of tmAb and refmAb is converted into a mass fraction in units of [g / g] by dividing the determined mass concentration by the brain tissue concentration of the sample. In a second step, the plasma carryover in the brain tissue sample, i.e. the plasma contamination, is calculated by dividing the mass fraction of the inert antibody obtained in the first step by the determined plasma concentration of refmAb. The volume of residual plasma per weight of sample (residual plasma volume / weight of brain sample) is thus obtained. In a third step, the mass fraction of tmAb in the brain tissue sample resulting from the plasma contamination is calculated by multiplying the plasma concentration of tmAb by the volume of residual plasma per weight of sample. In a fourth step (also the last step), the true brain concentration of tmAb is obtained by subtracting the mass fraction of tmAb in the plasma-contaminated brain tissue sample obtained in the third step from the determined mass fraction of tmAb in the first step.

[0450] According to the method of the present application, it has been applied to analyze two bispecific antibodies each binding to TfR and to a therapeutic target 1 or 2 in cynomolgus brain lysates. The structure of each of the antibodies is shown in Figure 2 The detection assays for the therapeutic antibody and the reference antibody are shown in Figure 3

[0451] As outlined in Example 1, the sensitivity of the assay for the determination of the inert antibody is 8 ng / ml, i.e. about 1.1-1.5 μL plasma per g cynomolgus brain (corresponding to about 2.2-3 μL blood per g cynomolgus brain) can be detected.

[0452] Five different brain regions have been analyzed: cerebellum, hippocampus, striatum, cortex and choroid plexus.

[0453] Four different animals have been analyzed, wherein animals 1 to 3 had no residual blood in the brain samples, but animal 4 did (data not shown) as determined by light analysis.

[0454] Using the method according to the present application, this contamination can be detected and the respective values can thus be corrected accordingly.

[0455]

[0456]

[0457] ​

[0458]

[0459] In a further study, 15 animals were dosed with 20 mg / kg of the anti-Abeta antibody and 15 animals were dosed with 10 mg / kg of the anti-Abeta / TfR antibody. After different time points after administration, the respective samples were analyzed. In all samples, residual blood in the respective brain tissue samples has been detected. Therefore, in these cases, the corrected values were obtained using the method as described in the present application.

[0460]

[0461]

[0462]

[0463]

[0464]

[0465]

[0466] To show the general applicability of the method according to the present application, the same analysis has been performed in C57BL / 6 wild type mice with a second antibody, i.e. an anti-TfR / target_2 antibody.

[0467] Figure 4 Overlay plot showing the corrected curve of the inert reference antibody detection assay in the presence of 1% cynomolgus monkey brain lysate (CBL; cynoBL) and 1% mouse brain lysate (MBL; muBL). It can be seen that the origin of the matrix does not affect the assay.

[0468] The working range of the assay is 8.4 ng / mL to 250 ng / mL in the presence of 1% MBL. Up to 10 pg / mL of therapeutic antibody can be present in the assay without causing interference in the presence of 1% MBL.

[0469] The working range of the assay is 11 ng / mL to 220 ng / mL in the presence of 1% mouse mixed plasma (MPP). Up to 20 pg / mL of therapeutic antibody can be present in the assay without causing interference in the presence of 1% MPP.

[0470] A single dose of 20 mg / ml of the antibody was administered and samples were analyzed at 24 hours, 48 hours, 96 hours, 168 hours, 336 hours, 504 hours and 672 hours after administration. The respective concentrations in brain lysates and plasma were determined. In Figure 5In the present case, the concentration of the administered antibody determined in the brain lysate is shown as a ratio of the uncorrected to the corrected brain concentration. That is, if the correction does not affect the value, the ratio is 1. If the determined second antibody concentration is decreased due to the correction for the residual plasma values, the value will be less than 1. This difference increases as more antibody is transported across the blood-brain barrier over time. From Figure 5 It can be seen that the ratio becomes smaller and smaller over time. It can thus be seen that the correction performed in the method according to the present application eliminates the interference from residual blood in the brain sample. Figure 6 The corresponding assay for determining the second antibody is shown.

[0471] Inert reference monoclonal antibody for the method according to the present application

[0472] In the method according to the present application, the inert reference monoclonal antibody useful is preferably a human immunoglobulin molecule, in particular a human immunoglobulin molecule which is not capable of specifically binding to an antigen.

[0473] An exemplary inert reference monoclonal antibody is antibody DP47GS. DP47GS comprises a heavy chain variable region sequence based on the human VH3-23 germline sequence and a light chain variable region sequence based on the human Vk3-20 germline sequence.

[0474] In one embodiment, the inert reference monoclonal antibody is an IgG class immunoglobulin molecule, in particular an IgG1 subclass immunoglobulin molecule. In one embodiment, the inert reference monoclonal antibody is a human immunoglobulin molecule. In one embodiment, the inert reference monoclonal antibody is a monoclonal antibody. In one embodiment, the inert reference monoclonal antibody is not capable of specifically binding to an antigen. In one embodiment, the inert reference monoclonal antibody comprises a heavy chain variable region sequence based on the human VH3-23 germline sequence. In a particular embodiment, the inert reference monoclonal antibody comprises the heavy chain variable region sequence of SEQ ID NO: 67. In one embodiment, the inert reference monoclonal antibody comprises a light chain variable region sequence based on the human Vk3-20 germline sequence. In a particular embodiment, the inert reference monoclonal antibody comprises the light chain variable region sequence of SEQ ID NO: 68. In an even more particular embodiment, the inert reference monoclonal antibody comprises the heavy chain variable region sequence of SEQ ID NO: 67 and the light chain variable region sequence of SEQ ID NO: 68. In one embodiment, the inert reference monoclonal antibody is not capable of specifically binding to an antigen and comprises a heavy chain variable region sequence based on the human VH3-23 germline sequence and a light chain variable region sequence based on the human Vk3-20 germline sequence.

[0475] In one embodiment, the inert reference monoclonal antibody comprises a heavy chain variable region sequence based on the human VH3-23 germline sequence. In a specific embodiment, the inert reference monoclonal antibody comprises a heavy chain variable region sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 67. In one embodiment, the inert reference monoclonal antibody comprises a light chain variable region sequence based on the human Vk3-20 germline sequence. In a specific embodiment, the inert reference monoclonal antibody comprises a light chain variable region sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 68. In an even more specific embodiment, the inert reference monoclonal antibody comprises the heavy chain variable region sequence of SEQ ID NO: 67 and the light chain variable region sequence of SEQ ID NO: 68. Immunoglobulin molecules comprising these variable region sequences do not specifically bind to antigens, in particular human antigens. They lack binding to normal tissues as well as PBMCs, are not polyreactive, and do not show non-specific accumulation in vivo by imaging (data not shown). The variable region sequences are entirely based on human germline sequences, except for the heavy chain CDR 3, where a GSG sequence has been introduced to generate a non-binding immunoglobulin.

