Method for determining the amount of therapeutic antibody in the brain

Abstract

Provided herein 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 interference from residual blood in the tissue sample of the experimental animal used to determine the concentration of the therapeutic antibody in said tissue is reduced, and the concentration of the therapeutic antibody in the tissue of the experimental animal is determined according to the following formula: TIFF2023508596000021.tif18170 (In the formula, C tmAb、組織、det. = C, obtained by determining the concentration of therapeutic antibody in tissue samples from experimental animals tmAb、血漿、det. = obtained by determining the concentration of therapeutic antibody in a blood sample of an experimental animal immediately before taking a tissue sample, C refmAb、組織、det. = C, obtained by determining the concentration of inactive reference antibody in tissue samples from experimental animals refmAb、血漿、det. = obtained by determining the concentration of the inactive reference antibody in a blood sample of the experimental animal immediately before taking the tissue sample, C 組織、サンプル A method is reported in which the inactive reference antibody is administered 2 to 10 minutes before obtaining tissue and blood samples, whereby the inactive reference antibody does not penetrate into the tissue and the inactive reference antibody is calculated by (obtained by determining the tissue concentration in the tissue sample).

Description

Technical Field

[0001] The present invention belongs to the field of immunoassay. More specifically, the present specification reports a method for determining the amount of a therapeutic antibody in brain tissue, more specifically, the amount of a therapeutic antibody transported from blood to the brain through the blood-brain barrier.

Background Art

[0002] Background Analysis of therapeutic monoclonal antibodies (tmAbs) in samples of in vitro or in vivo origin requires respective assays.

[0003] Determination of the amount of a therapeutic antibody is generally performed by determining the amount of the therapeutic antibody in a sample. Thus, for example, immunoassays such as ELISA, RIA, protein blot (western blot) assays, etc. can be used.

[0004] The role of antibodies and their receptors in the protection against ordered protein assemblies in neurodegeneration was reviewed by Katsinelos et al. (Front. Immunol. 10 (2019) A1139). Katsinelos et al. reviewed that the IgG level is maintained at about 10 mg / ml in human serum. The brain is separated from serum by the blood-brain barrier (BBB), which does not permit the passage of large macromolecules including IgG, and is bathed in cerebrospinal fluid (CSF), which is produced after filtration of blood and transport of ions across the choroid plexus. Thus, the resulting concentration of IgG in CSF is about 500 - 1,000 times lower than in serum.

[0005] Hanzatian et al. (mAbs 10(2018)765-777) reported that therapeutic monoclonal antibodies and endogenous IgG antibodies have restricted uptake into the central nervous system (CNS) due to the blood-brain barrier (BBB) that regulates and controls the selective and specific transport of both exogenous and endogenous substances into the brain. The use of natural transport mechanisms such as receptor-mediated transcytosis (RMT) to deliver antibody therapeutics to the brain has been studied in rodents and monkeys. After systemic administration of each DVD-Ig, Hanzatian et al. used two independent methods in parallel to observe specific uptake into the brain: a highly sensitive quantitative assay based on electrochemiluminescence and a semi-quantitative immunohistochemical technique were used for brain concentration determination and biodistribution / localization in the brain, respectively. Regardless of the CNS target or the selected systemic administration route, enhanced brain uptake and retention were observed for all TfR1 DVD-Ig proteins. To prepare brain samples for analysis, the C57BL / 6N mice used were perfused transcardially for 10 minutes at a rate of 2 ml / min with cold Dulbecco's phosphate-buffered saline (PBS) containing heparin via a programmable peristaltic pump.

[0006] An equivalent approach was used by Zuchero et al. (Neuron 89(2016)70-82; wild-type mice, which were IV injected with the target antibody followed by collection of the whole blood and PBS perfusion) and Janowicz et al. (Nature Sci.Rep.9(2019)9255; P301L tau transgenic pR5 mice, to which Alexa-647-labeled IgG, Fab or scFv had been administered by retro-orbital injection, were perfused following treatment to remove the antibody from their vasculature).

[0007] In International Publication No. WO 2018 / 152359, the target binding of chimeric IgG anti-Tau antibodies clone 1C7 and 1A1 was evaluated using mice overexpressing human Tau derived from the PS19 strain. Accordingly, control IgG, chimeric IgG clone 1C7, or chimeric IgG clone 1A1 was injected into mice either i.v. (at 35 mg / kg) or i.p. (at 50 mg / kg). Two or seven days after injection, cerebrospinal fluid (CSF) was collected via a cisterna magna, visually inspected for potential blood contamination, and after perfusion of the heart with ice-cold PBS, brain tissue was removed and snap-frozen.

[0008] Ayabe, M. et al. reported that anti-human interleukin-6 receptor (hIL-6R) antibody or control antibody was intravenously administered to hIL-6R transgenic mice bearing cancer, and bovine serum albumin (BSA) was intravenously administered as a marker of the amount of residual blood in tissues. Lysate samples were processed by immunoprecipitation using anti-BSA antibody and protein A magnetic beads followed by trypsin digestion. Each surrogate peptide was simultaneously analyzed by LC / ESI-MS / MS. Corrected tissue concentrations were calculated.

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

[0010] Shah et al. reported the antibody bio distribution coefficient, particularly the estimated concentration of monoclonal antibodies in tissues based on plasma concentrations in several preclinical species and humans (MABS, 5 (2013) 297-305).

[0011] Lavezzi et al. reported on the MPBPK-TMDD model of mAb, particularly alternative models, comparison, and identifiability issues (J. Pharmacokin. Pharmcodyn. 45 (2018) 787-802).

Summary of the Invention

[0012] Summary of the Invention This specification reports a method for determining the amount of a therapeutic antibody transported from the blood to the brain through the blood-brain barrier of an experimental animal. This amount is preferably determined in a brain lysate sample. The gist of the present invention lies in further applying an inactivated antibody that is not transported through the blood-brain barrier immediately before obtaining a brain sample for which the amount of the therapeutic antibody transported through the blood-brain barrier must be determined. By applying the inactivated antibody, a corrected value of the therapeutic antibody present in the residual blood in the brain sample is obtained. The amount derived from this residual blood is used to correct the amount determined for the antibody not located in the brain. Without correction, the total amount of the therapeutic antibody in the sample, i.e., the amount transported through the blood-brain barrier to the brain and the amount in the residual blood in the sample, is determined. Since only about 0.1% of the antibody in the blood passes through the blood-brain barrier, the amount of the therapeutic antibody in the residual blood cannot be ignored. Therefore, the concentration of the therapeutic antibody in the blood is at least two orders of magnitude, and at most three orders of magnitude higher than the concentration of the therapeutic antibody in the brain. As a result, the obtained results are too high if not corrected by the method according to the present invention.

[0013] The present invention is based, at least in part, on the finding that in order to robustly and accurately determine the amount of a therapeutic antibody transported into the brain through the blood-brain barrier in a brain lysate, correction, i.e., reduction, by the amount of the therapeutic antibody in the residual blood in the brain lysate sample must be performed.

[0014] 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 applying a correction antibody immediately before the brain sample is taken. It has been found that it is particularly advantageous to use an antibody that does not specifically bind to any target of the experimental animal from which the brain sample is obtained, most preferably a human germline antibody, as a reference antibody.

[0015] One aspect of the present invention is a method / assay for determining the concentration of a therapeutic antibody in the tissue of an experimental animal, wherein the tissue has a barrier to the blood circulation of the animal, the therapeutic antibody has been administered to the experimental animal, and 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, and the method comprises the following: i) determining the concentration of the therapeutic antibody in a blood sample of the experimental animal; ii) determining the concentration of the therapeutic antibody in a tissue sample of the experimental animal; iii) determining the concentration of an inert reference antibody in a blood sample of the experimental animal; iv) determining the concentration of the inert reference antibody in a tissue sample of the experimental animal; v) determining the tissue concentration in the tissue sample; and the following formula:

Number

[0016] Another embodiment of the same aspect is a method for determining the concentration of a therapeutic antibody in the tissue of an experimental animal to which the therapeutic antibody has been administered, wherein interference from residual blood in the tissue sample of the experimental animal used to determine the concentration of the therapeutic antibody in the tissue is reduced, The following formula: [Number] (wherein, C tmAb、組織、det. = Obtained by determining the concentration of the therapeutic antibody in the tissue sample of the experimental animal, C tmAb、血漿、det. = Obtained by determining the concentration of the therapeutic antibody in the blood sample of the experimental animal, C refmAb、組織、det. = Obtained by determining the concentration of the inert reference antibody in the tissue sample of the experimental animal, C refmAb、血漿、det. = Obtained by determining the concentration of the inert reference antibody in the blood sample of the experimental animal, C 組織、サンプル = Obtained by determining the tissue concentration in the tissue sample) The step of determining the concentration of the therapeutic antibody in the tissue of the experimental animal using includes - The inert reference antibody does not penetrate into the tissue, - The inert reference antibody is administered 2 to 10 minutes before obtaining the tissue sample.

[0017] The following are all individual embodiments of each and every aspect of the present invention. Accordingly, for any individual aspect according to the present invention, all possible substitutions of the embodiments are disclosed.

[0018] 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.

[0019] In one embodiment, the tissue is brain tissue and the therapeutic antibody can cross the blood-brain barrier, or the tissue is eye tissue and the therapeutic antibody can cross the blood-eye barrier, either one.

[0020] One aspect of the present invention is a method / assay for determining the concentration of a therapeutic antibody in the brain tissue or a brain tissue sample of an experimental animal, wherein the brain tissue has a barrier to the blood circulation of the animal, the therapeutic antibody has been administered to the experimental animal, and interference from residual blood in the brain tissue sample of the experimental animal used to determine the concentration of the therapeutic antibody in the brain tissue is reduced, and the method comprises the following: i) determining the concentration of the therapeutic antibody in a blood sample of the experimental animal; ii) determining the concentration of the therapeutic antibody in a brain tissue sample of the experimental animal; iii) determining the concentration of an inert reference antibody in a blood sample of the experimental animal; iv) determining the concentration of the inert reference antibody in a brain tissue sample of the experimental animal; v) determining the brain tissue concentration in the tissue sample; and the following formula:

Number

[0021] In the same manner in another expression, it is a method for determining the concentration of a therapeutic antibody in the brain tissue or a brain tissue sample of an experimental animal to which the therapeutic antibody has been administered, and the interference from residual blood in the brain tissue sample of the experimental animal used for determining the concentration of the therapeutic antibody in the brain tissue is reduced. The concentration of the therapeutic antibody in the brain tissue of the experimental animal is given by the following formula:

Number

[0022] In one embodiment, the therapeutic antibody is a bispecific antibody.

[0023] In one embodiment, the therapeutic antibody specifically binds to the human transferrin receptor and the brain target.

[0024] In one embodiment, the brain target is human CD20 or human A beta or human alpha-synuclein or human tau or human glucocerebrosidase or human ring-1 or human huntingtin.

[0025] In one embodiment, the experimental animal is selected from mice, rats, rabbits, dogs, sheep, apes and monkeys.

[0026] In one embodiment, the experimental animal is a non-human experimental animal with a body weight of more than 100 g and less than 15 kg.

[0027] In one embodiment, the experimental animal is a cynomolgus monkey.

[0028] In one embodiment, the inert reference antibody is a human germline antibody.

[0029] 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.

[0030] In one embodiment, the inert reference antibody does not pass through the barrier in a detectable amount within 15 minutes after its application.

[0031] In one embodiment, the inert reference antibody does not pass through the barrier in a detectable amount within 10 minutes after its application.

[0032] In one embodiment, the inert antibody is administered 5 to 10 minutes before collecting the tissue sample.

[0033] In one embodiment, the tissue is perfused with an aqueous solution immediately after collecting the blood sample and before collecting the tissue sample.

[0034] In one embodiment, the determination of the concentration is by cross-linking ELISA.

BEST MODE FOR CARRYING OUT THE INVENTION

[0035] Detailed Description of Embodiments of the Invention This specification reports a method for determining the amount of a therapeutic antibody transported from the blood to the brain through the blood-brain barrier of an experimental animal. This amount is preferably determined in a brain lysate sample. The gist of the present invention lies in applying an inert antibody that is not transported through the blood-brain barrier immediately before obtaining a brain sample for which the amount of the therapeutic antibody transported through the blood-brain barrier must be determined. By applying the inert antibody, a correction value for the therapeutic antibody present in the residual blood in the brain sample is obtained. The amount derived from this residual blood is used to correct the amount determined for the antibody not located in the brain. The determination without correction determines the total amount of the therapeutic antibody in the sample, i.e., the amount transported through the blood-brain barrier to the brain and the amount in the residual blood in the sample. Since only about 0.1% of the antibody in the blood passes through the blood-brain barrier, the amount of the therapeutic antibody in the residual blood cannot be ignored. Therefore, the concentration of the therapeutic antibody in the blood is at least two orders of magnitude, and at most three orders of magnitude higher than the concentration of the therapeutic antibody in the brain. As a result, the obtained results are too high if not corrected by the method according to the present invention.

[0036] The present invention is based, at least in part, on the finding that in order to robustly and accurately determine the amount of a therapeutic antibody transported into the brain through the blood-brain barrier in a brain lysate, correction, i.e., reduction, by the amount of the therapeutic antibody in the residual blood in the brain lysate sample must be performed.

[0037] 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 applying a correction antibody immediately before the brain sample is taken. It has been found to be particularly advantageous to use an antibody that does not specifically bind to any target of the experimental animal from which the brain sample is obtained, most preferably a human germline antibody, as a reference antibody.

[0038] One aspect of the present invention is a method / assay for determining the concentration of a therapeutic antibody in the tissue of an experimental animal, wherein the tissue has a barrier to the blood circulation of the animal, the therapeutic antibody has been administered to the experimental animal, and interference from residual blood in the tissue sample of the experimental animal used to determine the concentration of the therapeutic antibody in the tissue is reduced. The method is as follows: i) determining the concentration of the therapeutic antibody in a blood serum sample of the experimental animal; ii) determining the concentration of the therapeutic antibody in a tissue sample of the experimental animal; iii) determining the concentration of an inert reference antibody in a blood serum sample of the experimental animal; iv) determining the concentration of the inert reference antibody in a tissue sample of the experimental animal; v) determining the tissue concentration in the tissue sample, and the following formula:

Number

[0039] Definition The "knob-into-hole" dimerization module and its use in antibody engineering are described in Carter P.; Ridgway J.B.B.; Presta L.G.: Immunotechnology, Volume 2, Number 1, February 1996, pp. 73-73(1).

[0040] General information related to the nucleotide sequences of the light and heavy chains of human immunoglobulins is given in Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0041] As used herein, the amino acid positions of all constant regions and domains of the heavy and light chains are numbered according to the Kabat numbering system as described in Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), and are referred to herein as "Kabat numbering." Specifically, the Kabat numbering system of Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) (see pages 647-660) is used for the kappa and lambda isotype light chain constant domains CL, and the Kabat EU index numbering system (see pages 661-723) is used for the heavy chain constant domains (CH1, hinge, CH2 and CH3, which is further clarified herein by referring to this case as "numbering according to the Kabat EU index").

[0042] The term "about" means a range of ±20% of the numerical value that follows. In one embodiment, the term "about" means a range of ±10% of the numerical value that follows. In one embodiment, the term "about" means a range of ±5% of the numerical value that follows.

