Prediction method for immunoglobulin-binding glycans

Abstract

The present invention analyzes an N-linked glycan linked to a protein in a blood-derived sample collected from a subject, and, on the basis of the structure of the N-linked glycan, predicts the structure of a glycan linked to immunoglobulin in the subject's blood after administration when the immunoglobulin has been administered to the subject.

Description

[Technical Field]

[0001] The present invention relates to a method for predicting the structure of a sugar chain bound to an immunoglobulin such as an antibody drug in the blood of a subject after the administration of the immunoglobulin to the subject. [Background technology]

[0002] In recent years, pharmaceuticals containing immunoglobulins (antibodies) (antibody drugs) have been used to treat cancer, immune diseases, etc. The antibodies used in antibody drugs are created using genetic engineering techniques, and are produced by culturing cells capable of expressing the antibodies (e.g., Chinese hamster ovary (CHO) cells), and then purifying them to a high purity using column chromatography or other methods.

[0003] However, recent research has revealed that antibodies obtained by the above-described production process are aggregates of various molecules due to modifications such as oxidation, reduction, isomerization, and glycosylation, raising concerns about their impact on efficacy and safety. In particular, it has been reported that the glycan structure attached to an antibody has a significant impact on the activity, kinetics, and safety of antibody drugs, making detailed analysis of the glycan structure important (Non-Patent Document 1). Furthermore, Non-Patent Document 2 reports that, depending on the case of a patient receiving an antibody drug, the glycan structure attached to the administered antibody drug may change in the blood, and this change in glycan structure may affect the blood retention of the antibody drug. Non-Patent Document 3 reports that changes in the glycan structure of an antibody drug affect the blood retention of the antibody drug, and in particular, it is also mentioned that the glycan structure involved in the binding ability to FcγR can affect blood retention. Furthermore, as shown in Table 5 of Non-Patent Document 4, etc., it has also been shown that mannose, galactose, GlcNAc (N-acetylglucosamine), and sialic acid NANA (N-acetylneuraminic acid) are involved in antibody clearance.

[0004] Given these findings, if we could predict in advance the changes in the glycan structure of antibody drugs administered to disease patients, it would lead to predicting the impact on drug efficacy and safety. However, until now, it has been difficult to predict in advance the changes in the bound glycans of immunoglobulins such as antibody drugs. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] CHROMATOGRAPHY, 34(2), 83-88(2013) [Non-patent document 2] Pharmaceutical Research, 36, 82(2019) [Non-patent document 3] Cancer Research, 70, 4481-4489(2010) [Non-patent document 4] Journal of Pharmaceutical Sciences, 104, 1866-1884(2015) Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to provide a method for predicting the sugar chain structure of immunoglobulins such as antibody pharmaceuticals administered to subjects such as disease patients. [Means for solving the problem]

[0007] As a result of intensive research aimed at solving the above-mentioned problems, the present inventors have found that, when immunoglobulin is administered to a subject, the structure of glycans bound to endogenous proteins in a blood-derived sample correlates with the structure of glycans bound to the immunoglobulin in the blood after the administration. In other words, the present inventors have found that by analyzing the glycan structures of endogenous proteins contained in a blood-derived sample, it is possible to predict the glycans bound to immunoglobulins such as antibody drugs administered to the subject, and have completed the present invention.