[0476] In one embodiment, the inert reference monoclonal antibody comprises a heavy chain having a variable domain with the amino acid sequence of SEQ ID NO: 67 and a human IgGl constant region, and a light chain having a variable domain with the amino acid sequence of SEQ ID NO: 68 and a human kappa light chain constant domain. In one embodiment, the inert reference monoclonal antibody comprises the mutations L234A, L235A and P329G (numbering according to the Kabat EU index) in the heavy chain Fc region.

[0477] In one embodiment, the inert reference monoclonal antibody comprises a heavy chain with the amino acid sequence of SEQ ID NO: 69, and a light chain with the amino acid sequence of SEQ ID NO: 70.

[0478] Comparison methods and results

[0479] Comparative technical method:

[0480] Correction by residual blood volume without perfusion

[0481] Friden et al. (J. Cerebral Blood Flow & Met 30 (2010) 150-161) collected available information on the cerebral vascular space from the literature (see Table 1 of Friden et al.).

[0482] According to the most commonly used 14C-Dextran method, the brain plasma value would be approximately 18.1 μL / g brain tissue. Applying this for correction, all determined values become negative.

[0483] Thus, it is incorrect to assume that only the total brain plasma value would be present in the brain tissue sample.

[0484] Thus, absolute values cannot be applied, but correction factors for co-determination are needed.

[0485] Thus, different correction factors are needed.

[0486] Since perfusion would be performed, control of residual blood contamination is needed. This is especially important for the comparison of IgG or brain shuttle tools with close clearance to IgG - why? Because slowly cleared molecules remain in high concentration in the blood, and if the clearance in the brain is relatively small, a small amount of blood contamination can cover the determination of the brain concentration.

[0487] Thus, a quantitative blood correction marker is needed that does not significantly diffuse to the brain during the administration and perfusion phase.

[0488] Comparative marker:

[0489] Different other non-antibody inert reference molecules, which are other endogenous proteins with high molecular weight and high endogenous blood levels, are considered equally suitable as correction means in the method of the present invention before testing.

[0490] Determination of complement factor H

[0491] Different publications indicate that complement factor H is absent in the cerebrospinal fluid of cynomolgus monkeys and in cynomolgus monkey brain lysates, where complement factor H is normally found in non-CSF or non-brain tissue. Thus, it has been hypothesized that detection of complement factor H is a feasible surrogate marker for the determination of residual contaminating blood in cCSF and CBL samples.

[0492] As positive controls, human pooled serum (HPS; 200-800 μg / mL complement factor H) and human pooled plasma (HPP; about 300 μg / mL complement factor H) can be used.

[0493] The assay was set up as Elecsys assay (Roche Diagnostics GmbH, Mannheim, Germany). The corresponding calibration curve is shown in Figure 7 The working range of the assay is 7.8 μg / mL to 2000 μg / mL.

[0494]

[0495] Therefore, it has been found that the determination of complement factor H is not suitable as a surrogate marker for residual contaminating blood, because the assay is not sensitive enough.

[0496] Determination of alpha-2-macroglobulin

[0497] Different publications indicate that alpha2-macroglobulin is absent in CSF of cynomolgus monkeys and in cynomolgus monkey brain lysates, whereas alpha2-macroglobulin is found in non-CSF or non-brain tissues (1500-2000 μg / mL). Therefore, it has been hypothesized that the detection of alpha2-macroglobulin is a feasible surrogate marker for the determination of residual contaminating blood in cCSF and CBL samples.

[0498] The principle of the assay for the determination of alpha2-macroglobulin by means of an ELISA assay is shown in Figure 8 and the corresponding calibration curve is shown in Figure 9

[0499] The working range of the assay is 0.62 ng / mL (LLOQ) to 39 ng / mL (ULOQ).

[0500] Human pooled serum 2.8g / l Cynomolgus monkey pooled serum 1 36 ng / ml Cynomolgus monkey pooled serum 2 44 ng / ml Cynomolgus monkey brain lysate sample 0 ng / ml

[0501] The expected values could be confirmed for human serum and plasma, whereas only 1 / 25,000 of the expected amount could be detected in cynomolgus monkey pooled serum. Therefore, the values are too low to be quantified in diluted forms of cynomolgus monkey CSF and brain lysates. Thus, the determination of alpha2-macroglobulin is not suitable as a surrogate marker.

[0502] Determination of complement component 5a (C5a)

[0503] Different publications indicate that complement component 5a is absent in CSF of cynomolgus monkeys and in cynomolgus monkey brain lysates, whereas complement component 5a is found in non-CSF or non-brain tissues (60-110 μg / mL in human serum). Therefore, it has been hypothesized that the detection of complement component 5a is a feasible surrogate marker for the determination of residual contaminating blood in cCSF and CBL samples.

[0504] Like alpha2-macroglobulin, an ELISA has been established with a murine anti-human C5a antibody as capture antibody and a biotinylated murine anti-human C5a antibody as detection antibody, both of which bind to non-interfering epitopes of human C5a. The corresponding calibration curve is shown in Figure 10

[0505] The working range of the assay is 0.03 ng / mL (LLOQ) to 2 ng / mL (ULOQ).

[0506] ​​

[0507]

[0508] Thus, it was found that C5a can be determined in CBL samples.

[0509] Thus, the determination of C5a is not suitable as a surrogate marker.

[0510] Using

[0511] Gadopentetate dimeglumine (Magnevist®) is an MRT contrast agent. It is assumed not to cross the blood brain barrier.

[0512] Pharmacokinetic studies showed that only for a time of up to 15 minutes, the measured brain concentrations can correctly represent the blood compartment. After this time, diffusion into the brain tissue occurs, making the application for correction confusing. The corresponding time-course is shown in Figure 11 At the 5-minute time point, the plasma volume was estimated to be 14.1 μL / g brain.

[0513] This method cannot be used with perfusion, because the time spent for perfusion would lead to diffusion of Magnevist across the blood brain barrier - confusing residual blood correction.

[0514] Determination of cynomolgus monkey IgG in cynomolgus monkey cerebrospinal fluid (cCSF)

[0515] Different publications show that only small amounts of cynomolgus IgG are present in the cerebrospinal fluid of cynomolgus monkeys. Therefore, it was assumed that the detection of total Ig in cCSF is a feasible surrogate marker for the direct determination of transported therapeutic antibodies. Therefore, a bridging ELISA was established, as shown in Figure 12 To exclude matrix effects, human IgG-depleted cCSF was generated by incubation of cCSF with anti-human CH1 / Kappa antibodies bound to magnetic beads.

[0516] The corresponding calibration curves for buffer and human IgG-depleted cCSF are shown in Figure 13 It can be seen that no matrix effects occurred.

[0517] The working range of this assay is 120 ng / ml to 7.2 ng / mL IgG.

[0518] Using this assay, it was found that in cynomolgus plasma pool samples (CPP) about 11-19 mg / mL IgG could be detected, whereas in cCSF samples about 4-18 μg / mL cynomolgus IgG could be detected.

[0519] ***

[0520] The following examples, sequences, and figures are provided to aid in understanding the invention, the true scope of which is set forth in the appended claims. It should be understood that modifications may be made to the proposed steps without departing from the spirit of the invention.