[0043] The term "antibody-dependent cell-mediated cytotoxicity (ADCC)" is a function mediated by Fc receptor binding and refers to the lysis of target cells by the antibodies reported herein in the presence of effector cells. In one embodiment, ADCC is measured by treating a preparation of CD19-expressing erythrocytes (e.g., K562 cells expressing recombinant human CD19) with an antibody comprising a fusion polypeptide reported herein in the presence of effector cells such as freshly isolated PBMCs (peripheral blood mononuclear cells) or effector cells purified from buffy coats, such as monocytes or NK (natural killer) cells. The target cells are labeled with 51Cr and then incubated with the antibody. The labeled cells are incubated with the effector cells, and the supernatant is analyzed for released 51Cr. Controls include the incubation of target endothelial cells with effector cells, but without the antibody. The ability of an antibody to induce the initial steps mediating ADCC is investigated by measuring the binding of the antibody to cells expressing Fcγ receptors, such as cells recombinantly expressing FcγRI and / or FcγRIIA or NK cells (which essentially express FcγRIIIA). In a preferred embodiment, the binding of the antibody to FcγR on NK cells is measured.

[0044] The term "amplifier" represents an entity or process that enhances a signal in a detection method such as ELISA (e.g., an enzyme amplifier used in ELISA).

[0045] The terms "anti-human A-beta antibody" and "antibody that specifically binds to human A-beta" refer to antibodies that can bind to human A-beta peptide with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent when targeting the A-beta peptide.

[0046] Human A-beta has several naturally occurring forms, and it should be noted that the human forms are called Aβ39, Aβ40, Aβ41, Aβ42, and Aβ43. The most important form, Aβ42, has the amino acid sequence of SEQ ID NO: 01. In Aβ41, Aβ40, and Aβ39, the C-terminal amino acids A, IA, and VIA are missing respectively. In the Aβ43 form, an additional threonine residue is included at the C-terminus of SEQ ID NO: 01 (33106).

[0047] Accordingly, this term also encompasses antibodies that bind to shortened fragments of the human A-β polypeptide.

[0048] The term "antibody" as used 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).

[0049] Antibodies generally comprise two so-called light chain polypeptides (light chains) and two so-called heavy chain polypeptides (heavy chains). Each of the heavy and light chain polypeptides contains a variable domain (variable region) (generally the amino-terminal portion of the polypeptide chain) that can interact with an antigen and contains a binding region. Each of the heavy and light chain polypeptides contains a constant region (generally the carboxyl-terminal portion). The constant region of the heavy chain mediates the binding of the antibody to cells having an Fc gamma receptor (FcγR), such as phagocytes, or to cells having a neonatal Fc receptor (FcRn), also known as the Brambell receptor. The constant region of the heavy chain also mediates binding to several factors, including factors of the classical complement system such as component (C1q). The constant domain of the antibody heavy chain contains a CH1 domain, a CH2 domain, and a CH3 domain, whereas the light chain contains only one constant domain CL, which can be of the kappa isotype or the lambda isotype.

[0050] The variable domain of an immunoglobulin light or heavy chain contains distinct segments, namely four framework regions (FRs) and three hypervariable regions (HVRs).

[0051] “Antibody fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds an 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 (dAb); and multispecific antibodies formed from antibody fragments. For a review of certain antibody fragments, see Holliger and Hudson, Nature Biotechnology 23:1126-1136 (2005).

[0052] The "blood-brain barrier" or "BBB" refers to a physiological barrier between the peripheral circulation and the brain and spinal cord, formed by tight junctions within the membranes of brain capillary endothelial cells, which forms a tight barrier that restricts the transport of molecules into the brain, even very small molecules such as urea (60 Daltons). The BBB in the brain, the blood-spinal cord barrier in the spinal cord, and the blood-retinal barrier in the retina are continuous capillary barriers within the CNS and are collectively referred to herein as the blood-brain barrier or BBB. The BBB also encompasses the blood-CSF barrier (choroid plexus), which is composed of epithelial cells rather than capillary endothelial cells.

[0053] A "blood-brain barrier receptor" (abbreviated herein as "BBBR") is an extracellular membrane-bound receptor protein expressed on brain endothelial cells that can transport molecules across the BBB or is used to transport exogenously administered molecules. Examples of BBBRs herein include, but are not limited to, the transferrin receptor (TfR), insulin receptor, insulin-like growth factor receptor (IGF-R), 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).

[0054] The term "brain effector entity" refers to a molecule that is transported to the brain across the BBB. Effector entities typically have a characteristic therapeutic activity that is desired to be delivered to the brain. Effector entities include neurotoxic and cytotoxic agents, such as polypeptides and antibodies, particularly monoclonal antibodies or fragments thereof directed against brain targets.

[0055] The term "capture antibody" refers to an antibody used in a sandwich ELISA format to bind (i.e., capture) a target substance present in a sample for detection. A secondary antibody (i.e., a detection antibody) then binds to the captured target, enabling detection of the antibody-target-antibody complex (forming an "antibody-target-antibody" "sandwich").

[0056] "Central nervous system" or "CNS" refers to the complex of nerve tissues that control body functions and includes the brain and spinal cord.

[0057] 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 (A beta), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, 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.

[0058] A "conjugate" is a fusion protein of the invention conjugated to one or more heterologous molecules including, but not limited to, a label, a neurotoxic agent, or a cytotoxic agent.

[0059] The term "detection antibody" refers to an antibody having means for visualization or quantification. Such means are typically enzymes (which catalyze a colorimetric or fluorescent reaction product upon addition of an appropriate substrate), such as horseradish peroxidase, urease, alkaline phosphatase, glucoamylase, and β-galactosidase. In some embodiments, the detection antibody is specific for 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 is detected and / or quantified using this label.

[0060] The term "detection reagent" refers to a reagent that enables the detection and / or quantification of an antibody bound to an antigen. In some embodiments, the detection reagent is a colorimetric substrate for an enzyme conjugated to an antibody. Addition of an appropriate substrate to the antibody-enzyme conjugate results in the generation of a colorimetric or fluorescence measurement signal (e.g., after binding of the conjugated antibody to the antigen of interest). This definition also encompasses the use of biotin and avidin-based compounds (including, but not limited to, neutravidin and streptavidin) as part of the detection system.

[0061] As used herein, the term "immediately following" refers to the time span between the collection of a first sample and the collection of a second sample, which includes only the time for changing the sampling device and the actual time for collecting the sample. In one embodiment, the term immediately following refers to a period of 5 minutes or less, in a further embodiment 3 minutes or less, and in one preferred embodiment 2 minutes or less.

[0062] "Effector function" refers to biological activities resulting from the Fc region of an antibody, which vary depending on the class of the antibody. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0063] Fc receptor-binding-dependent effector functions can be mediated by the interaction of the Fc region of an antibody with a dedicated cell surface receptor in hematopoietic cells, the Fc receptor (FcR). Fc receptors belong to the immunoglobulin superfamily and have been shown to mediate both the removal of antibody-coated pathogens by phagocytosis of immune complexes and the lysis of erythrocytes and other cell targets (e.g., tumor cells) coated with the corresponding antibody by antibody-dependent cell cytotoxicity (ADCC) (see, for example, 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 called FcγRs. Fc receptor binding is described, for example, in Ravetch, J.V. and Kinet, J.P., Annu. Rev. 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.

[0064] Cross-linking of receptors for the Fc region of IgG antibodies (FcγRs) induces a variety of effector functions, including phagocytosis, antibody-dependent cell cytotoxicity, and the release of inflammatory mediators, as well as the control of immune complex clearance and antibody production. In humans, three classes of FcγRs have been characterized and are as follows. - FcγRI (CD64) binds monomeric IgG with high affinity and is expressed on macrophages, monocytes, neutrophils, and eosinophils. Modification within the Fc region at at least one of amino acid residues E233 - G236, P238, D265, N297, A327, and P329 (numbering according to Kabat's EU index) results in reduced binding to FcγRI. The IgG2 residues at positions 233 - 236, when substituted with IgG1 and IgG4, reduced binding to FcγRI by 103 - fold and abrogated the human mononuclear cell response to antibody - sensitized erythrocytes (Armour, K.L., et al., Eur. J. Immunol. 29 (1999) 2613 - 2624). - FcγRII (CD32) binds aggregated IgG with moderate 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 (e.g., macrophages, monocytes, neutrophils) and appears to be able to activate the killing process. FcγRIIB is thought to play a role in the inhibitory process and is found on B - cells, macrophages, as well as mast cells and eosinophils. On B - cells, FcγRIIB appears to have the function of further suppressing immunoglobulin production and, for example, isotype switching to the IgE class. On macrophages, FcγRIIB plays a role in inhibiting phagocytosis, as mediated by FcγRIIA. On eosinophils and mast cells, the B form may assist in suppressing the activation of these cells by the binding of IgE to its individual receptor. A decrease in binding to FcγRIIA has been found, for example, for antibodies containing an IgG Fc region with a mutation at at least one of amino acid residues E233 - G236, P238, D265, N297, A327, P329, D270, Q295, A327, R292, and K414 (numbering according to Kabat's EU index). - FcγRIII (CD16) binds to IgG with moderate to low affinity and exists as 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. Reduction in binding to FcγRIIIA is observed for antibodies that contain an IgG Fc region with a mutation in at least one of, for example, 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 the Kabat EU index).

[0065] Methods for mapping the binding site on human IgG1 for Fc receptors, measuring the mutations sites described above, and binding to FcγRI and FcγRIIA are described in Shields, R.L., et al. J. Biol. Chem. 276 (2001) 6591 - 6604.

[0066] A "effective amount" of a drug, e.g., a pharmaceutical preparation, refers to an amount effective at the dosage and for the period of time necessary to achieve the desired therapeutic or prophylactic result.

[0067] The term "ELISA" refers to enzyme-linked immunosorbent assay. A variety of ELISA formats and uses 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, Ch. 11, John Wiley and Sons, Inc., New York (1994)).

[0068] One specific ELISA format is the so-called "direct ELISA." In this ELISA format, a target present in a sample, such as a polypeptide, is detected. In direct ELISA, a sample containing the target is contacted with a solid phase, such as a fixed or immobilized support (e.g., a microtiter plate well). If the target is present in the sample, it is immobilized on the solid phase and then directly detected 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.

[0069] Another specific 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). Subsequently, an antigen-specific antibody (or antigen) is added, followed by the addition of a detection antibody specific for the antibody that specifically binds to the antigen. This detection antibody can be a "species-specific" antibody (e.g., goat anti-rabbit antibody).

[0070] Another specific ELISA format is the so-called "sandwich ELISA". In this format, the antigen is immobilized on a solid phase (e.g., a microtiter plate well) via capture by an antibody that specifically binds to the antigen (i.e., the capture antibody), which is immobilized on the solid phase (either via covalent bonding or specific binding pairs). Generally, after adding a sample containing the antigen to the solid phase, it is washed. If the antigen of interest is present in the sample, the antigen of interest binds to the solid phase by the capture antibody.

[0071] The above ELISA formats can be combined. The sandwich ELISA can be a "direct sandwich ELISA", where the captured antigen is detected directly by using an enzyme-conjugated antibody against the antigen. The sandwich ELISA can also be an "indirect sandwich ELISA", where the captured antigen is detected indirectly by using an antibody against the antigen, and then detected by another enzyme-conjugated antibody that binds either directly or via a label conjugated to the antigen-specific antibody. A third antibody is detected using a reporter reagent.

[0072] As used herein, the term "Fc receptor" refers to an activating receptor characterized by the presence of a cytoplasmic ITAM sequence associated with the receptor (see, for example, Ravetch, J.V. and Bolland, S., Annu. Rev. Immunol. 19 (2001) 275-290). Such receptors are FcγRI, FcγRIIA, and FcγRIIIA. The term "no FcγR binding" means that at an antibody concentration of 10 μg / ml, the binding of the antibodies reported herein to NK cells is 10% or less of the binding observed for the anti-OX40L antibody LC.001 reported in International Publication No. WO 2006 / 029879.

[0073] IgG4 shows reduced FcR binding, while antibodies of other IgG subclasses show strong binding. However, Pro238, Asp265, Asp270, Asn297 (loss of Fc carbohydrate), Pro329 and 234, 235, 236 and 237Ile253, Ser254, Lys288, Thr307, Gln311, Asn434 and His435 are residues whose modification also results in reduced FcR binding (see 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 EP0307434). In one embodiment, the antibodies reported herein are of the IgG1 or IgG2 subclass and contain the mutations PVA236, GLPSS331 and / or L234A / L235A. In one embodiment, the antibodies described herein are of the IgG4 subclass and contain the mutation L235E. In one embodiment, the antibody further contains the mutation S228P.

[0074] The term "Fc region" as used herein is used to define 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, the human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system (also called the Kabat 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.

[0075] The antibodies described herein include, as the Fc region, in one embodiment, an Fc region derived from a human origin. In one embodiment, the Fc region includes all parts of the human constant region. The Fc region of an antibody is directly involved in complement activation, C1q binding, C3 activation, and Fc receptor binding. The effect of an antibody on the complement system depends on specific conditions, but binding to C1q is caused by defined binding sites in the Fc region. Such binding sites are known in the prior art and are 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 European Patent No. 0307434. Such binding sites are, for example, L234, L235, D270, N297, E318, K320, K322, P331 and P329 (the numbering follows the Kabat EU index. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is the EU numbering system as described in Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242, also referred to as the EU index). Antibodies of subclasses IgG1, IgG2, and IgG3 usually exhibit complement activation, C1q binding, and C3 activation, whereas IgG4 does not activate the complement system, does not bind to C1q, and does not activate C3. "The Fc region of an antibody" is a term well known to those skilled in the art and is defined based on papain cleavage of the antibody.In one embodiment, the Fc region is a human Fc region. In one embodiment, the Fc region is of the human IgG4 subclass and includes the mutations S228P and / or L235E (numbering according to the Kabat EU index). In one embodiment, the Fc region is of the human IgG1 subclass and includes the mutations L234A and L235A (numbering is according to the Kabat EU index).

[0076] The terms "full-length antibody", "intact antibody", and "whole antibody" are used interchangeably herein and refer to an antibody having a structure substantially similar to the native antibody structure, i.e., an antibody comprising two light chains and two heavy chains.

[0077] A "human antibody" is an antibody produced by a human or human cell, or an antibody having an amino acid sequence corresponding to a non-human-derived antibody that utilizes a human antibody repertoire or other human antibody-encoding sequence. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues.

[0078] The term "in vitro" indicates either such an artificial environment or that a process or reaction is carried out within such an artificial environment.

[0079] The term "in vivo" represents the natural environment of a compound (e.g., an animal or a cell), or that a process or reaction is carried out within its natural environment.

[0080] The term "immunoassay" refers to any technique that utilizes specifically binding molecules such as antibodies to capture and / or detect a specific target for qualitative or quantitative analysis. Generally, an immunoassay is a method characterized by 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, on any solid surface, such as a membrane, a plastic plate, or any other solid surface.