[0008] That is, the present invention includes the following aspects [1] to [9]. [1] A method for predicting the structure of a sugar chain bound to an exogenous immunoglobulin in the blood of a subject after administration of the exogenous immunoglobulin, the method comprising the following steps (1) and (2): (1) analyzing the structure of N-linked glycans bound to endogenous proteins in a blood-derived sample collected from the subject; (2) A method for determining the structure of the sugar chain bound to the exogenous immunoglobulin based on the structure of the N-linked sugar chain obtained in (1). [2] The method according to [1], wherein the sugar chain bound to the exogenous immunoglobulin is a sugar chain involved in regulating cell function or immune activation. [3] The method according to [1] or [2], wherein the endogenous protein is an endogenous immunoglobulin. [4] The method according to any one of [1] to [3], wherein the structure of the N-linked sugar chain is analyzed using a column packed with an insoluble carrier on which an Fc-binding protein has been immobilized. [5] A method for predicting the blood retention of the exogenous immunoglobulin in the subject based on the sugar chain structure predicted by the method according to any one of [1] to [4]. [6] The method according to any one of [1] to [5], wherein the exogenous immunoglobulin is an antibody drug. [7] A method for predicting the efficacy of the antibody drug in the subject based on the sugar chain structure predicted by the method described in [6]. [8] The method according to any one of [4] to [7], wherein the Fc-binding protein is FcγRIIIa. [9] The method according to any one of [4] to [7], wherein the Fc-binding protein is a polypeptide selected from the group consisting of the following (1) to (3): (1) A polypeptide comprising a sequence consisting of amino acid residues 17 to 192 set forth in SEQ ID NO: 1, in which at least valine at position 176 set forth in SEQ ID NO: 1 is substituted with phenylalanine; (2) A polypeptide comprising a sequence consisting of amino acid residues 17 to 192 set forth in SEQ ID NO: 1, in which at least valine at position 176 set forth in SEQ ID NO: 1 is substituted with phenylalanine, and further having substitutions, deletions, insertions, and / or additions of one or several amino acid residues at one or several positions other than position 176, and having antibody-binding activity; (3) A polypeptide having an amino acid sequence having 70% or more homology with the sequence consisting of amino acid residues 17 to 192 of SEQ ID NO: 1, in which the amino acid residue corresponding to valine at position 176 of SEQ ID NO: 1 is substituted with phenylalanine, and having antibody binding activity. [Effects of the Invention]

[0009] According to the present invention, when immunoglobulin is administered to a subject, it is possible to predict the structure of the sugar chain bound to the immunoglobulin in the subject's blood after the administration.

[0010] Because the bound glycans of immunoglobulins administered to a subject change in their blood, it has been difficult to predict the activity, kinetics, and safety of immunoglobulins (antibody drugs, etc.) after administration simply by analyzing the bound glycans of immunoglobulins before administration. However, according to the present invention, by analyzing the N-linked glycans bound to proteins contained in a patient's blood sample before administration, it is possible to predict the bound glycans of antibody drugs in the sample after administration, making it possible to predict the activity, kinetics, and safety of antibody drugs after administration, which was previously difficult. This can also be used as an important biomarker for personalized medicine for patients and as data to assist in determining treatment strategies. For example, predicting the activity, kinetics, and safety of antibody drugs based on the predicted changes in the bound glycans of antibody drugs in the patient's body before administration can be used to determine the dosage and frequency of antibody drugs, as well as the selection of different dosages and treatments. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 is a schematic diagram showing the purification process of rituximab contained in serum using anti-rituximab antibody-modified magnetic particles. [Figure 2] 1 is a chromatogram showing an example of the separation patterns of a standard substance and a measurement sample obtained by analyzing an antibody using a column packed with an Fc-binding protein-immobilized gel. [Figure 3] 1 is a dot plot showing the correlation between the separation patterns of serum gamma globulins before administration of an antibody pharmaceutical and purified rituximab after administration. [Figure 4] These are chromatograms showing the separation patterns obtained when analyzing antibodies using a column packed with an Fc-binding protein-immobilized gel. (a) shows the results when rituximab was analyzed as the standard substance. (b) shows the results when serum gamma globulin and purified rituximab were analyzed for each of the patients with the diseases listed in Figure 3, specimens A to D. [Figure 5] 1 is a graph showing the amount of rituximab purified after administration to patients with each disease. [Figure 6]FIG. 1 shows the correlation between the third peak area % and blood retention of purified rituximab solutions. [Figure 7] 1 is a graph showing the third peak area % in the separation pattern of serum gamma globulin before or after administration of rituximab, the number of days from administration of the rituximab to blood collection, and the blood rituximab concentration at the time of blood collection. DETAILED DESCRIPTION OF THE INVENTION

[0012] The present invention will be described in detail below. As shown in the Examples below, the present inventors have found that, when immunoglobulin is administered to a subject, the structure of a sugar chain bound to an endogenous protein in a blood-derived sample collected from the subject correlates with the structure of a sugar chain bound to the immunoglobulin in the blood after the administration. The present invention was completed based on this finding, and relates to a method for predicting the structure of a sugar chain bound to an exogenous immunoglobulin in the blood of a subject after the administration of the exogenous immunoglobulin, comprising the following steps (1) and (2): (1) analyzing the structure of N-linked glycans bound to endogenous proteins in a blood-derived sample collected from the subject; (2) A method for determining the structure of the sugar chain bound to the exogenous immunoglobulin based on the structure of the N-linked sugar chain obtained in (1).