[0521] Sequence Description

[0522]

[0523]

[0524] General method:

[0525] Preparation of cynomolgus monkey brain tissue homogenate

[0526] 300 mg of frozen cynomolgus / mouse brain tissue sample was thawed at room temperature for 2 hours. 800 μL of lysis buffer and one tablet of cOmplete protease inhibitor mixture (Roche Diagnostics GmbH) were dissolved in 50 mL of Tissue Extraction Reagent I (Invitrogen) and added to the thawed brain tissue. The sample was then homogenized at 6500 rpm for 20 seconds using a MagNA Lyser instrument (Roche Diagnostics). The homogenate was then centrifuged at 12,000 rpm for 10 minutes using a 5430 centrifuge (Eppendorf). Finally, the supernatant was transferred to 1.5 mL vials for further analysis or stored at -80°C.

[0527] Example 1

[0528] ELISA for quantifying DP47GS-PGLALA in brain lysates

[0529] To quantify the inert reference monoclonal antibody DP47GS-PGLALA (SEQ ID NO: 69 and 70) in cynomolgus monkey lysate samples, a series of sandwich enzyme-linked immunosorbent assays (ELISAs) were used. In the ELISA steps, all samples and controls were initially pre-diluted 1:100 in assay diluent to achieve the required final detection concentration of 1%.

[0530] The capture antibody (anti-DP47GS antibody, biotinylated), the diluted calibrators (DP47GS-PGLALA) and the diluted quality controls and samples, the detection reagent (anti-PGLALA antibody clone M-1.7.24, digoxigenylated) and the anti-digoxigenin-antibody-POD-conjugate are added in this order to the streptavidin-coated microtiter plate wells (SA-MTP). The reagents are incubated on the MTP shaker for 1 hour at a temperature of 500 rpm, then after each step the MTP is washed 3 times with 300 μL of washing buffer (1 x PBS, 0.05% Tween) and the residual liquid is removed. Thereafter, the formed immobilized immune complexes are visualized by adding the ABTS solution, the horseradish peroxidase POD substrate, which is converted into a color change reaction product. Finally, the color intensity is determined using the luminosity method (absorption values at 405 nm - 490 nm reference wavelength). The signal is proportional to the analyte concentration in the brain lysate sample. The quantification of DP47GS-PGLALA is performed by back-calculation of the absorption values using the corresponding calibration curve with a weighted non-linear 4-parameter Wiemer-Rodbard curve fitting function.

[0531]

[0532] Coating of the capture reagent is achieved by pipetting 100 μL of a solution containing 500 ng / mL biotinylated anti-DP47GS antibody into each SA-MTP well. Thereafter, the MTP is covered with an adhesive foil and incubated on the MTP shaker (500 rpm) for 1 hour. The supernatant is removed and each well of the MTP is washed 3 times with 300 μL of washing buffer (PBS, 0.05% Tween). The residual washing buffer is carefully removed.

[0533] Then 100 μl of the respective calibrators, quality controls and samples are pipetted into the designated wells of the coated MTP. Thereafter, the MTP is covered with an adhesive foil and incubated on the MTP shaker (500 rpm) for 1 hour. The supernatant is removed and each well of the MTP is washed 3 times with 300 μL of washing buffer (PBS, 0.05% Tween). The residual washing buffer is carefully removed.

[0534] Then 100 μL of the long digoxigenylated anti-PGLALA antibody clone M-1.7.24 at a concentration of 125 ng / mL is added to each MTP well. Thereafter, the MTP is covered with an adhesive foil and incubated on the MTP shaker (500 rpm) for 1 hour. The supernatant is removed and each well of the MTP is washed 3 times with 300 μL of washing buffer (PBS, 0.05% Tween). The residual washing buffer is carefully removed.

[0535] 100 μΐ of anti-digoxin-antibody-POD-conjugate was added to each MTP well in a concentration of 50 mU / mL. After that, the MTP was covered with an adhesive foil and incubated on a MTP shaker (500 rpm) for 1 h. The supernatant was removed and each well of the MTP was washed 3 times with 300 μΐ of washing buffer (PBS, 0.05% Tween). Residual washing buffer was carefully removed.

[0536] Then 100 μΐ of ABTS solution was added to each MTP well. The optical density was measured at a measurement wavelength of 405 nm (reference wavelength 490 nm) until the average signal of the replicates of the calibrator sample 1 reached 1.8 - 2.2 AU.

[0537] Example 2

[0538] ELISA for quantification of Cynomolgus IgG in CSF

[0539] For the quantification of Cynomolgus IgG in Cynomolgus brain spinal fluid a series of sandwich enzyme-linked immunosorbent assays (ELISA) was used. In the ELISA procedure all samples and quality controls were pre-diluted initially in assay diluent to reach the required final assay concentration of 1%.

[0540] Sequentially, capture antibody (anti-Cynomolgus IgG antibody 1 ; antigen epitope 1 ; biotinylated), diluted calibrators as well as diluted quality controls and samples, detection reagent (anti-Cynomolgus IgG antibody 2; antigen epitope 2, does not interfere with antigen epitope 1 ; digoxinylated) and anti-digoxin-antibody-POD-conjugate were added to streptavidin-coated microtiter plate wells (SA-MTP). The reagents were incubated on a MTP shaker for 1 h at 500 rpm, followed by washing the MTP 3 times with 300 μΐ of washing buffer (1 x PBS, 0.05% Tween) after each step and removing residual liquid. Thereafter, the formed immobilized immune complexes were visualized by adding ABTS solution, the horseradish peroxidase POD substrate, which was converted into a color change reaction product. Finally, the color intensity was determined using the luminosity method (absorbance at 405 nm - 490 nm reference wavelength). The signal is directly proportional to the analyte concentration in the brain lysate samples. The quantification of Cynomolgus IgG was performed by back-calculation of the absorbance values using the respective calibration curve with a weighted non-linear 4-parameter Wiemer-Rodbard curve fitting function.

[0541]

[0542] Coating capture reagent was achieved by pipetting 100 μL of a solution containing 250 ng / mL biotinylated anti-Cynomolgus IgG antibody 1 into each SA-MTP well. Thereafter, the MTP was covered with an adhesive cover foil and incubated on a MTP shaker (500 rpm) for 1 hour. The supernatant was removed and each well of the MTP was washed 3 times with 300 μL of wash buffer (PBS, 0.05% Tween). Residual wash buffer was carefully removed.

[0543] Then 100 μL of each of the calibrators, quality controls and samples were pipetted into the designated wells of the coated MTP. Thereafter, the MTP was covered with an adhesive cover foil and incubated on a MTP shaker (500 rpm) for 1 hour. The supernatant was removed and each well of the MTP was washed 3 times with 300 μL of wash buffer (PBS, 0.05% Tween). Residual wash buffer was carefully removed.

[0544] Then 100 μL of anti-digoxin-antibody-POD-conjugate was added to each MTP well at a concentration of 25 mU / mL. Thereafter, the MTP was covered with an adhesive cover foil and incubated on a MTP shaker (500 rpm) for 1 hour. The supernatant was removed and each well of the MTP was washed 3 times with 300 μL of wash buffer (PBS, 0.05% Tween). Residual wash buffer was carefully removed.