[0081] The term "linker" refers to a chemical linker or single-chain peptide linker that covalently links the various entities of the blood-brain barrier shuttle module and / or fusion polypeptide and / or conjugate described herein. The linker, for example, links a brain effector entity to a monovalent binding entity. For example, when the monovalent binding entity includes a CH2-CH3 Ig entity and an scFab directed to a blood-brain barrier receptor, the linker conjugates the scFab to the C-terminus of the CH3-CH2 Ig entity. The linker (the first linker) that conjugates the brain effector entity to the monovalent binding entity and the linker (the second linker) that links the scFab to the C-terminus of the CH2-CH3 Ig domain may be the same or different.

[0082] A single-chain peptide linker containing 1 to 20 amino acid residues linked by peptide bonds may be used. In certain embodiments, the amino acids are selected from the 20 natural amino acids. In certain other embodiments, one or more of the amino acids are selected from glycine, alanine, proline, asparagine, glutamine, and lysine. In some embodiments, the linker is a chemical linker. In certain embodiments, the linker is a single-chain peptide linker having an amino acid sequence with a length of at least 25 amino acid residues, and in a preferred embodiment, a length of 32 to 50 amino acid residues. In one embodiment, the peptide linker is G = glycine, S = serine, (x = 3, n = 8, 9, or 10) or (x = 4 and n = 6, 7, or 8), and in one embodiment, x = 4, n = 6, or 7, and in a preferred embodiment, x = 4, n = 7, which is a (GxS)n linker. In one embodiment, the linker is (G4S)4 (SEQ ID NO: 02). In one embodiment, the linker is (G4S)6G2 (SEQ ID NO: 03).

[0083] Conjugation can be carried out using a variety of chemical linkers. For example, a monovalent binding entity or fusion polypeptide and a brain effector entity can be conjugated using various bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azide compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). The linker can be a "cleavable linker" that promotes the release of the effector entity upon delivery to the brain. For example, an acid-labile linker, a peptidase-sensitive linker, a photo-labile linker, a dimethyl linker or a disulfide-containing linker (Chari et al., Cancer Res. 52 (1992) 127-131; U.S. Patent No. 5,208,020) can be used.

[0084] The conjugate conjugation can be either direct or via a linker. In certain embodiments, the direct conjugation is by construction of a polypeptide fusion (i.e., a genetic fusion of two genes encoding a monovalent binding entity to a BBBR and an effector entity and expressed as a single polypeptide (chain)). In certain embodiments, the direct conjugation is by formation of a covalent bond between a reactive group in one of the two parts of a monovalent binding entity to a BBBR and a corresponding group or acceptor on the brain effector entity. In certain embodiments, the direct conjugation is by modifying (i.e., genetically modifying) one of the two molecules to be conjugated to contain a reactive group (non-limiting examples include a sulfhydryl group or a carboxyl group) that forms a covalent bond to the other molecule to be conjugated under appropriate conditions. As one non-limiting example, a molecule (i.e., an amino acid) having a desired reactive group (i.e., a cysteine residue) can be introduced, for example, into a disulfide bond formed by a monovalent binding entity to a BBBR antibody and a neurotherapeutic antibody. Methods for covalent conjugation of nucleic acids and proteins are also known in the art (i.e., photo-crosslinking, see, e.g., Zatsepin et al., Russ. Chem. Rev. 74 (2005) 77-95). The conjugation can also be carried out using various linkers.For example, a monovalent binding entity and an effector entity can be conjugated using various bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azide compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate) and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). A peptide linker consisting of 1 to 20 amino acid residues linked by peptide bonds can also be used. In certain such embodiments, the amino acid residues are selected from the 20 naturally occurring amino acids. In certain other such embodiments, one or more of the amino acid residues are selected from glycine, alanine, proline, asparagine, glutamine and lysine. The linker can be a "cleavable linker" that promotes the release of the effector entity upon delivery to the brain. For example, an acid-labile linker, a peptidase-sensitive linker, a photo-labile linker, a dimethyl linker or a disulfide-containing linker (Chari et al., Cancer Res. 52 (1992) 127-131; U.S. Patent No. 5,208,020) can be used.

[0085] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations or variant antibodies that may arise during the production of the monoclonal antibody preparation, and such variants are generally 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 characteristic of the antibody being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring that the antibody be produced by any particular method. For example, monoclonal antibodies according to the present invention can be made by a variety of techniques including, but not limited to, the hybridoma method, recombinant DNA methods, phage display methods, and methods that utilize transgenic animals that contain all or part of the human immunoglobulin loci, and such methods and other exemplary methods for making monoclonal antibodies described herein are included but not limited to.

[0086] The term "monovalent binding entity" refers to a molecule that can bind to BBBR specifically in a monovalent binding mode. The blood-brain shuttle module and / or conjugate described herein is characterized by the presence of a single unit of the monovalent binding entity, i.e., the blood-brain shuttle module and / or conjugate of the present invention contains exactly one unit of the monovalent binding entity. Examples of the monovalent binding entity include, but are not limited to, polypeptides, full-length antibodies, antibody fragments including Fab, Fab', Fv fragments, single-chain antibody molecules such as single-chain Fab, scFv. The monovalent binding entity can be a scaffold protein engineered using state-of-the-art techniques such as phage display or immunization, for example. 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 to a blood-brain barrier receptor. The scFab is coupled to the C-terminus of the CH2-CH3 Ig domain by a linker. In certain embodiments, the scFab targets the transferrin receptor.

[0087] The term "monovalent binding mode" refers to specific binding to BBBR in which the interaction between the monovalent binding entity and BBBR occurs via a single epitope. The monovalent binding mode prevents dimerization / multimerization of BBBR resulting from a single epitope interaction point. The monovalent binding pattern prevents changes in intracellular sorting of BBBR.

[0088] A "naked antibody" refers to an antibody that is not bound to a heterologous moiety (e.g., a cytotoxic moiety) or a radiolabel. The naked antibody may be present in a pharmaceutical composition.

[0089] The term "native antibody" refers to immunoglobulin molecules that exist natively with various structures. For example, a natural IgG antibody is a heterotetrameric glycoprotein of approximately 150,000 daltons composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From the N-terminus to the C-terminus, each heavy chain has a variable domain (VH), also called the variable heavy domain or heavy chain variable region, followed by three constant heavy domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable domain (VL), also called the variable light domain or light chain variable region, followed by a constant light (CL) domain.

[0090] The term "pharmaceutical composition" or "pharmaceutical preparation" refers to a preparation that is in a form such that the biological activity of the active ingredient contained therein is effective and that does not contain additional ingredients that are unacceptably toxic to the subject to which the pharmaceutical composition will be administered.

[0091] "Pharmaceutically acceptable carrier" refers to a component in a pharmaceutical composition or preparation other than the active ingredient that is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0092] The term "sample" includes, but is not limited to, any amount of substance from a biological or a former biological. Such biologicals include mice, monkeys, rats, rabbits, and other animals. In one embodiment, the sample is obtained from a monkey, particularly a cynomolgus monkey, or a rabbit, or a mouse, or a rat.

[0093] As used herein, the term "signal" 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. A signal in the form of a fluorescent or colorimetric product / reagent is a particular form of signal and is intended to be usable in the methods according to the invention. In some embodiments of the invention, the signal is evaluated quantitatively.

[0094] "Solid phase" represents 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 substances such as silica, alumina, and polymer gels; capillaries which may be made of polymers, metals, glass, and / or ceramics; zeolites and other porous substances; electrodes; microtiter plates; solid strips; and cuvettes, tubes or other spectrometer sample containers. The "solid support" is distinguished from the solid phase component of the assay in that it contains on its surface at least one portion that is intended to interact chemically with molecules. The solid phase may be a stationary component such as a chip, tube, strip, cuvette, or microtiter plate, or a non-stationary component such as beads and microparticles. Various microparticles that allow either non-covalent or covalent binding of proteins and other substances may be used. Such particles include polymer particles such as polystyrene and poly(methyl methacrylate); gold particles such as gold nanoparticles, gold colloids; ceramic particles such as silica, glass, metal oxide particles, etc. See, for example, Martin, C.R., et al., Analytical Chemistry - News & Features, 70(1998)322A - 327A, or Butler, J.E., Methods 22(2000)4 - 23.

[0095] The terms "therapeutic (monoclonal) antibody" and "drug" are used interchangeably herein. The term "antibody" as used herein is used in the broadest sense and encompasses various antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies, and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0096] The "transferrin receptor" ("TfR") is a transmembrane glycoprotein (with a molecular weight of approximately 180,000 Da) composed of two disulfide-linked subunits (each with an apparent molecular weight of approximately 90,000 Da) involved in iron uptake in vertebrates. In one embodiment, the TfR referred to herein is a human TfR that includes an amino acid sequence such as that of Schneider et al. (Nature 311 (1984) 675-678).

[0097] Bispecific antibody In one embodiment, 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.

[0098] In certain embodiments, the therapeutic antibody is a multispecific antibody, such as a bispecific antibody. A multispecific antibody is a monoclonal antibody having binding specificities for at least two different antigens. In certain embodiments, one of the binding specificities is for a first antigen and the other is for a different second antigen. The bispecific antibody can be prepared as a full-length antibody or an antibody fragment. In one embodiment, the antibody is a bispecific antibody that specifically binds to a first and a second antigen. In one embodiment, the bispecific antibody has i) a first binding specificity that specifically binds to a first antigen and ii) a second binding specificity that specifically binds to a second antigen. In one embodiment, the antibody is a bispecific trivalent antibody. In a preferred embodiment, the antibody is a monoclonal bispecific trivalent antibody.

[0099] In one embodiment, one of the binding sites specifically binds to the BBBR.

[0100] Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy-chain-light-chain pairs having different specificities (see Milstein, C. and Cuello, A.C., Nature 305 (1983) 537-540, WO 93 / 08829, and Traunecker, A. et al., EMBO J. 10 (1991) 3655-3659), and "knob-in-hole" engineering (see, e.g., U.S. Patent No. 5,731,168). Multispecific antibodies can also be made by engineering the electrostatic steering effect for making antibody Fc-heterodimeric molecules (WO 2009 / 089004), cross-linking two or more antibodies or fragments (see, e.g., U.S. Patent No. 4,676,980, and Brennan, M. et al., Science, 229 (1985) 81-83), producing bispecific antibodies using leucine zippers (see, e.g., Kostelny, S.A. et al., J. Immunol. 148 (1992) 1547-1553), using the "diabody" technology for making bispecific antibody fragments (see, e.g., Holliger, P. et al., Proc. Natl. Acad. Sci. USA, 90 (1993) 6448-6444), and using single-chain Fv (scFv) dimers (see, e.g., Gruber, M. et al., J. Immunol. 152 (1994) 5368-5374, and, e.g., Tutt, A. et al., J. Immunol. 147: (1991) 60-69), and can also be made by preparing trispecific antibodies as described therein.

[0101] The antibody or fragment can also be a multispecific antibody described in WO 2009 / 080251, WO 2009 / 080252, WO 2009 / 080253, WO 2009 / 080254, WO 2010 / 112193, WO 2010 / 115589, WO 2010 / 136172, WO 2010 / 145792, or WO 2010 / 145793.

[0102] Different bispecific antibody formats are known.

[0103] Exemplary bispecific antibody formats that can be used in the methods as reported herein are as follows. - CrossMab format (=CrossMab): A multispecific IgG antibody comprising a first Fab fragment and a second Fab fragment, wherein in the first Fab fragment, a) C ori (i.e., the light chain of the first Fab fragment comprises VL and CH1 domains, and the heavy chain of the first Fab fragment comprises VH and CL domains); b) Only the VH and VL domains are replaced with each other (i.e., the light chain of the first Fab fragment comprises VH and CL domains, and the heavy chain of the first Fab fragment comprises VL and CH1 domains); or c) The CH1 and CL domains are replaced with each other, and the VH and VL domains are replaced with each other (i.e., the light chain of the first Fab fragment comprises VH and CH1 domains, and the heavy chain of the first Fab fragment comprises VL and CL domains); and The second Fab fragment comprises a light chain comprising a VL domain and a CL domain and a heavy chain comprising a VH domain and a CH1 domain, CrossMab may comprise a first heavy chain comprising a CH3 domain and a second heavy chain comprising a CH3 domain, wherein both CH3 domains are operatively modified by respective amino acid substitutions to be complementary to assist in the heterodimerization of the first heavy chain and the modified second heavy chain as disclosed, for example, in WO 96 / 27011, WO 98 / 050431, EP 1870459, WO 2007 / 110205, WO 2007 / 147901, WO 2009 / 089004, WO 2010 / 129304, WO 2011 / 90754, WO 2011 / 143545, WO 2012 / 058768, WO 2013 / 157954, or WO 2013 / 096291 (incorporated herein by reference). - Arm single-chain format (= single-arm single-chain antibody): An antibody comprising a first binding site that specifically binds to a first epitope or antigen and a second binding site that specifically binds to a second epitope or antigen, wherein the individual chains are as follows. - Light chain (variable light chain domain + kappa light chain constant domain) - Light chain / heavy chain combination (variable light chain domain + light chain constant domain + peptide linker + variable heavy chain domain + CH1 + hinge + CH2 + CH3 with knob mutation); - Heavy chain (variable heavy chain domain + CH1 + hinge + CH2 + CH3 with hole mutation); - Two-arm single-chain format (= two-arm single-chain antibody): An antibody comprising a first binding site that specifically binds to a first epitope or antigen and a second binding site that specifically binds to a second epitope or antigen, wherein the individual chains are as follows - Light chain / heavy chain combination 1 (variable light chain domain + light chain constant domain + peptide linker + variable heavy chain domain + CH1 + hinge + CH2 + CH3 with hole mutation); - Light chain / heavy chain combination 2 (variable light chain domain + light chain constant domain + peptide linker + variable heavy chain domain + CH1 + hinge + CH2 + CH3 with knob mutation); - General light chain bispecific format (= general light chain bispecific antibody): An 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 individual chains are as follows: - Light chain (variable light chain domain + light chain constant domain) - Heavy chain 1 (variable heavy chain domain + CH1 + hinge + CH2 + CH3 with hole mutation) - Heavy chain 2 (variable heavy chain domain + CH1 + hinge + CH2 + CH3 with knob mutation); - Bispecific Fab format: A Fab that contains two (non-overlapping) paratopes in complementary pairs of VH and VL domains, wherein the first paratope comprises (consists of) amino acid residues derived from CDR1 and CDR3 of the VL domain and CDR2 of the VH domain, and the second paratope comprises (consists of) residues derived from CDR1 and CDR3 of the VH domain and CDR2 of the VL domain (the term "non-overlapping" in this context indicates that the amino acid residues contained within the first paratope of the bispecific Fab are not contained within the second paratope, and the amino acids contained within the second paratope of the bispecific Fab are not contained within the first paratope): - TCB format: - A first and a second Fab fragment, wherein each binding site of the first and second Fab fragments specifically binds to a second antigen, the first and second Fab fragments, - A third Fab fragment, wherein the binding site of the third Fab fragment specifically binds to a first antigen and comprises a domain swap such that the variable light chain domain (VL) and the variable heavy chain domain (VH) are replaced with each other, the third Fab fragment, - An Fc region, comprising an Fc region that contains a polypeptide of a first Fc region and a polypeptide of a second Fc region, The first Fab fragment and the second Fab fragment each comprise a heavy chain fragment and a full-length light chain, 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, 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, a bispecific antibody. - Brain-shuttle (BS) format: a) One (full-length) antibody that contains two pairs each of a (full-length) antibody light chain and a (full-length) antibody heavy chain, wherein the binding site formed by each pair of the (full-length) heavy chain and the (full-length) light chain specifically binds to a first antigen, one (full-length) antibody, b) A further Fab fragment, which is fused to the C-terminus of any one of the heavy chains of the (full-length) antibody, and the binding site of the further Fab fragment specifically binds to a second antigen, The further Fab fragment that specifically binds to the second antigen includes domain cross-over such that the constant light chain domain (CL) and the constant heavy chain domain 1 (CH1) are substituted for each other. The first antigen is a brain target and the second antigen is the human transferrin receptor.