[0013] <Subjects and blood-derived samples collected from subjects> The "subject" of the present invention refers to an individual subject to the method of the present invention and is not particularly limited, and may include not only humans but also non-human animals. Examples of such subjects include vertebrates, preferably mammals, more preferably primates (e.g., humans, monkeys, chimpanzees, orangutans, gorillas, etc.), ungulates (e.g., cattle, horses, sheep, goats, etc.), and rodents (e.g., mice, rats, guinea pigs), but are typically humans. Subjects may be male or female. They may be of any age, including children, young people, middle-aged people, and elderly people. They may also be healthy individuals, or individuals suffering from a disease, those undergoing or having undergone treatment for a disease, those at risk of disease recurrence, or those suspected of suffering from a disease. Such diseases include, but are not limited to, diseases that are the target of antibody pharmaceuticals, as described below, such as cancer, autoimmune diseases, infectious diseases, allergies, and inflammatory diseases.

[0014] In the present invention, a "blood-derived sample" refers to a solution containing blood (whole blood) or a component thereof, which contains or may contain a protein having an N-linked glycan, as described below. The solution may be in the form of a body fluid itself, such as blood or urine, as described below. Blood samples such as whole blood, diluted blood, serum, plasma, cerebrospinal fluid, cord blood, and apheresis; Specimens that may contain blood-derived components, such as urine, saliva, semen, feces, sputum, amniotic fluid, and peritoneal fluid; Furthermore, the protein may be in the form of a buffer solution that can contain the protein separated from these body fluids or the protein contained therein. These blood-derived samples may be used directly in the method of the present invention described below, or may be used after appropriate pretreatment. Pretreatment can be carried out by an appropriate method selected from conventional methods. Conventional methods include centrifugation, column purification, etc.

[0015] <Endogenous proteins> In the present invention, "endogenous protein" refers to a protein having a sugar chain bound to an asparagine side chain of the protein via an amide β-linked N-acetylglucosamine as the starting point. Note that "endogenous protein" includes not only proteins that are naturally expressed and present in the blood of the subject as described above, but also proteins that are present in the blood before or at the time of administration of exogenous immunoglobulin, as described below. In other words, it also includes exogenous proteins that were administered to the subject before administration of exogenous immunoglobulin and were already present in the blood.

[0016] There are no particular restrictions on the proteins with N-linked glycans to be subjected to glycan analysis, but immunoglobulins contained in blood-derived samples are preferred, as they resemble the protein structure of the exogenous immunoglobulins whose glycan structures are to be predicted.

[0017] "Immunoglobulin" is also called gamma globulin or antibody, and is sufficient as long as it contains at least a glycosylated Fc region. The immunoglobulin may be any of IgG, IgM, IgA, IgD, and IgE. However, when an Fc receptor is used as the Fc-binding protein used to separate immunoglobulins, as described below, the immunoglobulin must be compatible with the receptor. For example, when a human Fcγ receptor is used as the Fc-binding protein, the target antibody is human IgG. The IgG may be any of IgG1, IgG2, IgG3, and IgG4.

[0018] <Exogenous immunoglobulin> In the present invention, "exogenous immunoglobulin" refers to an immunoglobulin administered to a subject. Immunoglobulins are as described above, but exogenous immunoglobulins may contain amino acids to which N-linked glycans can be attached, regardless of whether the N-linked glycans are modified. Exogenous immunoglobulins may also be monoclonal or polyclonal antibodies. Furthermore, exogenous immunoglobulins include chimeric antibodies, humanized antibodies, and human antibodies. Exogenous immunoglobulins also include functional fragments of immunoglobulins. Examples of "functional fragments" include Fab, Fab', F(ab')2, variable region fragments (Fv), disulfide-linked Fv, single-chain Fv (scFv), sc(Fv)2, diabodies, multispecific antibodies, and polymers thereof.