[0545] Then 100 μL of anti-digoxin-antibody-POD-conjugate was added to each MTP well at a concentration of 25 mU / mL. Thereafter, the MTP was covered with an adhesive cover foil and incubated on a MTP shaker (500 rpm) for 1 hour. The supernatant was removed and each well of the MTP was washed 3 times with 300 μL of wash buffer (PBS, 0.05% Tween). Residual wash buffer was carefully removed.

[0546] Then 100 μL of ABTS solution was added to each MTP well. The optical density was measured at a measurement wavelength of 405 nm (reference wavelength 490 nm) until the average signal of the replicates of the calibrator sample 1 reached 1.8 - 2.2 AU.

[0547] Example 3

[0548] Generation of brain tissue lysates

[0549] First, fresh lysis buffer was prepared according to the manufacturer's instructions (Invitrogen; tissue extraction reagent I; cat. no. FNN0071). One tablet of Complete (Roche Diagnostics GmbH, Mannheim, Germany; cat. no.: 11697498001) was added per 50 ml lysis buffer.

[0550] Second, between 600 μL and 800 μL of lysis buffer was added to approximately 100-300 mg of brain tissue sample. MagNA Lyser Green Beads were optionally added.

[0551] Third, the sample was placed in a MagNA Lyser (Roche Diagnostics GmbH, Mannheim, Germany) and run at 6500 rpm for 20 seconds.

[0552] Fourth, after incubation in the MagNA Lyser, the sample was centrifuged at 12,000 rpm for 10 minutes (Eppendorf Centrifuge 5430).

[0553] Fifth, the supernatant (500-700 μL) was recovered and stored at -80°C until further analysis.

Claims

1. A method for determining the concentration of a therapeutic antibody in a tissue of an experimental animal, wherein the tissue serves as a barrier to the blood circulation of the animal, and wherein the experimental animal has been administered the therapeutic antibody, wherein interference from residual blood in a tissue sample of the experimental animal used to determine the concentration of the therapeutic antibody in the tissue is reduced, the method comprising the following steps: i) Determine the concentration of the therapeutic antibody in the blood sample of the experimental animal. ii) Determine the concentration of the therapeutic antibody in the tissue sample of the experimental animal. iii) Determine the concentration of the inert reference antibody in the blood sample of the experimental animal. iv) Determine the concentration of the inert reference antibody in the tissue sample of the experimental animal. v) Determine the tissue concentration in the tissue sample. The concentration of the therapeutic antibody in the tissues of the experimental animal is determined using the following formula: - wherein the inert reference antibody does not cross the barrier between the tissue and the blood circulation. - Wherein the inert reference antibody has been i) administered together with the therapeutic antibody, if the sample is to be collected within 5 minutes after administration of the therapeutic antibody, or ii) administered 2 to 10 minutes before collection of the tissue sample. - The blood sample was collected before the tissue sample.

2. The method of claim 1, wherein the blood sample is collected up to 5 minutes before the tissue sample.

3. The method of claim 1, wherein the blood sample is collected directly before the tissue sample.

4. The method of claim 1, wherein the tissue is brain tissue and the therapeutic antibody is capable of crossing the blood-brain barrier, or the eye tissue and the therapeutic antibody is capable of crossing the blood-eye barrier.

5. The method of claim 3, wherein the tissue is brain tissue and the therapeutic antibody can cross the blood-brain barrier or eye tissue, and the therapeutic antibody can cross the blood-eye barrier.

6. The method according to claim 1, wherein the therapeutic antibody is a bispecific antibody.

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

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

9. The method of claim 5, wherein the therapeutic antibody is a bispecific antibody.

10. The method according to any one of claims 6-9, wherein the bispecific antibody comprises a first Fab fragment and a second Fab fragment, wherein in the first Fab fragment... a) Only the CH1 and CL domains are replaced by each other, i.e., the light chain of the first Fab segment contains the VL and CH1 domains, while the heavy chain of the first Fab segment contains the VH and CL domains. b) Only the VH and VL domains are substituted for each other, i.e., the light chain of the first Fab segment contains the VH and CL domains, while the heavy chain of the first Fab segment contains the VL and CH1 domains; or c) The CH1 and CL domains are substituted for each other, and the VH and VL domains are substituted for each other, i.e., the light chain of the first Fab segment contains the VH and CH1 domains, while the heavy chain of the first Fab segment contains the VL and CL domains; and The second Fab fragment contains a light chain with VL and CL domains and a heavy chain with VH and CH1 domains.

11. The method according to any one of claims 1 to 9, wherein the therapeutic antibody specifically binds to human transferrin receptor and brain targets.

12. The method of claim 10, wherein the therapeutic antibody specifically binds to human transferrin receptor and brain targets.

13. The method according to any one of claims 6 to 9, wherein the bispecific antibody comprises a) A full-length antibody comprising two pairs of full-length antibody light chains and two pairs of full-length antibody heavy chains, wherein each pair of full-length heavy chains and full-length light chains forms a binding site that specifically binds to a first antigen. b) An additional Fab fragment, wherein the additional Fab fragment is fused to the C-terminus of any heavy chain of the full-length antibody, wherein the binding site of the additional Fab fragment specifically binds to the second antigen. The additional Fab fragment that specifically binds to the second antigen contains domain crossovers, such that the constant light chain domain CL and the constant heavy chain domain 1CH1 are substituted for each other. The first antigen is a brain target, while the second antigen is the human transferrin receptor.

14. The method of claim 11, wherein the therapeutic antibody is a bispecific antibody, the bispecific antibody comprising... a) A full-length antibody comprising two pairs of full-length antibody light chains and two pairs of full-length antibody heavy chains, wherein each pair of full-length heavy chains and full-length light chains forms a binding site that specifically binds to a first antigen. b) An additional Fab fragment, wherein the additional Fab fragment is fused to the C-terminus of any heavy chain of the full-length antibody, wherein the binding site of the additional Fab fragment specifically binds to the second antigen. The additional Fab fragment that specifically binds to the second antigen contains domain crossovers, such that the constant light chain domain CL and the constant heavy chain domain 1CH1 are substituted for each other. The first antigen is a brain target, while the second antigen is the human transferrin receptor.

15. The method according to any one of claims 6 to 9, wherein the bispecific antibody comprises a) A full-length antibody comprising two pairs of full-length antibody light chains and two pairs of full-length antibody heavy chains, wherein each pair of full-length heavy chains and full-length light chains forms a binding site that specifically binds to a first antigen. b) An additional Fab fragment, wherein the additional Fab fragment is fused to the C-terminus of a heavy chain of the full-length antibody, wherein the binding site of the additional Fab fragment specifically binds to a second antigen. Each full-length antibody light chain contains arginine at position 123 and lysine at position 124 in the constant light chain domain CL, according to Kabat numbering. Each full-length antibody heavy chain contains 147 glutamic acid residues in the first constant heavy chain domain CH1, and 213 glutamic acid residues, according to the Kabat EU index number. The additional Fab fragment that specifically binds to the second antigen contains domain crossovers, such that the constant light chain domain CL and the constant heavy chain domain 1CH1 are substituted for each other. The first antigen is a brain target, while the second antigen is the human transferrin receptor.