[0104] In one embodiment, the bispecific antibody is a CrossMab.

[0105] In one embodiment, the bispecific antibody is a one-arm single-chain antibody.

[0106] In one embodiment, the bispecific antibody is a two-arm single-chain antibody.

[0107] In one embodiment, the bispecific antibody is a common light chain bispecific antibody.

[0108] In one embodiment, the bispecific antibody is a bispecific Fab.

[0109] In one embodiment, the bispecific antibody is a TCB.

[0110] In one embodiment, the bispecific antibody is a BS.

[0111] Multivalent multispecific antibodies are most likely to specifically bind to different targets and have different affinities and complex stabilities for each target. Only fully active multivalent multispecific antibodies can bind to all targets and exhibit complete biological activity in the corresponding assays.

[0112] A. Exemplary bispecific antibody: anti-human A-beta / human transferrin receptor antibody In certain embodiments, the therapeutic antibody determined by the method according to the invention is an antibody that binds to human A-β and the human transferrin receptor. This antibody is a bispecific antibody consisting of a full-length core antibody and a fusion Fab fragment in which certain domains are tolerantly exchanged. Thus, the resulting bispecific antibody is asymmetric. Thus, the bispecific antibody is produced using a heterodimerization technique called knobs-into-holes, using a first heavy chain having a so-called knob mutation (HC knob) and a second heavy chain having a so-called hole mutation (HC hole).

[0113] Exemplary antibody 0012 is composed of four polypeptides having the amino acid sequences of SEQ ID NOs: 04-07.

[0114] Exemplary antibody 0015 is composed of four polypeptides having the amino acid sequences of SEQ ID NOs: 08-11.

[0115] Exemplary antibody 0020 is composed of three polypeptides having the amino acid sequences of SEQ ID NOs: 12-14.

[0116] Exemplary antibody 0024 is composed of four polypeptides having the amino acid sequences of SEQ ID NOs: 15-18.

[0117] In one aspect, the bispecific antibody comprises a) one full-length antibody comprising two pairs each of a full-length antibody light chain and a full-length antibody heavy chain, wherein the binding site formed by each pair of the full-length heavy chain and the full-length light chain specifically binds to a first antigen, one full-length antibody, and b) a further Fab fragment fused to the C-terminus of one heavy chain of the full-length antibody, the binding site of the further Fab fragment specifically binding to a second antigen, a further Fab fragment. Each full-length antibody light chain contains the amino acid residue arginine at position 123 of the constant light chain domain (CL) (instead of the wild-type glutamic acid residue; E123R mutation) and the amino acid residue lysine at position 124 (instead of the wild-type glutamine residue; Q124K mutation) (numbering follows Kabat), Each full-length antibody heavy chain contains a glutamic acid residue at position 147 of the first constant heavy chain domain (CH1) (instead of the wild-type lysine residue; K147E mutation) and a glutamic acid residue at position 213 (instead of the wild-type lysine amino acid residue; K213E mutation) (numbering follows the Kabat EU index), A further Fab fragment that specifically binds to the second antigen contains a domain crossover such that the constant light chain domain (CL) and the constant heavy chain domain 1 (CH1) are replaced with each other, The first antigen is human A-beta protein, and the second antigen is human transferrin receptor.

[0118] In another embodiment, the therapeutic antibody is a) One full-length antibody comprising two pairs each of a full-length antibody light chain and a full-length antibody heavy chain, wherein the binding site formed by each pair of the full-length heavy chain and the full-length light chain specifically binds to the first antigen, one full-length antibody, b) A further Fab fragment fused to the C-terminus of one heavy chain of the full-length antibody, and a further Fab fragment wherein the binding site of the further Fab fragment specifically binds to the second antigen, Each full-length antibody light chain contains the amino acid residue arginine at position 123 of the constant light chain domain (CL) (instead of the wild-type glutamic acid residue; E123R mutation) and the amino acid residue lysine at position 124 (instead of the wild-type glutamine residue; Q124K mutation) (numbering follows Kabat), Each of the full-length antibody heavy chains contains a glutamic acid residue at position 147 of the first constant heavy chain domain (CH1) (instead of the wild-type lysine residue; K147E mutation) and a glutamic acid residue at position 213 (instead of the wild-type lysine amino acid residue; K213E mutation) (numbering follows the Kabat EU index), A further Fab fragment that specifically binds to a second antigen is a bispecific antibody that includes a domain crossover such that the constant light chain domain (CL) and the constant heavy chain domain 1 (CH1) are substituted for each other, The first antigen is human A-beta protein and the second antigen is human transferrin receptor, The human A-beta binding site includes a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 19, and a light chain variable domain (VL) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 20, The human transferrin receptor binding site includes a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 21, and a light chain variable domain (VL) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 22.

[0119] In certain embodiments, VH sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity include substitutions (e.g., conservative substitutions), insertions or deletions relative to a reference sequence, but the binding site comprising such a sequence retains the ability to bind its antigen. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 19 or 21. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside of the HVRs (i.e., in the FRs).

[0120] In certain embodiments, VL sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity include substitutions (e.g., conservative substitutions), insertions or deletions as compared to a reference sequence, but the binding site comprising such a sequence retains the ability to bind its antigen. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 20 or 22. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside of the HVRs (i.e., in the FRs).

[0121] In one embodiment, the human A-beta binding site comprises a VH sequence such as that in SEQ ID NO: 19 and a VL sequence such as that in SEQ ID NO: 20, including post-translational modifications of the sequences.

[0122] In one embodiment, the human transferrin receptor binding site comprises a VH sequence such as SEQ ID NO: 21 and a VL sequence such as SEQ ID NO: 22, including post-translational modifications of the sequences.

[0123] In one embodiment, the bispecific antibody is i) a light chain having at least 70%, at least 80%, at least 90%, or 95% or more sequence identity with SEQ ID NO: 23, and ii) a heavy chain having at least 70%, at least 80%, at least 90%, or 95% or more sequence identity with SEQ ID NO: 24. (iii) a light chain having at least 70%, at least 80%, at least 90%, or 95% or more sequence identity with SEQ ID NO: 25, (iv) a heavy chain Fab fragment having at least 70%, at least 80%, at least 90%, or 95% or more sequence identity with SEQ ID NO: 26, in the above sequences, SEQ ID NO: 23 has the amino acid sequence DIVLTQSPATLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGVPARFSGSGSGTDFTLTISSLEPEDFATYYCLQIYNMPITFGQGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC; SEQ ID NO: 24 has the amino acid sequence QVELVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAINASGTRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGKGNTHKPYGYVRYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG; SEQ ID NO: 25 has the amino acid sequence AIQLTQSPSSLSASVGDRVTITCRASQSISSYLAWYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNYASSNVDNTFGGGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC, SEQ ID NO: 26 has the amino acid sequence QSMQESGPGLVKPSQTLSLTCTVSGFSLSSYAMSWIRQHPGKGLEWIGYIWSGGSTDYASWAKSRVTISKTSTTVSLKLSSVTAADTAVYYCARRYGTSYPDYGDASGFDPWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0124] In another embodiment, the therapeutic antibody is a) one full-length antibody comprising two pairs each of a full-length antibody light chain and a full-length antibody heavy chain, wherein the binding site formed by each pair of the full-length heavy chain and the full-length light chain specifically binds to a first antigen; b) a further Fab fragment fused to the C-terminus of one heavy chain of the full-length antibody, wherein the binding site of the further Fab fragment specifically binds to a second antigen. Each of the full-length antibody light chains comprises the amino acid residue arginine at position 123 (instead of the wild-type glutamic acid residue; E123R mutation) and the amino acid residue lysine at position 124 (instead of the wild-type glutamine residue; Q124K mutation) of the constant light chain domain (CL) (numbering according to Kabat), Each of the full-length antibody heavy chains is a bispecific antibody that contains a glutamic acid residue at position 147 of the first constant heavy chain domain (CH1) (instead of the wild-type lysine residue; K147E mutation) and a glutamic acid residue at position 213 (instead of the wild-type lysine amino acid residue; K213E mutation) (numbering follows the Kabat EU index). A further Fab fragment that specifically binds to a second antigen is a bispecific antibody that includes a domain crossover such that the constant light chain domain (CL) and the constant heavy chain domain 1 (CH1) are substituted for each other. The first antigen is human A-beta protein and the second antigen is human transferrin receptor. The human A-beta binding site includes 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. The human transferrin receptor binding site includes 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.

[0125] In another embodiment, the therapeutic antibody is a) One full-length antibody comprising two pairs each of a full-length antibody light chain and a full-length antibody heavy chain, wherein the binding site formed by each pair of the full-length heavy chain and the full-length light chain specifically binds to a first antigen, and the full-length antibody includes an Fc region formed by the Fc region polypeptides of the two full-length heavy chains, each of which includes CH1, CH2, and CH3 domains. b) A further Fab fragment that is fused to the C-terminus of one of the heavy chains of the full-length antibody, and the binding site of the further Fab fragment specifically binds to a second antigen. Each of the full-length antibody light chains contains an arginine amino acid residue at position 123 of the constant light chain domain (CL) (instead of the wild-type glutamic acid residue; E123R mutation) and a lysine amino acid residue at position 124 (instead of the wild-type glutamine residue; Q124K mutation) (numbering follows Kabat). Each of the full-length antibody heavy chains is a bispecific antibody that contains a glutamic acid residue at position 147 of the first constant heavy chain domain (CH1) (instead of the wild-type lysine residue; K147E mutation) and a glutamic acid residue at position 213 (instead of the wild-type lysine amino acid residue; K213E mutation) (numbering follows the Kabat EU index). A further Fab fragment that specifically binds to a second antigen is a bispecific antibody that includes a domain crossover such that the constant light chain domain (CL) and the constant heavy chain domain 1 (CH1) are replaced with each other. The first antigen is human A-beta protein, and the second antigen is human transferrin receptor. The human A-beta binding site includes 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. The human transferrin receptor binding site includes 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. The Fc region polypeptide is as follows: a) Human subclass IgG1 b) Human subclass IgG4 c) Human subclass IgG1 with mutations L234A, L235A, and P329G d) Human subclass IgG4 with mutations S228P, L235E, and P329G e) Human subclass IgG1 with mutations L234A, L235A, and P329G in both Fc region polypeptides, mutations T366W and S354C in one Fc region polypeptide, and mutations T366S, L368A, Y407V, and Y349C in each of the other Fc region polypeptides. f) Human subclass IgG4 with mutations S228P and P329G in both Fc region polypeptides, mutations T366W and S354C in one Fc region polypeptide, and mutations T366S, L368A, Y407V, and Y349C in each of the other Fc region polypeptides. g) Mutations L234A, L235A, P329G, I253A, H310A, and H435A in both Fc region polypeptides, and mutations T366W and S354C in one Fc region polypeptide, and mutations T366S, L368A, Y407V, and Y349C in each of the other Fc region polypeptides, in human subclass IgG1, h) 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 each of the other Fc region polypeptides, in human subclass IgG1.

[0126] B. Exemplary anti-transferrin receptor antibodies The anti-transferrin receptor binding site of the therapeutic antibody determined by the method according to the present invention has an off-rate of binding to human transferrin receptor within a certain range to ensure appropriate BBB shuttling. This range is defined at one end by the off-rate of the mouse anti-transferrin receptor antibody 128.1 (variable domain amino acid sequences given in SEQ ID NOs: 27 and 28) determined by surface plasmon resonance against cynomolgus transferrin receptor, and at the other end by 5% of that off-rate (i.e., 20-fold slower dissociation). The off-rate of human transferrin receptor should be 0.1 1 / s to 0.005 1 / s.

[0127] One aspect described herein is an anti-transferrin receptor antibody that specifically binds to human transferrin receptor and cynomolgus transferrin receptor, 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, and The off-rate of the antibody for the human transferrin receptor is equal to or less than (i.e., at most) the off-rate of anti-transferrin receptor antibody 128.1 for the cynomolgus monkey transferrin receptor, and the off-rate is determined by surface plasmon resonance, and 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.

[0128] In one embodiment, the off-rate of the human transferrin receptor is 0.1 1 / s to 0.005 1 / s.

[0129] In one embodiment, the antibody has a proline amino acid residue (P) (numbering according to Kabat) at position 80 of the light chain variable domain.

[0130] In one embodiment, the antibody has an asparagine amino acid residue (N) (numbering according to Kabat) at position 91 of the light chain variable domain.

[0131] In one embodiment, the antibody has an alanine amino acid residue (A) (numbering according to Kabat) at position 93 of the light chain variable domain.

[0132] In one embodiment, the antibody has a serine amino acid residue (S) (numbering according to Kabat) at position 100g of the heavy chain variable domain.

[0133] In one embodiment, the antibody has a glutamine amino acid residue (Q) (numbering according to Kabat) at position 100g of the heavy chain variable domain.

[0134] In one embodiment, the antibody has a serine amino acid residue (S) (numbering according to Kabat) at position 65 of the heavy chain variable domain.

[0135] In one embodiment, the antibody has a glutamine amino acid residue (Q) (numbering according to Kabat) at position 105 of the heavy chain variable domain.

[0136] 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 according to Kabat).

[0137] 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 according to Kabat)

[0138] Such anti-transferrin receptor bispecific antibodies can be used as a blood-brain barrier shuttle module for delivering a brain effector entity into the brain through the blood-brain barrier. The blood-brain barrier shuttle module is a monovalent binding entity that specifically binds to the human transferrin receptor. The anti-transferrin receptor bispecific antibody when used as a blood-brain barrier shuttle module is useful for the diagnosis or treatment of neurological disorders such as Alzheimer's disease, Parkinson's disease, and co-existing conditions of Alzheimer's disease and Parkinson's disease.

[0139] 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 reflect the binding characteristics of the mouse antibody 128.1 to cynomolgus transferrin receptor with respect to the binding off-rate for human transferrin receptor.

[0140] In one embodiment, the anti-transferrin receptor binding site of the therapeutic antibody specifically binds to 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 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 (numbering according to Kabat).

[0141] In one embodiment, the anti-transferrin receptor binding site of the therapeutic antibody further has a serine amino acid residue (S) at position 100 in the heavy chain variable domain (numbering according to Kabat).

[0142] In one embodiment, the anti-transferrin receptor binding site of the therapeutic antibody further has a serine amino acid residue (S) at position 65 in the heavy chain variable domain (numbering according to Kabat).

[0143] In one embodiment, the anti-transferrin receptor binding site of the therapeutic antibody further has a glutamine amino acid residue (Q) at position 105 in the heavy chain variable domain (numbering according to Kabat).