[0019] Furthermore, as shown in the examples of antibody drugs described below (trastuzumab emtansine, brentuximab vedotin, emtuzumab ozogamicin, ibritumomab tiuxetan, tositumomab, etc.), the exogenous immunoglobulin may take the form of an antibody-drug conjugate. An "antibody-drug conjugate (ADC)" is an immunoglobulin covalently bound to a chemotherapeutic agent (drug) via a linker. Such chemotherapeutic agents are not particularly limited, but examples include alkylating agents such as deruxtecan, irinotecan (CPT-11), irinotecan metabolite SN-38 (10-hydroxy-7-ethylcamptothecin), adriamycin, taxol, 5-fluorouracil, nimustine, and laministine; metabolic antagonists such as gemcitabine and hydroxycarbamide; plant alkaloids such as etoposide and vincristine; anticancer antibiotics such as calicheamicin, calicheamicin derivatives (ozogamicin, etc.), mitomycin, and bleomycin; platinum-based agents such as cisplatin; molecularly targeted agents such as sorafenib and erlotinib; methotrexate, cytosine arabinoside, 6-thioguanine, 6-mercaptopurine, cyclophosphamide, ifosfamide, busulfan, MMAE (monomethylauristatin E), DM-1 (mertansine), and calicheamicin. Also, copper 64( 64 Cu), Iodine-131( 131 I), Indium 111 ( 111 In), Yttrium 90 ( 90 Y), Boron-10 ( 10 Radioisotopes such as B) are also included as chemotherapeutic agents.

[0020] The new version of the newspaper has been updated or updated. You can also subscribe to abagovo mab、abatacept、abciximab、ABT-414、adalimumab、adalimumab、ad alimumab-atto、aducanumab、afelimomab、aflibercept、aflibercept、alefacept、alemtuzumab、alemtuzumab、alirocumab、altumom ab、ALX-0061、amatuximab、anifrolumab、arcitumomab、atezolizumab、bapineuzumab、basiliximab、bavituximab、begelomab、belat accept、belimumab、benralizumab、besilesomab、bevacizumab、bezlotoxumab、bimagrumab、blinatumomab、bococizumab、brentuximab vedotin、Briakinumab、brodalumab、canakinumab、capromab、catumaxomab、certolizumab pegol、cetuximab、crenezumab、daclizumab、daclizumab、daratumumab、demcizumab、denosumab、denosumab、dupilumab、durvalumabzuliedzu、ectumab colomab、efalizumab、efungumab、elotuzumab、epratuzumab、etanercept、etanercept、etanercept-szzs、etaracizumab、etrolizumab、evolocumab、fresolimumab、bgene ozogamicin、gevokizumab、girentuximab、golimumab、GSK2398852、guselkumab、ibritumab tiuxetan、idarucizumab、igovomab、imciromabpentetate、infliximab、infliximab、infliximab、infliximab-dyyb、inotuzumab ozogamicin、ipilimumab、ixekizumab、labetuzumab、lampalizumab、lebrizumab、lifastuzumab vedotin、lintuzumab、lorvotuzumab mertansine、lulizumab pegol、margetuximab、mavrilimumab、mepolizumab、milatuzumab、mitumomab、mogamulizumab、motavizumab、moxetumomab pasudotox、muromonab-CD3、natalizumab、natalizumab、necitumumab、necitumumab、nesvacumab、nimotuzumab、nivolumab、nofetumomab、obiltoxaximab、obinutuzumab、tocrelizumab b、ofatumumab、olaratumab、omalizumab、otelixizumab、ozanezumab、palivizumab、panitumumab、pascolizumab、pembrolizumab、pemtumomab、pertuzumab、pidilizumab、polatuzumab vedotin、racotumomab、ramucirumab、ranibizumab、raxibacumab、reslizumab、rilonacept、rilotumumab、rituximab、romiplostim、romosozumab、sacituzumab govitecan、satumomab、secukinumab、seribantumab、cefalimumab、silutuximab、simtuzumab、sirukumab、solanezumab、sulesomab、ta balumab、tanezumab、tarextumab、tildrakizumab、tilmanocept、tocilizumab、tositumomab、tralokinumab、trastuzumab、trastuzumabExamples include emtansine, trastuzumab deruxtecan, tremelimumab, ustekinumab, vantictumab, vedolizumab, veltuzumab, votumumab, yttrium (90Y) clivatuzumab tetraxetan, etc.

[0021] There are no particular limitations on the diseases that can be treated with such antibody drugs, but examples include cancer, autoimmune diseases, infectious diseases, allergies, and inflammatory diseases, and more specifically, the following:

[0022] "Cancer" includes brain tumors, breast cancer, uterine cancer, cervical cancer, ovarian cancer, esophageal cancer, stomach cancer, appendix cancer, colon cancer, liver cancer, gallbladder cancer, bile duct cancer, pancreatic cancer, adrenal cancer, gastrointestinal stromal tumor (GIST), mesothelioma, head and neck cancer, kidney cancer, lung cancer, osteosarcoma, Ewing's sarcoma, chondrosarcoma, prostate cancer, testicular tumors, renal cell carcinoma, bladder cancer, rhabdomyosarcoma, skin cancer, and anal cancer.