16. The method of claim 11, wherein the therapeutic antibody is a bispecific antibody, the bispecific antibody comprising... a) A full-length antibody comprising two pairs of full-length antibody light chains and two pairs of full-length antibody heavy chains, wherein each pair of full-length heavy chains and full-length light chains forms a binding site that specifically binds to a first antigen. b) An additional Fab fragment, wherein the additional Fab fragment is fused to the C-terminus of a heavy chain of the full-length antibody, wherein the binding site of the additional Fab fragment specifically binds to a second antigen. Each full-length antibody light chain contains arginine at position 123 and lysine at position 124 in the constant light chain domain CL, according to Kabat numbering. Each full-length antibody heavy chain contains 147 glutamic acid residues in the first constant heavy chain domain CH1, and 213 glutamic acid residues, according to the Kabat EU index number. The additional Fab fragment that specifically binds to the second antigen contains domain crossovers, such that the constant light chain domain CL and the constant heavy chain domain 1CH1 are substituted for each other. The first antigen is a brain target, while the second antigen is the human transferrin receptor.

17. The method of claim 11, wherein the brain target is human CD20 or human Aβ or human α-synuclein or human τ or human glucocerebrosidase or human lingo-1 or human huntingtin protein.

18. The method of claim 12, wherein the brain target is human CD20 or human Aβ or human α-synuclein or human τ or human glucocerebrosidase or human lingo-1 or human huntingtin protein.

19. The method of claim 13, wherein the brain target is human CD20 or human Aβ or human α-synuclein or human τ or human glucocerebrosidase or human lingo-1 or human huntingtin.

20. The method of claim 15, wherein the brain target is human CD20 or human Aβ or human α-synuclein or human τ or human glucocerebrosidase or human lingo-1 or human huntingtin protein.

21. The method according to any one of claims 17 to 20, wherein the therapeutic antibody is a bispecific antibody, the bispecific therapeutic antibody comprising... i) A pair of first antibody light chains and first antibody heavy chains, comprising a first light chain variable domain and a first heavy chain variable domain, forming a first binding site specifically binding to a brain target selected from the group consisting of human CD20, human τ protein, phosphorylated human τ protein, human α-synuclein, and human amyloid-β protein. ii) A pair of second antibody light chains and second antibody heavy chains, comprising variable domains of the second light chain and the second heavy chain, which form a second binding site that specifically binds to the same brain target as the first binding site. iii) Other antibody fragments, selected from the group consisting of scFv, Fab, scFab, dAb fragments, DutaFab, and CrossFab, containing a third light chain variable domain and a third heavy chain variable domain, specifically bind to the third binding site of the human transferrin receptor, and iv) The Fc region capable of performing effector functions, The additional antibody fragment of iii) is directly or via a peptide linker attached to the C-terminus of the antibody heavy chain of i) or ii).

22. The method of claim 21, wherein the transferrin receptor is transferrin receptor 1, or the Fc region is the Fc region of a human IgG1 subclass.

23. The method of claim 13, wherein the additional antibody fragment is a Fab fragment that specifically binds to the second antigen and is fused via a peptide linker to the C-terminus of one of the heavy chains of a) or i) or ii), wherein the constant domains CL and CH1 of the second light chain and the second heavy chain are replaced by each other, comprising a variable domain of the third light chain and a variable domain of the third heavy chain, which form a third binding site specifically binding to the human transferrin receptor.

24. The method of claim 15, wherein the additional antibody fragment is a Fab fragment that specifically binds to the second antigen and is fused via a peptide linker to the C-terminus of one of the heavy chains of a) or i) or ii), wherein the constant domains CL and CH1 of the second light chain and the second heavy chain are substituted for each other, comprising a variable domain of the third light chain and a variable domain of the third heavy chain, which form a third binding site specifically binding to the human transferrin receptor.

25. The method of claim 17, wherein the additional antibody fragment is a Fab fragment that specifically binds to the second antigen and is fused via a peptide linker to the C-terminus of one of the heavy chains of a) or i) or ii), wherein the constant domains CL and CH1 of the second light chain and the second heavy chain are substituted for each other, comprising a variable domain of the third light chain and a variable domain of the third heavy chain, which form a third binding site specifically binding to the human transferrin receptor.

26. The method according to any one of claims 23-25, wherein the transferrin receptor is transferrin receptor 1.

27. The method of claim 13, wherein the binding site specifically binding to the human transferrin receptor comprises (a) HVR-H1, which comprises the amino acid sequence SEQ ID NO: 33 or 62; (b) HVR-H2, which comprises the amino acid sequence SEQ ID NO: 34, 63 or 35; (c) HVR-H3, which comprises the amino acid sequence SEQ ID NO: 36, 37 or 64; (d) HVR-L1, which comprises the amino acid sequence SEQ ID NO: 38 or 65; (e) HVR-L2, which comprises the amino acid sequence SEQ ID NO: 39; and (f) HVR-L3, which comprises the amino acid sequence SEQ ID NO: 66 or 40.

28. The method of claim 15, wherein the binding site specifically binding to the human transferrin receptor comprises (a) HVR-H1, which comprises the amino acid sequence SEQ ID NO: 33 or 62; (b) HVR-H2, which comprises the amino acid sequence SEQ ID NO: 34, 63 or 35; (c) HVR-H3, which comprises the amino acid sequence SEQ ID NO: 36, 37 or 64; (d) HVR-L1, which comprises the amino acid sequence SEQ ID NO: 38 or 65; (e) HVR-L2, which comprises the amino acid sequence SEQ ID NO: 39; and (f) HVR-L3, which comprises the amino acid sequence SEQ ID NO: 66 or 40.

29. The method of claim 21, wherein the binding site specifically binding to the human transferrin receptor comprises (a) HVR-H1, which comprises the amino acid sequence SEQ ID NO: 33 or 62; (b) HVR-H2, which comprises the amino acid sequence SEQ ID NO: 34, 63 or 35; (c) HVR-H3, which comprises the amino acid sequence SEQ ID NO: 36, 37 or 64; (d) HVR-L1, which comprises the amino acid sequence SEQ ID NO: 38 or 65; (e) HVR-L2, which comprises the amino acid sequence SEQ ID NO: 39; and (f) HVR-L3, which comprises the amino acid sequence SEQ ID NO: 66 or 40.

30. The method according to any one of claims 27-29, wherein the transferrin receptor is transferrin receptor 1.

31. The method of claim 13, wherein the binding site specifically binding to the human transferrin receptor comprises (a) HVR-H1, which comprises the amino acid sequence SEQ ID NO: 33; (b) HVR-H2, which comprises the amino acid sequence SEQ ID NO: 34; (c) HVR-H3, which comprises the amino acid sequence SEQ ID NO: 37; (d) HVR-L1, which comprises the amino acid sequence SEQ ID NO: 38; (e) HVR-L2, which comprises the amino acid sequence SEQ ID NO: 39; and (f) HVR-L3, which comprises the amino acid sequence SEQ ID NO:

40.