[0144] 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 includes 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. The off-rate of the therapeutic antibody expressed in units of 1 / s for the human transferrin receptor is at most (i.e., is) the off-rate of anti-transferrin receptor antibody 128.1 expressed in units of 1 / s for the cynomolgus monkey transferrin receptor. The off-rate is determined by surface plasmon resonance, and 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.

[0145] In one embodiment, the anti-transferrin receptor binding site of the therapeutic antibody has an off-rate expressed in units of 1 / s for the human transferrin receptor, and is (i) at most (i.e., is) the off-rate of anti-transferrin receptor antibody 128.1 expressed in units of 1 / s for the cynomolgus monkey transferrin receptor, and (ii) at least 5% of the off-rate of anti-transferrin receptor antibody 128.1 expressed in units of 1 / s for the cynomolgus monkey transferrin receptor.

[0146] In one embodiment, the anti-transferrin receptor binding site of the therapeutic antibody includes (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: 35, 36 or 37, and in a preferred embodiment, SEQ ID NO: 36; (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.

[0147] In any of the above embodiments, the anti-transferrin receptor binding site is humanized. In one embodiment, the anti-transferrin receptor binding site comprises an HVR of any of the above embodiments and further comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0148] 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 that form a binding site for the transferrin receptor, and at least one pair of a heavy chain variable domain of SEQ ID NO: 41 and a light chain variable domain binding site 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 position 11 according to Kabat with any amino acid other than leucine. In one embodiment, the substitution comprises a substitution of the amino acid residue at position 11 according to Kabat with a nonpolar amino acid. In a preferred embodiment, the substitution comprises substituting the amino acid residue at position 11 according to Kabat of 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.

[0149] 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 that form a binding site for the transferrin receptor, and at least one pair of a heavy chain variable domain of SEQ ID NO: 43 and a light chain variable domain binding site of SEQ ID NO: 44 for human alpha-synuclein.

[0150] 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 that form a binding site for the transferrin receptor, and at least one pair of a humanized heavy chain variable domain derived from SEQ ID NO: 45 and a humanized light chain variable domain binding site derived from SEQ ID NO: 46 for human alpha-synuclein.

[0151] 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 that form a binding site for the transferrin receptor, and at least one pair of a humanized heavy chain variable domain derived from SEQ ID NO: 47 and a humanized light chain variable domain binding site derived from SEQ ID NO: 48 for human alpha-synuclein.

[0152] 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 that form a binding site for the 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 binding site derived from SEQ ID NO: 50 for human alpha-synuclein.

[0153] 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 that form a binding site for the 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 binding site derived from SEQ ID NO: 52 for human alpha-synuclein.

[0154] 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 that form a binding site for the 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 binding site derived from SEQ ID NO: 54 for human alpha-synuclein.

[0155] In one embodiment, the antibody is a bispecific antibody having 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 that form a binding site for the transferrin receptor, and a binding site for i) glucocerebrosidase having the amino acid sequence of SEQ ID NO: 55, or ii) a functional variant of SEQ ID NO: 55 having at least 70% sequence identity, or iii) a functional variant of SEQ ID NO: 55 having one or more amino acid mutations, deletions or insertions, or iv) a truncated functional variant of SEQ ID NO: 55 having at least one amino acid residue deleted in the N-terminus or C-terminus or within the amino acid sequence, or v) a combination of iii) and iv).

[0156] In another embodiment, the therapeutic antibody is a multispecific antibody. In such an 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 such an aspect, the brain antigen is selected from the group consisting of beta-secretase 1 (BACE1), A beta, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, 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 Tf and BACE1. In another embodiment, the multispecific antibody binds to both TfR and A beta. 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 an antibody for treating neuropathy.

[0157] In one embodiment, the effector function is reduced or eliminated by at least one modification of the Fc region. In one embodiment, the effector function or complement activation function is reduced or eliminated by deletion of all or part of the Fc region, or by designing the antibody to not contain an Fc region or non-Fc region eligible for the effector function or complement activation function. In one embodiment, at least one modification of the Fc region is selected from point mutations in the Fc region that inhibit binding to one or more Fc receptors selected from the following 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, 335, 338, 340, 373, 376, 382, 388, 389, 414, 416, 419, 434, 435, 437, 438, 439; point mutations in the Fc region that inhibit binding to C1q selected from the following positions: 270, 322, 329, 321; eliminating point mutations at positions in part or all of the Fc region and at position 132 of the CH1 domain. In one embodiment, the modification is a point mutation in the Fc region that inhibits binding to C1q selected from the following positions: 270, 322, 329, and 321. In another embodiment, the modification is removal of part or all of the Fc region. In another embodiment, the complement trigger function is reduced or eliminated by deletion of all or part of the Fc region, or by designing the antibody to not contain an Fc region that engages the complement pathway. In one embodiment, the antibody is selected from a Fab or 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 CH1 region that inhibits 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).

[0158] In one aspect of the above-described embodiment, the affinity of the antibody for TfR is reduced compared to a wild-type antibody of the same isotype that has not reduced affinity for TfR. In one such aspect, the antibody has a K D or IC 50 for TfR of about 1 pM to about 100 μM.

[0159] In one embodiment, the antibodies reported herein do not have effector function. In one embodiment, the antibody does not have effector function. In one embodiment, the antibody is of the human IgG1 subclass and has the mutations L234A, L235A, and P329G in both heavy chains (numbering follows the Kabat EU index).

[0160] In one embodiment, the antibody is a) a full-length antibody of the human subclass IgG1, or b) a full-length antibody of the human subclass IgG4, or c) a full-length antibody of the human subclass IgG1 with the mutations L234A, L235A, and P329G, d) a full-length antibody of the human subclass IgG4 with the mutations S228P, L235E, and P329G, e) a full-length antibody of the human subclass IgG1 with the mutations L234A, L235A, and P329G in both heavy chains and the mutations T366W and S354C in one heavy chain and the mutations T366S, L368A, Y407V, and Y349C in the other heavy chain, or f) a full-length antibody of the human subclass IgG4 having the mutations S228P in both heavy chains and optionally P329G, the mutations T366W and S354C in one heavy chain, and the mutations T366S, L368A, Y407V, and Y349C in the other heavy chain.

[0161] In one embodiment, the bispecific therapeutic antibody is i) optionally, a homodimeric Fc region of the human IgG1 subclass, including the mutations P329G, L234A, and L235A, or ii) Optionally, a homodimeric Fc region of the human IgG4 subclass, comprising the mutations P329G, S228P, and L235E, or iii) A heterodimeric Fc-region, wherein a) one Fc region polypeptide comprises the mutation T366W and the other Fc region polypeptide comprises the mutations T366S, L368A, and Y407V, or 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 c) a heterodimeric Fc-region, wherein one Fc region polypeptide comprises the mutations T366W and S354C and the other Fc region polypeptide comprises the mutations T366S, L368A, Y407V, and Y349C, or iv) A heterodimeric Fc region of the human IgG4 subclass, wherein both Fc region polypeptides comprise the mutations P329G, L234A and L235A, a) one Fc region polypeptide comprises the mutation T366W and the other Fc region polypeptide comprises the mutations T366S, L368A, and Y407V, or 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 c) a heterodimeric Fc region of the human IgG4 subclass, wherein one Fc region polypeptide comprises the mutations T366W and S354C and the other Fc region polypeptide comprises the mutations T366S, L368A, Y407V, and Y349C or v) A heterodimeric Fc region of the human IgG4 subclass, wherein both Fc region polypeptides comprise the mutations P329G, S228P, and L235E, a) one Fc region polypeptide comprises the mutation T366W and the other Fc region polypeptide comprises the mutations T366S, L368A, and Y407V, or b) one Fc region polypeptide contains the mutations T366W and Y349C, and the other Fc region polypeptide contains the mutations T366S, L368A, Y407V, and S354C, or c) one Fc region polypeptide contains the mutations T366W and S354C, and the other Fc region polypeptide contains the mutations T366S, L368A, Y407V, and Y349C, which is a heterodimeric Fc region of the human IgG4 subclass.

[0162] Immunoassay The principles of various immunoassays are described in the art. For example, Hage, D. S. (Anal. Chem. 71 (1999) 294R - 304R) and Lu, B. et al. (Analyst 121 (1996) 29R - 32R) have reported the oriented immobilization of antibodies for use in immunoassays. Avidin - biotin - mediated immunoassays are reported, for example, in Wilchek, M., and Bayer, E. A., in Methods Enzymol. 184 (1990) 467 - 469.

[0163] Monoclonal antibodies and their constant domains contain several reactive amino acid side chains for binding to members of a binding pair such as polypeptides / proteins, polymers (e.g., PEG, cellulose or polystyrene), or enzymes. Chemically reactive groups of amino acids are, for example, amino groups (lysine, alpha - amino group), thiol groups (cysteine, cysteine and methionine), carboxylic acid groups (aspartic acid, glutamic acid), and sugar alcohol groups. Such methods are described, for example, in "Bioconjugation", MacMillan Ref. Ltd., 1999, pages 50 - 100.

[0164] One of the most common reactive groups of antibodies is the aliphatic ε - amine of the amino acid lysine. Generally, almost all antibodies contain abundant lysine. Since lysine amine is a moderately good nucleophile when the pH exceeds 8.0 (pKa = 9.18), it reacts easily and cleanly with various reagents to form stable bonds. Amine - reactive reagents mainly react with lysine and the α - amino groups of proteins. Reactive esters, especially N - hydroxy - succinimide (NHS) esters, are one of the most commonly used reagents for the modification of amine groups. The optimal pH for the reaction in an aqueous environment is pH 8.0 - 9.0. Isothiocyanates are amine - modifying reagents that form thiourea bonds with proteins. They react with protein amines in aqueous solution (optimally at pH 9.0 - 9.5). Aldehydes react with aliphatic and aromatic amines, hydrazine, and hydrazide under mild aqueous conditions to form imine intermediates (Schiff bases). Schiff bases can be selectively reduced with mild or strong reducing agents (such as sodium borohydride or sodium cyanoborohydride) to induce stable alkylamine bonds. Other reagents used to modify amines are acid anhydrides. For example, diethylenetriaminepentaacetic anhydride (DTPA) is a bifunctional chelating agent containing two amine - reactive anhydride groups. This can react with the N - terminal and ε - amine groups of amino acids to form amide bonds. The anhydride ring can open to generate polyvalent metal - chelating arms that can strongly bind to metals in coordination complexes.

[0165] Another common reactive group in antibodies is the thiol residue from the sulfur-containing amino acid cysteine and its reduced product cysteine (or half-cysteine). Cysteine is more nucleophilic than amine and generally contains a free thiol group which is the most reactive functional group in proteins. Since thiols are generally reactive at neutral pH, they can selectively bind to other molecules in the presence of amines. Because free sulfhydryl groups are relatively reactive, proteins with these groups often exist in their oxidized form as disulfide groups or disulfide bonds. In such proteins, reduction of the disulfide bond by a reagent such as dithiothreitol (DTT) is necessary to generate reactive free thiols. Thiol-reactive reagents are reagents that bind to the thiol groups on polypeptides to form thioether bond products. These reagents react rapidly at slightly acidic to neutral pH and can thus react selectively in the presence of amine groups. The literature reports the use of several thiolation cross-linking reagents such as the Traut reagent (2-iminothiolane), succinimidyl (acetylthio)acetate (SATA) and sulfo-succinimidyl 6-[3-(2-pyridyldithio)propionamido]hexanoate (Sulfo-LC-SPDP) to provide an efficient method for introducing multiple sulfhydryl groups via reactive amino groups. Haloacetyl derivatives, such as iodoacetamide, form thioether bonds and are reagents for thiol modification. A further useful reagent is maleimide. The reaction of maleimide with thiol-reactive reagents is essentially the same as that of iodoacetamide. Maleimide reacts rapidly at slightly acidic to neutral pH.

[0166] Another common reactive group in antibodies is the carboxylic acid. Antibodies contain carboxylic acid groups at the C-terminal position and within the side chains of aspartic acid and glutamic acid. The relatively low reactivity of carboxylic acids in water usually makes it difficult to use these groups to selectively modify polypeptides and antibodies. At this time, the carboxylic acid groups are usually converted to reactive esters using water-soluble carbodiimides and react with nucleophiles such as amines, hydrazides, hydrazine, etc. Amine-containing reagents must be weakly basic in order to react selectively with activated carboxylic acids in the presence of the more basic ε-amines of lysine to form stable amide bonds. When the pH rises above 8.0, protein cross-linking can occur.

[0167] Sodium periodate can be used to oxidize the alcohol moieties of sugars within the carbohydrate moieties attached to the antibody to aldehydes. Each aldehyde group can be reacted with an amine, hydrazide, or hydrazine as described for carboxylic acids. Since the carbohydrate moieties are mainly found on the crystallizable fragment region (Fc region) of the antibody, conjugation can be achieved by site-specific modification of the carbohydrate away from the antigen-binding site. A Schiff base intermediate is formed that can be reduced to an alkylamine by reduction of the intermediate with sodium cyanoborohydride (mild and selective) or sodium borohydride (strong) water-soluble reducing agents.

[0168] The conjugation of the tracer and / or capture and / or detection antibody with its conjugation partner can be carried out by different methods such as chemical bonding or binding via a binding pair. As used herein, the term "conjugation partner" refers to, for example, a solid support, a polypeptide, a detectable label, a member of a specific binding pair. In one embodiment, the binding of the capture and / or tracer and / or detection antibody to its binding partner is effected by chemically bonding through the N-terminus and / or ε-amino group (lysine), the ε-amino groups of different lysines, the carboxy-, sulfhydryl-, hydroxyl-, and / or phenolic functional groups of the amino acid backbone of the antibody, and / or the sugar alcohol groups of the carbohydrate structure of the antibody. In one embodiment, the capture antibody is conjugated to its conjugation partner via a binding pair. In one preferred embodiment, the capture antibody is conjugated to biotin, and immobilization on the solid support is effected via a solid support to which avidin or streptavidin is immobilized. In one embodiment, the capture antibody is conjugated to its conjugation partner via a binding pair. In one preferred embodiment, the tracer antibody is conjugated to digoxigenin by covalent bonding as a detectable label.

[0169] Chromogens (fluorescent or luminescent groups and dyes), enzymes, NMR-active groups or metal particles, haptens such as digoxigenin are examples of "detectable labels". Detectable labels can also be photoactivatable crosslinking groups such as azide or azirine groups. Metal chelates that can be detected by electrochemiluminescence are also preferred signal-emitting groups, and ruthenium chelates such as ruthenium(bispyridyl)32+ chelate are particularly preferred. Suitable ruthenium labeling groups are described, for example, in EP0580979, WO 90 / 05301, WO 90 / 11511 and WO 92 / 14138. For direct detection, the labeling group can be selected from any known detectable marker group such as dyes, luminescent labeling groups such as chemiluminescent groups such as acridinium esters or dioxetanes, or fluorescent dyes such as fluorescein, coumarin, rhodamine, oxazine, resorufin, cyanine and their derivatives. Other examples of labeling groups are luminescent metal complexes such as ruthenium or europium complexes, enzymes used in, for example, ELISA or CEDIA (cloned enzyme donor immunoassay, for example, EP-A-0061888), and radioisotopes.