[0023] "Autoimmune diseases" include Guillain-Barré syndrome, myasthenia gravis, multiple sclerosis, chronic gastritis, chronic atrophic gastritis, autoimmune hepatitis, primary biliary cholangitis, ulcerative colitis, Crohn's disease, primary biliary cholangitis, autoimmune pancreatitis, Takayasu's arteritis, Goodpasture's syndrome, rapidly progressive glomerulonephritis, megaloblastic anemia, autoimmune hemolytic anemia, autoimmune neutropenia, idiopathic thrombocytopenic purpura, Graves' disease, Hashimoto's disease, primary hypothyroidism, idiopathic Addison's disease, type 1 diabetes, and chronic discoid erythema. These include erythematosus, localized scleroderma, pemphigus, pustular psoriasis, plaque psoriasis, pemphigoid, herpes gestationis, linear IgA bullous dermatosis, epidermolysis bullosa acquisita, alopecia areata, vitiligo vulgaris, Sutton's acquired vitiligo / Sutton's nevus, Harada's disease, autoimmune optic neuropathy, autoimmune inner ear disorder, idiopathic azoospermia, habitual abortion, rheumatism, systemic lupus erythematosus, antiphospholipid antibody syndrome, polymyositis, dermatomyositis, scleroderma, Sjögren's syndrome, IgG4-related disease, vasculitis syndrome, and mixed connective tissue disease.

[0024] Examples of "infectious diseases" include bacterial infections, fungal infections, parasitic protozoan infections, parasitic helminth infections, and viral infections. Examples of bacterial infections include infections caused by various bacteria such as streptococci, Staphylococcus aureus, Staphylococcus epidermidis, enterococci, Listeria, Neisseria meningitidis, Neisseria gonorrhoeae, pathogenic Escherichia coli, Klebsiella, Proteus, Bordetella pertussis, Pseudomonas aeruginosa, Serratia, Citrobacter, Acinetobacter, Enterobacter, Mycoplasma, Clostridium, Rickettsia, and Chlamydia; tuberculosis, nontuberculous mycobacterial diseases, cholera, plague, diphtheria, dysentery, scarlet fever, anthrax, syphilis, tetanus, leprosy, Legionnaires' disease, leptospirosis, Lyme disease, tularemia, and Q fever. Fungal infections include aspergillosis, candidiasis, cryptococcosis, tinea mycosis, histoplasmosis, and Pneumocystis carinii pneumonia. Parasitic protozoan infections include amebic dysentery, malaria, toxoplasmosis, leishmaniasis, and cryptosporidiosis. Parasitic helminth infections include echinococcosis, schistosomiasis japonicum, filariasis, ascariasis, and diphyllobothriasis. Viral infections include influenza, viral hepatitis, viral meningitis, viral gastroenteritis, viral conjunctivitis, acquired immunodeficiency syndrome (AIDS), adult T-cell leukemia, Ebola hemorrhagic fever, yellow fever, common cold syndrome, rabies, cytomegalovirus infection, severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), progressive multifocal leukoencephalopathy, chickenpox-shingles, herpes simplex, hand, foot, and mouth disease, dengue fever, Japanese encephalitis, erythema infectiosum, infectious mononucleosis, smallpox, rubella, acute poliomyelitis (polio), measles, pharyngoconjunctival fever (swimming pool fever), Marburg hemorrhagic fever, hemorrhagic fever with renal syndrome, Lassa fever, mumps, West Nile fever, herpangina, and chikungunya. The infectious disease may be, for example, an opportunistic infection.

[0025] "Allergies" include anaphylactic shock, allergic rhinitis (such as seasonal allergic rhinitis), conjunctivitis, bronchial asthma, urticaria, atopic dermatitis, hemolytic anemia, idiopathic thrombocytopenic purpura, drug-induced hemolytic anemia, granulocytopenia, thrombocytopenia, Goodpasture's syndrome, serum sickness, systemic lupus erythematosus (SLE), rheumatism, glomerulonephritis, hypersensitivity pneumonitis, allergic bronchopulmonary aspergillosis (ABPA), contact dermatitis, allergic encephalitis, transplant rejection, tuberculous cavities, epithelioid cell granuloma, eosinophilic granulomatosis with polyangiitis, and chronic sinusitis with nasal polyps.