32. The method of claim 15, wherein the binding site specifically binding to the human transferrin receptor comprises (a) HVR-H1, which comprises the amino acid sequence SEQ ID NO: 33; (b) HVR-H2, which comprises the amino acid sequence SEQ ID NO: 34; (c) HVR-H3, which comprises the amino acid sequence SEQ ID NO: 37; (d) HVR-L1, which comprises the amino acid sequence SEQ ID NO: 38; (e) HVR-L2, which comprises the amino acid sequence SEQ ID NO: 39; and (f) HVR-L3, which comprises the amino acid sequence SEQ ID NO:

40.

33. The method of claim 21, wherein the binding site specifically binding to the human transferrin receptor comprises (a) HVR-H1, which comprises the amino acid sequence SEQ ID NO: 33; (b) HVR-H2, which comprises the amino acid sequence SEQ ID NO: 34; (c) HVR-H3, which comprises the amino acid sequence SEQ ID NO: 37; (d) HVR-L1, which comprises the amino acid sequence SEQ ID NO: 38; (e) HVR-L2, which comprises the amino acid sequence SEQ ID NO: 39; and (f) HVR-L3, which comprises the amino acid sequence SEQ ID NO:

40.

34. The method according to any one of claims 31 to 33, wherein the transferrin receptor is transferrin receptor 1.

35. The method according to claim 27 or 31, wherein the bispecific antibody comprises a pair of heavy chain variable domains of SEQ ID NO: 31 and light chain variable domains of SEQ ID NO: 32 forming a binding site for transferrin receptor, and at least a pair of heavy chain variable domains of SEQ ID NO: 19 and light chain variable domains of SEQ ID NO: 20 forming a binding site for human amyloid β protein.

36. The method according to claim 28 or 32, wherein the bispecific antibody comprises a pair of heavy chain variable domains of SEQ ID NO: 31 and light chain variable domains of SEQ ID NO: 32 forming a binding site for transferrin receptor, and at least a pair of heavy chain variable domains of SEQ ID NO: 19 and light chain variable domains of SEQ ID NO: 20 forming a binding site for human amyloid β protein.

37. The method according to claim 29 or 33, wherein the bispecific antibody comprises a pair of heavy chain variable domains of SEQ ID NO: 31 and light chain variable domains of SEQ ID NO: 32 forming a binding site for transferrin receptor, and at least a pair of heavy chain variable domains of SEQ ID NO: 19 and light chain variable domains of SEQ ID NO: 20 forming a binding site for human amyloid β protein.

38. The method according to claim 27 or 31, wherein the bispecific antibody comprises a pair of heavy chain variable domains of SEQ ID NO: 31 and light chain variable domains of SEQ ID NO: 32 forming a binding site for human transferrin receptor, and two pairs of heavy chain variable domains of SEQ ID NO: 41 and light chain variable domains of SEQ ID NO: 42 forming binding sites for human CD20, respectively.

39. The method according to claim 28 or 32, wherein the bispecific antibody comprises a pair of heavy chain variable domains of SEQ ID NO: 31 and light chain variable domains of SEQ ID NO: 32 forming a binding site for human transferrin receptor, and two pairs of heavy chain variable domains of SEQ ID NO: 41 and light chain variable domains of SEQ ID NO: 42 forming binding sites for human CD20, respectively.

40. The method according to claim 29 or 33, wherein the bispecific antibody comprises a pair of heavy chain variable domains of SEQ ID NO: 31 and light chain variable domains of SEQ ID NO: 32 forming a binding site for human transferrin receptor, and two pairs of heavy chain variable domains of SEQ ID NO: 41 and light chain variable domains of SEQ ID NO: 42 forming binding sites for human CD20, respectively.

41. The method according to any one of claims 38 to 40, wherein the heavy chain variable region of the human CD20 binding site comprises replacing the amino acid residue at position 11 of Kabat with any amino acid other than leucine.

42. The method of claim 38, wherein the heavy chain variable region of the human CD20 binding site comprises an amino acid residue at position 11 of Kabat replaced by a nonpolar amino acid.

43. The method of claim 39, wherein the heavy chain variable region of the human CD20 binding site comprises an amino acid residue at position 11 of Kabat replaced by a nonpolar amino acid.

44. The method of claim 40, wherein the heavy chain variable region of the human CD20 binding site comprises an amino acid residue at position 11 of Kabat replaced by a nonpolar amino acid.

45. The method of claim 38, wherein the heavy chain variable region of the human CD20 binding site comprises replacing the amino acid residue at position 11 of the heavy chain variable domain of SEQ ID NO: 41 with the following amino acid residue selected from the group consisting of valine, leucine, isoleucine, serine and phenylalanine.

46. ​​The method of claim 39, wherein the heavy chain variable region of the human CD20 binding site comprises replacing the amino acid residue at position 11 of the heavy chain variable domain of SEQ ID NO: 41 with the following amino acid residue selected from the group consisting of valine, leucine, isoleucine, serine and phenylalanine.

47. The method of claim 40, wherein the heavy chain variable region of the human CD20 binding site comprises replacing the amino acid residue at position 11 of the heavy chain variable domain of SEQ ID NO: 41 with the following amino acid residue selected from the group consisting of valine, leucine, isoleucine, serine and phenylalanine.

48. The method according to claim 27 or 31, wherein the bispecific antibody comprises a pair of heavy chain variable domains of SEQ ID NO: 31 and light chain variable domains of SEQ ID NO: 32 forming a binding site for human transferrin receptor, and two pairs of heavy chain variable domains of SEQ ID NO: 43 and light chain variable domains of SEQ ID NO: 44 forming binding sites for human α-synuclein, respectively.

49. The method according to claim 28 or 32, wherein the bispecific antibody comprises a pair of heavy chain variable domains of SEQ ID NO: 31 and light chain variable domains of SEQ ID NO: 32 forming a binding site for human transferrin receptor, and two pairs of heavy chain variable domains of SEQ ID NO: 43 and light chain variable domains of SEQ ID NO: 44 forming binding sites for human α-synuclein, respectively.

50. The method according to claim 29 or 33, wherein the bispecific antibody comprises a pair of heavy chain variable domains of SEQ ID NO: 31 and light chain variable domains of SEQ ID NO: 32 forming a binding site for human transferrin receptor, and two pairs of heavy chain variable domains of SEQ ID NO: 43 and light chain variable domains of SEQ ID NO: 44 forming binding sites for human α-synuclein, respectively.

51. The method according to claim 27 or 31, wherein the bispecific antibody comprises a pair of heavy chain variable domains of SEQ ID NO: 31 and light chain variable domains of SEQ ID NO: 32, which form a binding site for human transferrin receptor, and two pairs of humanized heavy chain variable domains derived from SEQ ID NO: 45 and humanized light chain variable domains derived from SEQ ID NO: 46, which respectively form binding sites for human α-synuclein.

52. The method according to claim 28 or 32, wherein the bispecific antibody comprises a pair of heavy chain variable domains of SEQ ID NO: 31 and light chain variable domains of SEQ ID NO: 32, which form a binding site for human transferrin receptor, and two pairs of humanized heavy chain variable domains derived from SEQ ID NO: 45 and humanized light chain variable domains derived from SEQ ID NO: 46, which respectively form binding sites for human α-synuclein.