[0170] Indirect detection systems include, for example, the detection reagent, such as a detection antibody, being labeled with the first partner of a binding pair. Examples of suitable binding pairs are antigen / antibody, biotin or biotin analogs such as aminobiotin, iminobiotin or desthiobiotin / avidin or streptavidin, sugar / lectin, nucleic acid or nucleic acid analog / complementary nucleic acid, and receptor / ligand such as steroid hormone receptor / steroid hormone. In a preferred embodiment, the first binding pair member includes haptens, antigens and hormones. In a preferred embodiment, the hapten is selected from the group consisting of digoxin, digoxigenin and biotin and their analogs. The second partner of such a binding pair, such as an antibody, streptavidin, etc., is usually labeled to enable direct detection, for example, by the labels as described above.

[0171] Immunoassays can generally be performed in three different formats. One is by direct detection, one is by indirect detection, or by sandwich assay. Direct detection immunoassays use a detection (or tracer) antibody that can be directly measured. Enzymes or other molecules enable the generation of a signal that produces a color, fluorescence, or luminescence that enables the signal to be visualized or measured (radioisotopes can also be used, although not commonly used today). In an indirect assay, a primary antibody that binds to the analyte is used to provide a defined target for a secondary antibody (tracer antibody) that specifically binds to the target provided by the primary antibody (referred to as the detector or tracer antibody). The secondary antibody generates a measurable signal. Sandwich assays utilize two antibodies, a capture antibody and a tracer (detector) antibody. The capture antibody is used to bind (immobilize) the analyte from solution or to bind the analyte in solution. This enables the specific removal of the analyte from the sample. The tracer (detector) antibody is used in a second step to generate a signal (either directly or indirectly as described above). The sandwich format requires two antibodies that each have a different epitope on the target molecule. Further, since both antibodies must bind to the target simultaneously, they must not interfere with each other.

[0172] Different principles for the determination of bispecific antibodies in immunoassays are known to those skilled in the art. 1) Imaging - One of the antigens; - An anti-idiotype antibody against one of the binding sites; 2) Detection used - Each of the other antigens; - An anti-idiotype antibody against each of the other binding sites;

[0173] These can be combined independently of each other.

[0174] Blood-brain barrier permeable antibodies of the method according to the invention In one aspect, the present invention is the determination of the concentration of a bispecific antibody for use in the treatment of a disease in a patient in a brain tissue, wherein the bispecific therapeutic antibody is i) (effector function competent) Fc region, ii) two binding sites that specifically bind to a first (cell surface) target, and iii) one binding site that specifically binds to a second (cell surface) target the treatment reduces side effects after administration, the side effects are one or more selected from the group consisting of vasodilation, bronchoconstriction, laryngeal edema, decreased cardiac pressure, and hypothermia, and relates to the determination of the concentration of the antibody, which is a bispecific antibody.

[0175] 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.

[0176] In one embodiment, the first (cell surface) target and the second (cell surface) target are different.

[0177] 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, and the binding site that binds to the second target is at one of the C-termini of the (full-length) antibody heavy chain of the bispecific antibody).

[0178] In one aspect, 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 on opposite sides of the bispecific antibody, i.e., one of the binding sites that specifically binds to the first target is conjugated to the first N-terminus of the Fc region, the other is conjugated to the second N-terminus of the Fc region, and the binding site that specifically binds to the second target is conjugated to one of the C-termini of the Fc region.

[0179] In one embodiment, the binding site that specifically binds to the second (cell surface) target is linked by a peptide linker to one of the binding sites that specifically bind to the first (cell surface) target. In one embodiment, the peptidic linker has the amino acid sequence of SEQ ID NO: 56 or 57.

[0180] In one embodiment, the binding site that specifically binds to the second (cell surface) target is within the Fc region, and at least one structural loop region of any of the CH2 domain, CH3 domain, or CH4 domain contains at least one modification that enables binding of the at least one modified loop region to the second (cell surface) target, and the unmodified immunoglobulin constant domain does not bind to the target.

[0181] In one embodiment, the binding site is a pair of an antibody heavy chain variable domain and an antibody light chain variable domain.

[0182] In one embodiment, the bispecific therapeutic antibody i) a pair of a first antibody light chain and a first antibody heavy chain, ii) a pair of a second antibody light chain and a second antibody heavy chain, iii) further antibody fragments selected from the group consisting of scFv, Fab, scFab, dAb fragments, DutaFab and CrossFab, wherein the pairs of antibody chains of i) and ii) contain a binding site that specifically binds to the first (cell surface) target, and the further antibody fragment of iii) contains a binding site that specifically binds to the second (cell surface) target.

[0183] In one embodiment, the further antibody fragment of (iii) is conjugated either directly or via a peptide linker to either the first antibody heavy chain or the second antibody heavy chain. In one embodiment, the further antibody fragment of (iii) is conjugated either directly or via a peptide linker to the C-terminus of the antibody heavy chain of (i) or (ii). In one embodiment, the Fc region has 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, and 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 not conjugated to the further antibody fragment of (iii) does not contain (i) a C-terminal lysine residue or (ii) a C-terminal glycine-lysine dipeptide.

[0184] In one embodiment, the first target is a brain target and the second target is the human transferrin receptor. In one embodiment, the first target is a brain target and the second target is the human transferrin receptor 1.

[0185] In one embodiment, the brain target is selected from the group consisting of beta-secretase 1 (BACE1), human amyloid beta (A-beta), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), human tau protein, phosphorylated human tau protein, 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), and caspase 6. In a preferred embodiment, the brain target is selected from the group consisting of human CD20, human tau protein, phosphorylated human tau protein, 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.

[0186] In a preferred embodiment, the bispecific therapeutic antibody is 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 that form a first binding site that specifically binds to a brain target selected from the group consisting of human CD20, human tau protein, phosphorylated human tau protein, human alpha-synuclein, and human amyloid beta protein; 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 that form a second binding site that specifically binds to the same brain target as the first binding site; iii) an additional antibody fragment comprising a third light chain variable domain and a third heavy chain variable domain selected from the group consisting of scFv, Fab, scFab, dAb fragment, DutaFab, and CrossFab that form a third binding site that specifically binds to the human transferrin receptor (transferrin receptor 1); iv) comprising a (human) effector function competent Fc region of the (human) IgG1 subclass; iii) The further antibody fragment is conjugated either directly or via a peptide linker to the C-terminus of the heavy chain of the antibody of i) or ii).

[0187] In one embodiment, the further antibody fragment is a Fab fragment that specifically binds to a second antigen and is fused via a peptide linker to the C-terminus of one of the heavy chains of i) or ii), and the constant domains CL and CH1 of the second light chain and the second heavy chain are replaced with each other, and the third light chain variable domain and the third heavy chain variable domain that form a third binding site that specifically binds to the human transferrin receptor (transferrin receptor 1) are included.

[0188] In one embodiment, the binding site that specifically binds to the 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.

[0189] In one embodiment, the binding site that specifically binds to the 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; (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 40.

[0190] In one embodiment of the portable device, the therapeutic antibody comprises a pair of a heavy chain variable domain of SEQ ID NO: 31 and a light chain variable domain of SEQ ID NO: 32 that form a binding site for the transferrin receptor (transferrin receptor 1), and at least one (i.e., 1 or 2) pair of a heavy chain variable domain of SEQ ID NO: 19 and a light chain variable domain of SEQ ID NO: 20 that each form a binding site for human amyloid beta protein (Aβ).

[0191] In one embodiment, the therapeutic antibody comprises a pair of a heavy chain variable domain of SEQ ID NO: 31 and a light chain variable domain of SEQ ID NO: 32 that form a binding site for the human transferrin receptor (transferrin receptor 1), and two pairs of a heavy chain variable domain of SEQ ID NO: 41 and a light chain variable domain of SEQ ID NO: 42 that each form a binding site for human CD20. In one embodiment, the heavy chain variable region comprises a substitution of the amino acid residue at position 11 according to Kabat with any amino acid other than leucine. In one embodiment, the substitution comprises a substitution of the amino acid residue at position 11 according to Kabat with a nonpolar amino acid. In a preferred embodiment, the substitution comprises substituting the amino acid residue at position 11 according to Kabat of 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.

[0192] In one embodiment, the therapeutic antibody comprises a pair of a heavy chain variable domain of SEQ ID NO: 31 and a light chain variable domain of SEQ ID NO: 32 that form a binding site for the human transferrin receptor (transferrin receptor 1), and two pairs of a heavy chain variable domain of SEQ ID NO: 43 and a light chain variable domain of SEQ ID NO: 44 that each form a binding site for human alpha-synuclein.

[0193] In one embodiment, the therapeutic antibody comprises a pair of a heavy chain variable domain of SEQ ID NO: 31 and a light chain variable domain of SEQ ID NO: 32 that form a binding site for the human transferrin receptor (transferrin receptor 1), and two pairs of a humanized heavy chain variable domain derived from SEQ ID NO: 45 and a humanized light chain variable domain derived from SEQ ID NO: 46 that form a binding site for human alpha-synuclein.

[0194] In one embodiment, the therapeutic antibody comprises a pair of a heavy chain variable domain of SEQ ID NO: 31 and a light chain variable domain of SEQ ID NO: 32 that form a binding site for the human transferrin receptor, and two pairs of 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 that each form a binding site for human alpha-synuclein.

[0195] In one embodiment, the therapeutic antibody comprises a pair of a heavy chain variable domain of SEQ ID NO: 31 and a light chain variable domain of SEQ ID NO: 32 that form a binding site for the human transferrin receptor (transferrin receptor 1), and two pairs 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 that each form a binding site for human alpha-synuclein.

[0196] In one embodiment, the therapeutic antibody comprises a pair of a heavy chain variable domain of SEQ ID NO: 31 and a light chain variable domain of SEQ ID NO: 32 that form a binding site for the human transferrin receptor (transferrin receptor 1), and two pairs 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 that each form a binding site for human alpha-synuclein.

[0197] In one embodiment, the therapeutic antibody comprises a pair of a heavy chain variable domain of SEQ ID NO: 31 and a light chain variable domain of SEQ ID NO: 32 that form a binding site for the human transferrin receptor (transferrin receptor 1), and two pairs 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 that form binding sites for human alpha-synuclein, respectively.

[0198] In one embodiment, the disease is a neuropathy. In one embodiment, the disease is selected from the group of neuropathy diseases consisting of neuropathy, amyloidosis, cancer, eye diseases or disorders, viral or microbial infections, inflammation, ischemia, neurodegenerative diseases, seizures, behavioral disorders, lysosomal storage diseases, Lewy body disease, post-polio syndrome, Shy-Drager 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 familial insomnia, bulbar palsy, motor neuron disease, nervous system heterotrophic degenerative disorders, Canavan disease, Huntington's disease, neuronal lipidosis, Alexander disease, Tourette syndrome, Menkes kinky hair syndrome, Cockayne syndrome, Hallervorden-Spatz syndrome, Lafora disease, Rett syndrome, hepatocortical degeneration, Lesch-Nyhan syndrome, Unverricht-Lundborg syndrome, dementia, Pick's disease, spinocerebellar ataxia, cancers of the CNS and / or brain (including brain metastases resulting from cancers in other parts of the body). In one embodiment, the disease is selected from the group of neuropathies consisting of Alzheimer's disease, Parkinson's disease, cancers of the CNS and / or brain (including brain metastases resulting from cancers in other parts of the body), and tauopathy. In one embodiment, the disease is selected from the group of neuropathies consisting of Alzheimer's disease, Parkinson's disease and tauopathy.

[0199] In one embodiment, the therapeutic antibody comprises an effector function competent Fc region. In one embodiment, the effector function competent Fc region is an Fc region that specifically binds to human FcγR / can be specifically bound by human FcγR. In one embodiment, the effector function competent Fc region can induce ADCC.

[0200] In one embodiment, the ADCC induced by the bispecific antibody (while binding to the second (cell surface) target during injection) is lower than the ADCC induced 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., lacking one of the binding sites that specifically binds to the first (cell surface) target. In one embodiment, the ADCC is at least 10-fold lower.

[0201] In one embodiment, the administration is intravenous, subcutaneous, or intramuscular administration.

[0202] 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 the formation of a heterodimer.

[0203] In one embodiment, a) the antibody heavy chain is a full-length antibody heavy chain of human subclass IgG1, or b) the antibody heavy chain is a full-length antibody heavy chain of human subclass IgG4, or c) one of the antibody heavy chains is a full-length antibody heavy chain of human subclass IgG1 having the mutations T366W and optionally S354C or Y349C, and the other antibody heavy chain is a full-length antibody heavy chain of human subclass IgG1 having the mutations T366S, L368A, Y407V and optionally Y349C or S354C? d) Both heavy antibody chains have mutations I253A, H310A, and H435A, one of the heavy antibody chains has mutations T366W and optionally S354C or Y349C, and each of the other heavy antibody chains has mutations T366S, L368A, Y407V, and optionally Y349C or S354C, and is a full-length heavy antibody chain of human subclass IgG1, or e) Both heavy antibody chains have mutations M252Y, S254T, and T256E, one of the heavy antibody chains has mutations T366W and optionally S354C or Y349C, and each of the other heavy antibody chains has mutations T366S, L368A, Y407V, and optionally Y349C or S354C, and is a full-length heavy antibody chain of human subclass IgG1, or f) Both heavy antibody chains have mutations T307H and N434H, one of the heavy antibody chains has mutations T366W and optionally S354C or Y349C, and each of the other heavy antibody chains has mutations T366S, L368A, Y407V, and optionally Y349C or S354C, and is a heavy antibody chain of human subclass IgG1.

[0204] In one embodiment, a) Is the heavy antibody chain a heavy antibody chain of human subclass IgG1? b) Is the heavy antibody chain a heavy antibody chain of human subclass IgG4? c) One of the heavy antibody chains is a heavy antibody chain of human subclass IgG1 having mutations T366W and optionally S354C or Y349C, and the other heavy antibody chain is a heavy antibody chain of human subclass IgG1 having mutations T366S, L368A, Y407V, and optionally Y349C or S354C, or d) Both heavy antibody chains have mutations I253A, H310A, and H435A, one of the heavy antibody chains has mutations T366W and optionally S354C or Y349C, and each of the other heavy antibody chains has mutations T366S, L368A, Y407V, and optionally Y349C or S354C, and is a heavy antibody chain of human subclass IgG1, or e) The heavy chains of both antibodies have the mutations M252Y, S254T, and T256E, with the mutation T366W and optionally S354C or Y349C in one of the heavy chains of the antibody, and the mutations T366S, L368A, Y407V, and optionally Y349C or S354C in each of the other heavy chains of the antibody, and are heavy chains of human subclass IgG1, or f) The heavy chains of both antibodies have the mutations T307H and N434H, with the mutation T366W and optionally S354C or Y349C in one of the heavy chains of the antibody, and the mutations T366S, L368A, Y407V, and optionally Y349C or S354C in each of the other heavy chains of the antibody, and are heavy chains of human subclass IgG1, and The C-terminal lysine or glycine-lysine dipeptide is present or absent.

[0205] Specific embodiments of the method according to the invention The relationship between CSF, the blood-brain barrier, and blood is outlined as follows by Katsinelos, T., et al. (Front. Immunol. 10 (2019) 1139). IgG levels are maintained at approximately 10 mg / ml in human serum. The brain is isolated from serum by the blood-brain barrier (BBB), which is impermeable to large macromolecules 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 generated after filtration of blood and transport of ions across the choroid plexus. The resulting concentration of IgG in CSF is approximately 500-1,000 times lower than in serum. In fact, this low concentration of antibody within the brain makes CNS antigens unattractive targets for passive immunotherapy, which is usually administered peripherally. This is exacerbated by the fact that the mechanisms by which antibody steady-state levels are maintained are not well understood. CSF flows around the brain along spinal and cranial nerves before exiting the CNS via drainage into the lymphatic system (Louveau, A., et al., Nature 523 (2015) 337-341; Aspelund, A., J. Exp. Med. 212 (2015) 991-999). Intrathecally administered IgG is rapidly removed from the brain mainly via this bulk flow and may contribute to selective transport from the brain. The neonatal Fc receptor FcRn is abundantly expressed at the BBB (Schlachetzki, F. et al., J. Neurochem. 81 (2002) 203-206). Considering the role of FcRn in transcytosis of antibodies across the placenta, it has been suggested that FcRn may perform reverse transcytosis to help maintain the low IgG environment of the CNS. There is evidence that a portion of antibody clearance from the brain is mediated by the antibody Fc domain (Zhang, Y. and Pardridge, W.M., J. Neuroimmunol. 114 (2001) 168-172; Cooper, P.R., et al., Brain Res. 1534 (2013) 13-21), and antibody efflux of anti-Aβ monoclonal antibody was reduced in FcRn-deficient mice (Deane, R., et al., J. Neurosci. 25 (2005) 11495-11503).However, the brain concentration of peripherally administered IgG did not differ significantly between wild-type mice and mice lacking FcRn (Abuqayyas, L. and Balthasar, J.P., Mol. Pharma. 10 (2013) 1505-1513).

[0206] For small experimental animals, blood is removed from the brain prior to sampling by perfusion. For example, mice can be perfused intracardially with ice-cold PBS at a rate of 2 ml / min for 8 minutes, after which the brain is harvested.

[0207] Methods of transporting therapeutic antibodies across the blood-brain barrier via the receptor-mediated transcytosis pathway using multispecific antibodies, such as bispecific or trispecific antibodies that include one or more carrier molecules and one or more cargo molecules, are currently being studied. For example, transferrin receptor (TfR)-binding antibodies (and variants thereof) can be used as carriers and, when fused to cargo molecules, produce bispecific antibodies that can cross the blood-brain barrier (see, for example, Zuchero, Y.J., Y., et al., Neuron 89 (12016) 70-82; Bien-Ly, N., et al., J. Exp. Med. 211 (2014) 233-244; U.S. Patent Application Publication No. 2018 / 8002433; Canadian Patent No. 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, when fused to cargo molecules, can produce bispecific antibodies that cross the blood-brain barrier (see, for example, International Publication No. 2015 / 131256; International Publication No. 2015 / 131257; International Publication No. 2015 / 131258, which are incorporated herein by reference).

[0208] For the determination of the amount in the brain in a robust and accurate manner, the lysate of the therapeutic antibody transported into the brain through the blood-brain barrier needs to exclude interference from residual blood in the sample. As outlined above, the resulting concentration of IgG in the CSF is about 500 to 1,000 times lower than in serum, and the brain is spanned by an intertwined network of blood vessels. Thus, the possibility of residual blood in the brain tissue sample cannot be ignored. Furthermore, even a small amount of residual blood can significantly interfere with the quantification of antibodies in brain tissue.

[0209] Therefore, correction, i.e., reduction, must be made for the amount of the therapeutic antibody in the residual blood in the brain lysate sample.

[0210] Therefore, the use of a quantitative blood correction marker that does not significantly diffuse into the brain during the perfusion phase is required. However, when these are determined in the steady state, a certain low concentration exists behind the BBB.

[0211] 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 applying a correction antibody immediately before the brain sample is taken. It has been found to be 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, most preferably a human germline antibody, as a reference antibody.

[0212] Accordingly, the present specification reports a method for determining the amount of a therapeutic antibody transported from the blood to the brain of an experimental animal through the blood-brain barrier. This amount is preferably determined in a brain lysate sample. The gist of the present invention lies in further applying an inactivated antibody that is not transported through the blood-brain barrier immediately before obtaining a brain sample in which the amount of the therapeutic antibody transported through the blood-brain barrier must be determined. By applying the inactivated antibody, a correction value for the amount of the therapeutic antibody present in the residual blood in the brain sample is obtained. The amount derived from this residual blood is used to correct the amount determined for the antibody not located in the brain. The determination without correction determines the total amount of the therapeutic antibody in the sample, that is, the amount transported through the blood-brain barrier to the brain and the amount in the residual blood in the sample. Since only about 0.1% of the antibody in the blood passes through the blood-brain barrier, the amount of the therapeutic antibody in the residual blood cannot be ignored. Therefore, the concentration of the therapeutic antibody in the blood is at least two orders of magnitude, up to three orders of magnitude higher than the concentration of the therapeutic antibody in the brain. As a result, the obtained results are too high if not corrected by the method according to the present invention.

[0213] This is particularly important for control IgG or brain shuttles having a clearance close to that of IgG clearance. Because when the molecule is slowly removed, the concentration in the blood is maintained high, and when the amount is relatively very small in the brain, a small amount of blood contamination can overwhelm the determination of the brain concentration. The method according to the present invention can be applied to any brain tissue sample regardless of the method used to remove the blood.

[0214] The method according to the present invention has the availability of cross-species assays, sufficient assay robustness, accuracy and precision, and a wide sensitivity range.

[0215] Briefly described, the present invention is a method for determining residual blood in a brain sample of an experimental animal, - providing a method of administering a second inactivated IgG, collecting a plasma sample, perfusing, measuring and correcting the brain and plasma concentrations immediately before perfusion, i.e., up to 5 minutes before, - The advantage is that the amount passing through the blood-brain barrier is limited, whereby the concentration of the second inert antibody reflects only the plasma volume, - A first specific assay for the therapeutic antibody and a second specific assay for the second inert antibody are used, - Problems regarding anti-therapeutic antibody-positive animals that could confound the measurement could be prevented.

[0216] Figure 1 provides an exemplary calculation utilizing the use of an inert antibody to determine blood contamination in a (brain) tissue sample.

[0217] For example, conventional ELISA is used to determine the concentrations of a therapeutic monoclonal antibody (tmAb) and an inert reference monoclonal antibody (refmAb) in plasma as well as in a homogenized brain tissue sample. The results of the ELISA are usually obtained as mass concentrations using SI units [g / L]. In a first step, each of the mass concentrations determined for tmAb and refmAb is converted to a mass fraction having the unit [g / g] by dividing the determined mass concentration by the brain tissue concentration of the sample. In a second step, the amount of residual plasma 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 plasma concentration of the determined refmAb. Thereby, the volume of residual plasma per weight of the brain sample is obtained. In a third step, the mass fraction of tmAb in the brain tissue sample due to plasma contamination is calculated by multiplying the plasma concentration of tmAb by the volume of residual plasma per weight of the brain sample. In a fourth and final step, the true brain concentration of tmAb is obtained by subtracting the mass fraction of tmAb in the brain tissue sample due to plasma contamination obtained in the third step from the mass fraction determined for tmAb in the first step.

[0218] The method according to the invention was applied to the analysis of two bispecific antibodies that bind to TfR and therapeutic target 1 or 2, respectively, in cynomolgus monkey brain lysates. The respective structures of the antibodies are shown in Figure 2. The detection assays for the therapeutic antibody and the reference antibody are shown in Figure 3, respectively.

[0219] As outlined in Example 1, the assay for determining the inert antibody has a sensitivity of 8 ng / ml, i.e., it can detect about 1.1 - 1.5 μL plasma / 1 g of cynomolgus monkey brain (this corresponds to about 2.2 - 3 μL blood / 1 g of cynomolgus monkey brain).

[0220] Five different brain regions: cerebellum, hippocampus, striatum, cortex and choroid plexus have been analyzed.

[0221] When four different animals were analyzed, the brain samples of animals 1 - 3 did not contain residual blood, while animal 4 was as determined by optical analysis (data not shown).

[0222] With the method according to the invention, this contamination can be detected and thus the respective values can be corrected accordingly. [Table 1] [Table 2]

[0223] In further studies, 15 animals were administered with 20 mg / kg of anti-A beta antibody and 15 animals were administered with 10 mg / kg of anti-A beta / TfR antibody. After various time points after administration, the respective samples were analyzed. Residual blood was detected in each brain tissue sample in all samples. Thus, also in these cases, corrected values were obtained using the method according to the invention. [Table 3] TIFF0007692916000009.tif252161 TIFF0007692916000010.tif252161 TIFF0007692916000011.tif196161

[0224] To demonstrate the general applicability of the method according to the present invention, the same analysis was performed in C57BL / 6 wild-type mice using a second antibody, an anti-TfR / target_2 antibody.

[0225] Figure 4 shows the overlay of calibration curves of the detection assay of an inactivated reference antibody in the presence of 1% cynomolgus 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.

[0226] The measurement range of the assay in the presence of 1% MBL is 8.4 ng / mL to 250 ng / mL. Therapeutic antibodies up to 10 μg / mL can be present in the assay system without interference in the presence of 1% MBL.

[0227] The measurement range of the assay in the presence of 1% mouse pooled plasma (MPP) is 11 ng / mL to 220 ng / mL. Therapeutic antibodies up to 20 μg / mL can be present in the assay system without interference in the presence of 1% MPP.

[0228] A single dose of 20 mg / ml of antibody was applied, and samples were analyzed 24 hours, 48 hours, 96 hours, 168 hours, 336 hours, 504 hours, and 672 hours after application. The respective concentrations in the brain lysate and plasma were determined. In Figure 5, the determined concentration of the treated antibody in the brain lysate is shown as the ratio of the uncorrected brain concentration to the corrected brain concentration. That is, if the correction does not affect the value, the ratio is 1. If the determined concentration of the second antibody decreases due to correction for the residual plasma value, the value becomes less than 1. This difference increases with time as more antibody is transported across the blood-brain barrier. It can be seen from Figure 5 that the ratio decreases with time. Thereby, it can be seen that the correction performed according to the method of the present invention eliminated the interference from residual blood in the brain sample. The respective assays used for the determination of the second antibody are shown in Figure 6.

[0229] Inactivated reference monoclonal antibody of the method according to the present invention An inert reference monoclonal antibody useful in the method according to the present invention is preferably a human immunoglobulin molecule, particularly a human immunoglobulin molecule having no specific binding ability to an antigen.

[0230] An exemplary inert reference monoclonal antibody is antibody DP47GS. DP47GS includes 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.

[0231] In one embodiment, the inert reference monoclonal antibody is an IgG class immunoglobulin molecule, particularly 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 has no specific binding ability to an antigen. In one embodiment, the inert reference monoclonal antibody includes a heavy chain variable region sequence based on the human VH3-23 germline sequence. In a particular embodiment, the inert reference monoclonal antibody includes the heavy chain variable region sequence of SEQ ID NO: 67. In one embodiment, the inert reference monoclonal antibody includes a light chain variable region sequence based on the human Vk3-20 germline sequence. In a particular embodiment, the inert reference monoclonal antibody includes the light chain variable region sequence of SEQ ID NO: 68. In an even more specific embodiment, the inert reference monoclonal antibody includes 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 has no specific binding ability to an antigen and includes 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.

[0232] In one embodiment, the inactivated reference monoclonal antibody comprises a heavy chain variable region sequence based on the human VH3-23 germline sequence. In certain embodiments, the inactivated 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 inactivated reference monoclonal antibody comprises a light chain variable region sequence based on the human Vk3-20 germline sequence. In certain embodiments, the inactivated 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 inactivated 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 are unable to specifically bind to antigens, particularly human antigens. They are unable to bind 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 based overall on human germline sequences, except for the heavy chain CDR3 into which a GSG sequence has been introduced to generate a non-binding immunoglobulin.

[0233] In one embodiment, the inactivated reference monoclonal antibody comprises a heavy chain having a variable domain with the amino acid sequence of SEQ ID NO: 67 and a human IgG1 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 inactivated reference monoclonal antibody comprises mutations L234A, L235A and P329G (numbering according to the KabatEU index) in the heavy chain Fc region.

[0234] In one embodiment, the inactivated reference monoclonal antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 69 and a light chain having the amino acid sequence of SEQ ID NO: 70.

[0235] Comparison methods and results Comparative technical approach: Correction for residual blood volume without perfusion Friden et al. (J. Cerebral Blood Flow & Met 30 (2010) 150 - 161) collected the information available from the literature on the cerebrovascular space (see Table 1 of Friden et al.).

[0236] Based on the most commonly used 14C - dextran method, the cerebral plasma value corresponds to approximately 18.1 μL per 1 g of brain tissue. Applying this correction, all the determined values became negative.

[0237] Therefore, it was not correct to simply consider that the total cerebral plasma value was indicated in the brain tissue sample.

[0238] Therefore, it was not possible to apply the absolute value, and a co - determined correction factor was required.

[0239] Therefore, various correction factors were required.

[0240] Since perfusion will be performed, control for the remaining blood contamination is required. This is particularly important for comparative IgG or brain shuttle having a clearance similar to that of IgG clearance - why? When the molecule is removed slowly, the concentration in the blood is maintained high, so when the amount in the brain is relatively very small, a small amount of blood contamination can overwhelm the determination of the brain concentration.

[0241] Therefore, it is necessary to use a quantitative blood correction marker that is applied into the brain and does not significantly diffuse into the perfusion phase.

[0242] Comparative markers: Prior to those tests, other different non - antibody inert reference molecules that were considered to be similarly suitable as correction means in the method according to the present invention are other endogenous proteins with high molecular weight and high endogenous blood levels.

[0243] Determination of Complement Factor H Rhesus publications have shown that complement factor H is absent in rhesus cerebrospinal fluid (CSF) as well as in rhesus brain lysates, which is common in non-CSF or non-brain tissues. Therefore, the detection of complement factor H is considered to be a viable surrogate marker for the determination of residual contaminated blood in cCSF and CBL samples.

[0244] As positive controls, human pooled serum (HPS; 200 μg / mL - 800 μg / mL complement factor H) and human pooled plasma (HPP; approximately 300 μg / mL of complement factor H) were available.

[0245] The assay was configured as an Elecsys assay (Roche Diagnostics GmbH, Mannheim, Germany). The respective calibration curves are shown in Figure 7. The measurement range of this assay was 7.8 μg / mL - 2000 μg / mL.

Table 4

[0246] Therefore, since the assay was not sensitive enough, it was found that the determination of complement factor H was not suitable as a surrogate marker for residual contaminated blood.

[0247] Determination of alpha-2-macroglobulin Various publications have shown that alpha2-macroglobulin is absent in rhesus cerebrospinal fluid (CSF) and rhesus brain lysates and is common in non-CSF or non-brain tissues (1500 - 2000 μg / mL). Therefore, the detection of alpha2-macroglobulin is considered to be a viable surrogate marker for the determination of residual contaminated blood in cCSF and CBL samples.

[0248] The assay principle of the ELISA assay for determining alpha2-macroglobulin is shown in Figure 8, and the respective calibration curves are shown in Figure 9.