53. The method according to claim 29 or 33, wherein the bispecific antibody comprises a pair of heavy chain variable domains of SEQ ID NO: 31 and light chain variable domains of SEQ ID NO: 32, which form a binding site for human transferrin receptor, and two pairs of humanized heavy chain variable domains derived from SEQ ID NO: 45 and humanized light chain variable domains derived from SEQ ID NO: 46, which respectively form binding sites for human α-synuclein.

54. The method according to claim 27 or 31, wherein the bispecific antibody comprises a pair of heavy chain variable domains of SEQ ID NO: 31 and light chain variable domains of SEQ ID NO: 32, which form a binding site for human transferrin receptor, and a humanized heavy chain variable domain derived from SEQ ID NO: 47 and a humanized light chain variable domain derived from SEQ ID NO: 48, which respectively form binding sites for human α-synuclein.

55. The method according to claim 28 or 32, wherein the bispecific antibody comprises a pair of heavy chain variable domains of SEQ ID NO: 31 and light chain variable domains of SEQ ID NO: 32, which form a binding site for human transferrin receptor, and a humanized heavy chain variable domain derived from SEQ ID NO: 47 and a humanized light chain variable domain derived from SEQ ID NO: 48, which respectively form binding sites for human α-synuclein.

56. The method according to claim 29 or 33, wherein the bispecific antibody comprises a pair of heavy chain variable domains of SEQ ID NO: 31 and light chain variable domains of SEQ ID NO: 32 forming a binding site for human transferrin receptor, and a humanized heavy chain variable domain derived from SEQ ID NO: 47 and a humanized light chain variable domain derived from SEQ ID NO: 48 forming a binding site for human α-synuclein, respectively.

57. The method according to claim 27 or 31, wherein the bispecific antibody comprises a pair of heavy chain variable domains of SEQ ID NO: 31 and light chain variable domains of SEQ ID NO: 32 forming a binding site for human transferrin receptor, and two pairs of humanized heavy chain variable domains derived from SEQ ID NO: 49 and humanized light chain variable domains derived from SEQ ID NO: 50 forming binding sites for human α-synuclein, respectively.

58. The method according to claim 28 or 32, wherein the bispecific antibody comprises a pair of heavy chain variable domains of SEQ ID NO: 31 and light chain variable domains of SEQ ID NO: 32, forming a binding site for human transferrin receptor, and two pairs of humanized heavy chain variable domains derived from SEQ ID NO: 49 and humanized light chain variable domains derived from SEQ ID NO: 50, respectively forming binding sites for human α-synuclein.

59. The method according to claim 29 or 33, wherein the bispecific antibody comprises a pair of heavy chain variable domains of SEQ ID NO: 31 and light chain variable domains of SEQ ID NO: 32 forming a binding site for human transferrin receptor, and two pairs of humanized heavy chain variable domains derived from SEQ ID NO: 49 and humanized light chain variable domains derived from SEQ ID NO: 50 forming binding sites for human α-synuclein, respectively.

60. The method according to claim 27 or 31, wherein the bispecific antibody comprises a pair of heavy chain variable domains of SEQ ID NO: 31 and light chain variable domains of SEQ ID NO: 32 forming a binding site for human transferrin receptor, and two pairs of humanized heavy chain variable domains derived from SEQ ID NO: 51 and humanized light chain variable domains derived from SEQ ID NO: 52 forming binding sites for human α-synuclein, respectively.

61. The method according to claim 28 or 32, wherein the bispecific antibody comprises a pair of heavy chain variable domains of SEQ ID NO: 31 and light chain variable domains of SEQ ID NO: 32 forming a binding site for human transferrin receptor, and two pairs of humanized heavy chain variable domains derived from SEQ ID NO: 51 and humanized light chain variable domains derived from SEQ ID NO: 52 forming binding sites for human α-synuclein, respectively.

62. The method according to claim 29 or 33, wherein the bispecific antibody comprises a pair of heavy chain variable domains of SEQ ID NO: 31 and light chain variable domains of SEQ ID NO: 32, which form a binding site for human transferrin receptor, and two pairs of humanized heavy chain variable domains derived from SEQ ID NO: 51 and humanized light chain variable domains derived from SEQ ID NO: 52, which respectively form binding sites for human α-synuclein.

63. The method according to claim 27 or 31, wherein the bispecific antibody comprises a pair of heavy chain variable domains of SEQ ID NO: 31 and light chain variable domains of SEQ ID NO: 32, which form a binding site for human transferrin receptor, and two pairs of humanized heavy chain variable domains derived from SEQ ID NO: 53 and humanized light chain variable domains derived from SEQ ID NO: 54, which respectively form binding sites for human α-synuclein.

64. The method according to claim 28 or 32, wherein the bispecific antibody comprises a pair of heavy chain variable domains of SEQ ID NO: 31 and light chain variable domains of SEQ ID NO: 32, which form a binding site for human transferrin receptor, and two pairs of humanized heavy chain variable domains derived from SEQ ID NO: 53 and humanized light chain variable domains derived from SEQ ID NO: 54, which respectively form binding sites for human α-synuclein.

65. The method according to claim 29 or 33, wherein the bispecific antibody comprises a pair of heavy chain variable domains of SEQ ID NO: 31 and light chain variable domains of SEQ ID NO: 32 forming a binding site for human transferrin receptor, and two pairs of humanized heavy chain variable domains derived from SEQ ID NO: 53 and humanized light chain variable domains derived from SEQ ID NO: 54 forming binding sites for human α-synuclein, respectively.

66. The method according to any one of claims 27-29, 31-33, 35-37, 38-40, 48-50, 51-53, 54-56, 57-59, 60-62 and 63-63, wherein the therapeutic antibody comprises an Fc region capable of performing effector functions.

67. The method of claim 66, wherein the therapeutic antibody comprises an Fc region capable of performing effector functions and specifically binds to human FcγR / can be specifically bound by human FcγR.

68. The method of claim 66, wherein the therapeutic antibody comprises an Fc region capable of inducing ADCC and performing effector functions.

69. The method according to any one of claims 27-29, 31-33, 35-37, 38-40, 48-50, 51-53, 54-56, 57-59, 60-62 and 63-63, wherein the first antibody heavy chain of the bispecific antibody and the second antibody heavy chain of the bispecific antibody form a heterodimer.

70. The method according to any one of claims 27-29, 31-33, 35-37, 38-40, 48-50, 51-53, 54-56, 57-59, 60-62 and 63-63, wherein the first antibody heavy chain and the second antibody heavy chain of the bispecific antibody contain mutations supporting heterodimer formation.