[0249] The assay had a measurement range of 0.62 ng / mL (LLOQ) to 39 ng / mL (ULOQ).

Table 5

[0250] Expected values for serum and plasma could be confirmed, but in cynomolgus monkey pooled serum, only 1 / 25,000 of the expected amount could be detected. Therefore, this value is too low to quantify cynomolgus monkey cerebrospinal fluid and brain lysates in a diluted form. Therefore, the determination of α2-macroglobulin is not suitable as a surrogate marker.

[0251] Determination of complement component 5a (C5a) Various publications have shown that complement component 5a is absent in cynomolgus monkey cerebrospinal fluid (CSF) and cynomolgus monkey brain lysates, which is common in non-CSF or non-brain tissues (60 - 110 μg / mL in human serum). Therefore, the detection of complement component C5a is considered to be a feasible surrogate marker for the determination of residual contaminated blood in cCSF and CBL samples.

[0252] Similar to α2-macroglobulin, ELISA was set up using a mouse anti-human C5a antibody as the capture antibody and a biotinylated mouse anti-human C5a antibody as the detection antibody, whereby both antibodies bind to non-interfering epitopes on human C5a. The respective calibration curves are shown in Figure 10.

[0253] The assay had a measurement range of 0.03 ng / mL (LLOQ) to 2 ng / mL (ULOQ).

Table 6

[0254] Therefore, it was found that C5a in CBL samples could be determined.

[0255] Therefore, the determination of C5a is not suitable as a surrogate marker.

[0256] Use of Magnevist (registered trademark) Magnevist (registered trademark) (gadopentetate dimeglumine) is an MRT contrast agent. Magnevist (registered trademark) was thought not to cross the blood-brain barrier.

[0257] Pharmacokinetic studies showed that only the measured brain concentration accurately represented the blood compartment up to 15 minutes. After this time, Magnevist (registered trademark) diffused into the brain tissue, confounding the applied corrections. The respective time courses are shown in Figure 11. At the 5-minute time point, the plasma volume was estimated to be 14.1 μL / brain 1 g.

[0258] Therefore, this approach cannot be used for perfusion because the time taken for perfusion results in the diffusion of Magnevist through the blood-brain barrier, confounding the residual blood correction.

[0259] Determination of cynomolgus IgG in cynomolgus cerebrospinal fluid (cCSF) Various publications have shown that cynomolgus cerebrospinal fluid (cerebrospinal fluid) contains only small amounts of cynomolgus IgG. Therefore, detection of total Ig in cCSF is considered to be a viable surrogate marker for the direct determination of the transported therapeutic antibody. Therefore, a cross-linked ELISA was constructed as shown in Figure 12. To eliminate matrix effects, human IgG-depleted cCSF was generated by incubating cCSF with anti-human CH1 / kappa antibodies bound to magnetic beads.

[0260] The respective calibration curves using buffer and human IgG-depleted cCSF are shown in Figure 13. It can be seen that no matrix effect occurred.

[0261] The assay had a measurement range of 120 ng / ml to 7.2 ng / mL IgG.

[0262] Using this assay, it was found that IgG can be detected at approximately 11 - 19 mg / mL in cynomolgus pooled plasma samples (CPP), while an equivalent amount of cynomolgus IgG can be detected at approximately 4 - 18 μg / mL in cCSF samples. ***

[0263] The following examples, sequences, and drawings are provided to assist in the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that changes can be made to the defined procedures without departing from the spirit of the present invention.

[0264] Description of Sequences [Table 7] TIFF0007692916000016.tif126161 [Brief Description of Drawings]

[0265]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

[0266] General method: Preparation of cynomolgus brain tissue homogenate 300 mg of frozen cynomolgus / mouse brain tissue samples were thawed at room temperature for 2 hours. 800 μL of lysis buffer and one tablet of cOmplete protease inhibitor cocktail (Roche Diagnostics GmbH) dissolved in 50 mL of tissue extraction reagent I (Invitrogen) were added to the thawed brain tissue. The samples were then homogenized in a MagNA Lyser device (Roche Diagnostics) at 6500 rpm for 20 seconds. The tissue homogenate was then centrifuged at 12,000 rpm for 10 minutes using a Centrifuge 5430 (Eppendorf). Finally, the supernatant was transferred to 1.5 mL vials for further analysis or stored at -80 °C.

[0267] Example 1 ELISA for quantification of DP47GS-PGLALA in brain lysates To quantify the inactive reference monoclonal antibody DP47GS-PGLALA (SEQ ID NOs: 69 and 70) in cynomolgus monkey brain lysate samples, a sequential sandwich enzyme-linked immunosorbent assay (ELISA) was used. In the ELISA procedure, all samples and controls were subjected to an initial 1:100 pre-dilution in assay diluent to the desired final assay concentration of 1%.

[0268] To the streptavidin-coated microtiter plate (SA-MTP), capture antibody (anti-DP47GS antibody, biotinylated), dilution standards (DP47GS-PGLALA), as well as diluted quality control and samples, detection reagent (anti-PGLALA antibody clone M-1.7.24, digoxigenylated) and anti-digoxigenin antibody-POD conjugate were added sequentially. The reagents were incubated for 1 hour at 500 rpm on an MTP shaker, and after each step, the MTP was washed 3 times with 300 μL of wash buffer (1× PBS, 0.05% Tween) to remove residual fluid. Thereafter, the formed immobilized immune complex was visualized by adding an ABTS solution, a horseradish POD substrate, which was converted to a colored reaction product. Finally, the color intensity was determined photometrically (absorption at a reference wavelength of 405 nm - 490 nm). The signal is proportional to the analyte concentration in the brain lysate sample. Quantification of DP47GS-PGLALA was performed by back-calculation of the absorbance values using the corresponding calibration curve with a weighted non-linear four-parameter Wiemer-Rodbard curve fitting function.

Table 8

[0269] Coating with the capture reagent was achieved by pipetting 100 μL of a solution containing 500 ng / mL of biotinylated anti-DP47GS antibody into each SA-MTP well. Thereafter, the MTP was covered with an adhesive cover foil and incubated for 1 hour on an MTP shaker (500 rpm). The supernatant was removed, and each well of the MTP was washed 3 times with 300 μL of wash buffer (PBS, 0.05% Tween). The residual wash buffer was carefully removed.

[0270] Next, 100 μl of each standard, quality control, and sample are added to the designated wells of the coated MTP. The MTP is then covered with an adhesive cover foil and incubated on an MTP shaker (500 rpm) for 1 hour. The supernatant is removed, and each well of the MTP is washed three times with 300 μL of wash buffer (PBS, 0.05% Tween). The residual wash buffer is carefully removed.

[0271] Next, 100 μL of digoxigenylated anti-PGLALA antibody clone M-1.7.24 at a concentration of 125 ng / mL is added to each MTP well. The MTP is then covered with an adhesive cover foil and incubated on an MTP shaker (500 rpm) for 1 hour. The supernatant is removed, and each well of the MTP is washed three times with 300 μL of wash buffer (PBS, 0.05% Tween). The residual wash buffer is carefully removed.

[0272] 100 μL of anti-digoxigenin antibody-POD-conjugate at a concentration of 50 mU / mL is added to each MTP well. The MTP is then covered with an adhesive cover foil and incubated on an MTP shaker (500 rpm) for 1 hour. The supernatant is removed, and each well of the MTP is washed three times with 300 μL of wash buffer (PBS, 0.05% Tween). The residual wash buffer is carefully removed.

[0273] Next, 100 μL of ABTS solution is added to each MTP well. The absorbance is measured until the average signal of the duplicate standard sample 1 reaches 1.8 - 2.2 AU at a measurement wavelength of 405 nm (reference wavelength 490 nm).

[0274] Example 2 ELISA for quantification of cynomolgus IgG in CSF To quantify cynomolgus IgG in cynomolgus cerebrospinal fluid, a sequential sandwich enzyme-linked immunosorbent assay (ELISA) was used. In the ELISA procedure, all samples and controls were subjected to an initial pre-dilution in assay diluent to the desired final assay concentration of 1%.

[0275] To the streptavidin-coated microtiter plate (SA-MTP), a capture antibody (anti-cynomolgus IgG antibody 1; epitope 1; biotinylated), diluted standards, as well as diluted quality controls and samples, a detection reagent (anti-cynomolgus IgG antibody 2; epitope 2, which does not interfere with epitope 1; digoxigenylated) and an anti-digoxigenin antibody-POD conjugate were added sequentially. The reagents were incubated for 1 hour at 500 rpm on an MTP shaker, and after each step, the MTP was washed three times with 300 μL of wash buffer (1× PBS, 0.05% Tween) to remove residual fluid. Subsequently, the formed immobilized immune complexes were visualized by adding an ABTS solution, a horseradish POD substrate, which was converted to a colored reaction product. Finally, the color intensity was determined photometrically (absorbance at a reference wavelength of 405 nm - 490 nm). The signal is proportional to the analyte concentration in the brain lysate sample. Quantification of cynomolgus IgG was performed by inverse calculation of absorbance values using a non-linear four-parameter Wiemer-Rodbard curve fitting function with weighting, using the corresponding calibration curve.

Table 9

[0276] Coating with the capture reagent was achieved by pipetting 100 μL of a solution containing 250 ng / mL of biotinylated anti-cynomolgus IgG antibody 1 into each SA-MTP well. Subsequently, the MTP was covered with an adhesive cover foil and incubated for 1 hour on an MTP shaker (500 rpm). The supernatant was removed and each well of the MTP was washed three times with 300 μL of wash buffer (PBS, 0.05% Tween). The residual wash buffer was carefully removed.

[0277] Next, 100 μl of each standard substance, quality control, and sample are added to the designated wells of the coated MTP. Subsequently, the MTP is covered with an adhesive cover foil and incubated on an MTP shaker for 1 hour (500 rpm). The supernatant is removed, and each well of the MTP is washed three times with 300 μL of wash buffer (PBS, 0.05% Tween). The residual wash buffer is carefully removed.

[0278] Next, 100 μL of digoxigenylated anti-cynomolgus monkey antibody 2 at a concentration of 250 ng / mL is added to each MTP well. Subsequently, the MTP is covered with an adhesive cover foil and incubated on an MTP shaker (500 rpm) for 1 hour. The supernatant is removed, and each well of the MTP is washed three times with 300 μL of wash buffer (PBS, 0.05% Tween). The residual wash buffer is carefully removed.

[0279] 100 μL of anti-digoxigenin antibody-POD-conjugate at a concentration of 25 mU / mL is added to each MTP well. Subsequently, the MTP is covered with an adhesive cover foil and incubated on an MTP shaker (500 rpm) for 1 hour. The supernatant is removed, and each well of the MTP is washed three times with 300 μL of wash buffer (PBS, 0.05% Tween). The residual wash buffer is carefully removed.

[0280] Next, 100 μL of ABTS solution is added to each MTP well. The absorbance is measured until the average signal of the duplicate standard substance sample 1 reaches 1.8 - 2.2 AU at a measurement wavelength of 405 nm (reference wavelength 490 nm).

[0281] Example 3 Preparation of brain tissue lysate First, the lysis buffer was freshly prepared according to the manufacturer's (Invitrogen; Tissue Extraction Reagent I; Catalog number. FNN0071) instructions. One tablet of Complete (Roche Diagnostics GmbH, Mannheim, Germany; Catalog number. 11697498001) was added per 50 ml of lysis buffer.

[0282] Second, add about 100 to 300 mg and 600 μL to 800 μL of lysis buffer to the brain tissue sample. Optionally, add MagNA Lyser Green Beads.

[0283] Third, place the sample in a MagNA Lyser (Roche Diagnostics GmbH, Mannheim, Germany) at 6500 rpm for 20 seconds.

[0284] Fourth, after incubation in the MagNA Lyser, centrifuge the sample at 12,000 rpm for 10 minutes (Eppendorf Centrifuge 5430).

[0285] Fifth, collect the supernatant (500 - 700 μL) and store it at -80 °C until further analysis.

Claims

1. A method or assay for determining the concentration of a therapeutic antibody in the tissue of an experimental animal, wherein the tissue has a barrier to the blood circulation of the animal, the therapeutic antibody has been administered to the experimental animal, and interference from residual blood in the tissue sample of the experimental animal used to determine the concentration of the therapeutic antibody in the tissue is reduced, the method comprising: i) determining the concentration of the therapeutic antibody in a blood sample of the experimental animal; ii) determining the concentration of the therapeutic antibody in the tissue sample of the experimental animal; iii) determining the concentration of an inert reference antibody in the blood sample of the experimental animal; iv) determining the concentration of the inert reference antibody in the tissue sample of the experimental animal; v) determining the tissue concentration in the tissue sample, and the following formula: [Equation 1] (wherein, C ｔｍＡｂ、血漿、ｄｅｔ． = the concentration of the therapeutic antibody in i) C ｔｍＡｂ、組織、ｄｅｔ． = the concentration of the therapeutic antibody in ii) C ｒｅｆｍＡｂ、組織、ｄｅｔ． = the concentration of the inert reference antibody in iii) C ｒｅｆｍＡｂ、血漿、ｄｅｔ． = the concentration of the inert reference antibody in iv) C 組織、サンプル = the tissue concentration in v)) using to determine the concentration of the therapeutic antibody in the tissue of the experimental animal and - the inert reference antibody does not pass through the barrier between the tissue and the blood circulation, - the inert reference antibody is administered either together with the therapeutic antibody if the sample is taken within 5 minutes after administration of the therapeutic antibody, or at any time between 2 and 10 minutes before taking the tissue sample, - the blood sample is taken immediately before the tissue sample, method or assay.

2. The method according to claim 1, wherein the tissue is brain tissue and the therapeutic antibody can pass through the blood-brain barrier, or the tissue is eye tissue and the therapeutic antibody can pass through the blood-eye barrier.

3. The method according to any one of claims 1 or 2, wherein the therapeutic antibody is a bispecific antibody.

4. The method according to any one of claims 1 to 3, wherein the therapeutic antibody specifically binds to the human transferrin receptor and a brain target.

5. The method according to claim 4, wherein the brain target is human CD20 or human Aβ or human alpha-synuclein or human tau or human glucocerebrosidase or human LINGO-1 or human huntingtin.

6. The method according to any one of claims 1 to 5, wherein the experimental animal is selected from mice, rats, rabbits, dogs, sheep, monkeys and apes.

7. 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.

8. The method according to any one of claims 1 to 7, wherein the experimental animal is a cynomolgus monkey.

9. The method according to any one of claims 1 to 8, wherein the inert reference antibody is a human germline antibody.

10. The method according to any one of claims 1 to 9, wherein the inert reference antibody is DP47GS.

11. The method according to any one of claims 1 to 10, wherein the inert reference antibody does not pass through the barrier in a detectable amount within 15 minutes after its application.

12. The method according to claim 11, wherein the inert reference antibody does not pass through the barrier in a detectable amount within 10 minutes after its application.

13. The method according to any one of claims 1 to 12, wherein the inactivated reference antibody is administered about 5 minutes before collecting the tissue sample.

14. The method according to any one of claims 1 to 10, wherein the tissue is perfused with an aqueous solution immediately after collecting the blood sample and before collecting the tissue sample.

15. The method according to any one of claims 1 to 11, wherein the determination of the concentration is by cross-linking ELISA.

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