71. The method according to any one of claims 27-29, 31-33, 35-37, 38-40, 48-50, 51-53, 54-56, 57-59, 60-62, and 63-63, wherein... a) The antibody heavy chain is the full-length antibody heavy chain of the human IgG1 subclass. b) The full-length antibody heavy chain of the human IgG4 subclass. c) One antibody heavy chain is a full-length antibody heavy chain of human subclass IgG1 with mutant T366W and optionally S354C or Y349C, while the other antibody heavy chain is a full-length antibody heavy chain of human subclass IgG1 with mutant T366S, L368A, Y407V and optionally Y349C or S354C. d) Both antibody heavy chains are full-length human subclass IgG1 antibody heavy chains. One antibody heavy chain has mutations I253A, H310A, and H435A, as well as mutation T366W and optional S354C or Y349C. The corresponding other antibody heavy chain has mutations T366S, L368A, Y407V and optional Y349C or S354C. e) Both antibody heavy chains are full-length human subclass IgG1 antibody heavy chains. One antibody heavy chain has mutations M252Y, S254T, and T256E, as well as mutation T366W and optional S354C or Y349C. The corresponding other antibody heavy chain has mutations T366S, L368A, Y407V and optional Y349C or S354C. f) Both antibody heavy chains are human subclass IgG1 antibody heavy chains, one of which has mutations T307H and N434H, as well as mutations T366W and optional S354C or Y349C, while the other antibody heavy chain has mutations T366S, L368A, Y407V and optional Y349C or S354C.

72. The method according to any one of claims 27-29, 31-33, 35-37, 38-40, 48-50, 51-53, 54-56, 57-59, 60-62, and 63-63, wherein... a) The antibody heavy chain is the antibody heavy chain of human subtype IgG1. b) The antibody heavy chain is the antibody heavy chain of human subtype IgG4. c) One antibody heavy chain is a human subclass IgG1 antibody heavy chain with the mutant T366W and optionally S354C or Y349C, while the other antibody heavy chain is a human subclass IgG1 antibody heavy chain with the mutant T366S, L368A, Y407V and optionally Y349C or S354C. d) Both antibody heavy chains are human subclass IgG1 antibody heavy chains. One antibody heavy chain has mutations I253A, H310A, and H435A, as well as mutation T366W and optional S354C or Y349C, while the corresponding other antibody heavy chain has mutations T366S, L368A, Y407V and optional Y349C or S354C. e) Both antibody heavy chains are human subclass IgG1 antibody heavy chains. One antibody heavy chain has mutations M252Y, S254T, and T256E, as well as mutation T366W and optional S354C or Y349C. The corresponding other antibody heavy chain has mutations T366S, L368A, Y407V and optional Y349C or S354C. f) Both antibody heavy chains are human subclass IgG1 antibody heavy chains. One antibody heavy chain has mutations T307H and N434H, as well as mutations T366W and optional S354C or Y349C, while the corresponding other antibody heavy chain has mutations T366S, L368A, Y407V and optional Y349C or S354C. It may or may not contain a C-terminal lysine or glycine-lysine dipeptide.

73. The method according to any one of claims 1 to 6, wherein the experimental animal is selected from mice, rats, rabbits, dogs, sheep, apes, and monkeys.

74. The method according to any one of claims 1 to 6, wherein the experimental animal is a non-human experimental animal weighing more than 100 g and less than 15 kg.

75. The method according to any one of claims 1 to 6, wherein the experimental animal is a cynomolgus monkey.

76. The method according to any one of claims 1 to 6, wherein the inert reference antibody is a human lineage antibody.

77. The method according to any one of claims 1 to 6, wherein the inert reference antibody is DP47GS.

78. The method of claim 77, wherein the inert reference antibody comprises the heavy chain variable domain of SEQ ID NO:67 and the light chain variable domain of SEQ ID NO:

68.

79. The method of claim 77, wherein the inert reference antibody comprises the heavy chain of SEQ ID NO:69 and the light chain of SEQ ID NO:

70.

80. The method according to any one of claims 1 to 6, wherein the inert reference antibody does not cross the barrier in a detectable amount within 15 minutes after its administration.

81. The method of claim 80, wherein the inert reference antibody does not cross the barrier in a detectable amount within 10 minutes after its administration.

82. The method according to any one of claims 1 to 6, wherein the inert reference antibody is applied about 5 minutes before the tissue sample is collected.

83. The method according to any one of claims 1 to 6, wherein the tissue is perfused with an aqueous solution directly after the blood sample is collected and before the tissue sample is collected.

84. The method according to any one of claims 1 to 6, wherein the concentration is determined by bridge ELISA.

85. The method according to any one of claims 27-29, 31-33, 35-37, 38-40, 48-50, 51-53, 54-56, 57-59, 60-62, 63-63 and 71-72, wherein the experimental animal is selected from mice, rats, rabbits, dogs, sheep, apes and monkeys.

86. The method according to any one of claims 27-29, 31-33, 35-37, 38-40, 48-50, 51-53, 54-56, 57-59, 60-62, 63-63 and 71-72, wherein the experimental animal is a non-human experimental animal weighing more than 100 g and less than 15 kg.

87. The method according to any one of claims 27-29, 31-33, 35-37, 38-40, 48-50, 51-53, 54-56, 57-59, 60-62, 63-63 and 71-72, wherein the experimental animal is a cynomolgus monkey.

88. The method according to any one of claims 27-29, 31-33, 35-37, 38-40, 48-50, 51-53, 54-56, 57-59, 60-62, 63-63 and 71-72, wherein the inert reference antibody is a human lineage antibody.

89. The method according to any one of claims 27-29, 31-33, 35-37, 38-40, 48-50, 51-53, 54-56, 57-59, 60-62, 63-63 and 71-72, wherein the inert reference antibody is DP47GS.

90. The method according to claims 27-29, 31-33, 35-37, 38-40, 48-50, 51-53, 54-56, 57-59, 60-62, 63-63 and 71-72, wherein the inert reference antibody comprises the heavy chain variable domain of SEQ ID NO:67 and the light chain variable domain of SEQ ID NO:

68.

91. The method according to claims 27-29, 31-33, 35-37, 38-40, 48-50, 51-53, 54-56, 57-59, 60-62, 63-63 and 71-72, wherein the inert reference antibody comprises the heavy chain of SEQ ID NO:69 and the light chain of SEQ ID NO:

70.

92. The method according to any one of claims 27-29, 31-33, 35-37, 38-40, 48-50, 51-53, 54-56, 57-59, 60-62, 63-63 and 71-72, wherein the inert reference antibody does not cross the barrier in a detectable amount within 15 minutes after its administration.

93. The method according to claims 27-29, 31-33, 35-37, 38-40, 48-50, 51-53, 54-56, 57-59, 60-62, 63-63 and 71-72, wherein the inert reference antibody does not cross the barrier in a detectable amount within 10 minutes after its administration.

94. The method according to any one of claims 27-29, 31-33, 35-37, 38-40, 48-50, 51-53, 54-56, 57-59, 60-62, 63-63 and 71-72, wherein the inert antibody is applied about 5 minutes before collecting the tissue sample.

95. The method according to any one of claims 27-29, 31-33, 35-37, 38-40, 48-50, 51-53, 54-56, 57-59, 60-62, 63-63 and 71-72, wherein the tissue is perfused with an aqueous solution directly after the blood sample is collected and before the tissue sample is collected.

96. The method according to any one of claims 27-29, 31-33, 35-37, 38-40, 48-50, 51-53, 54-56, 57-59, 60-62, 63-63 and 71-72, wherein the concentration is determined by a bridge ELISA.

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