[0026] "Inflammatory diseases" include diseases induced by inflammatory cytokines, such as IL-6 and TNF-α. Specific examples of inflammatory diseases include encephalitis, osteomyelitis, meningitis, neuritis, eye inflammation (dacryoadenitis, scleritis, episcleritis, keratitis, chorioretinitis, retinitis, chorioretinitis, blepharitis, conjunctivitis, uveitis, etc.), ear inflammation (otitis externa, otitis media, otitis interna, etc.), mastitis, carditis (endocarditis, myocarditis, pericarditis, etc.), vasculitis (arteritis, phlebitis, capillaritis, etc.), respiratory inflammation (sinusitis, rhinitis, pharyngitis, laryngitis, tracheitis, bronchitis, bronchiolitis, pneumonia, pleuritis, mediastinitis, etc.), oral inflammation (stomatitis, gingivitis, gingivostomatitis, glossitis, tonsillitis, siladenitis, parotitis, cheilitis, pulpitis, nasal congestion, etc.), and inflammation of the digestive tract (esophagitis, gastritis, gastroenteritis, enteritis, enteritis, colitis, duodenitis, ileitis, appendicitis, proctitis, etc.), dermatitis, cellulitis, hidradenitis, arthritis, dermatomyositis, myositis, synovitis, tendonitis, panniculitis, osteitis, osteomyelitis, periostitis, nephritis, ureteritis, cystitis, ureteritis, oophoritis, salpingitis, endometritis, cervicitis, vaginitis, vulvitis, orchitis, epididymitis, prostatitis, seminal vesicle cystitis, balanitis, presitis, chorioamnionitis, omphalitis, omphalitis, hepatitis, ascending cholangitis, cholecystitis, pancreatitis, peritonitis, hypophysitis, thyroiditis, parathyroiditis, adrenalitis, lymphangitis, and lymphadenitis.

[0027] <Analysis of N-linked glycans bound to endogenous proteins and, based on the results, prediction of glycan structures bound to exogenous immunoglobulins> Those skilled in the art can appropriately perform "analysis of N-linked glycans" bound to endogenous proteins using standard methods. Examples of such standard methods include mass spectrometry, particularly LC-MS analysis including cleavage of glycans (e.g., JP 2016-194500 A, JP 2016-099304 A), analysis using lectins or antibodies, electrophoretic analysis, and affinity chromatography analysis based on the affinity between an Fc-binding protein immobilized on an insoluble carrier and an antibody (e.g., Patent Document: WO 2019 / 244901). Among these, affinity chromatography analysis is a particularly preferred glycan analysis method because it allows analysis based on differences in the N-linked glycan structures bound to the Fc region of an antibody in a simple manner without complex procedures. For details of such glycan analysis methods, see <Affinity Chromatography Analysis Based on the Affinity Between an Fc-binding Protein Immobilized on an Insoluble Carrier and an Antibody>.

[0028] As will be shown in the Examples below, the structure of the N-linked glycan thus obtained correlates with the structure of the glycan bound to exogenous immunoglobulin in the blood after administration. Therefore, it is possible to determine the structure of the glycan bound to exogenous immunoglobulin based on the N-linked glycan bound to endogenous proteins.

[0029] For example, a correlation equation can be prepared in advance between the N-linked glycans bound to endogenous proteins and the glycans bound to exogenous immunoglobulins in the blood, and an estimated value of the bound glycans in the sample of the antibody drug can be determined from the analytical value of the N-linked glycans bound to the protein. Furthermore, the bound glycans of exogenous immunoglobulins in the blood can be quantitatively or qualitatively determined by comparing the N-linked glycans bound to endogenous proteins with the analytical value of a reference glycan.

[0030] Furthermore, sugar chain structures bound to exogenous immunoglobulins such as antibody drugs are known to be involved in immune activation, including antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent cellular phagocytosis (ADCP).

[0031] Sugar chain structures bound to exogenous immunoglobulins such as antibody drugs are known to be involved in regulating cellular functions, such as cellular uptake by sugar chain receptors and signal transduction.

[0032] Furthermore, as will be shown in the Examples below, the sugar chain structure bound to exogenous immunoglobulins such as antibody drugs is involved in their kinetics (e.g., pharmacokinetic control via fetal Fc receptor (FcRn) binding, blood retention, and dispersion stability).

[0033] Thus, according to the method of the present invention, by predicting the structure of the sugar chains bound to exogenous immunoglobulins in the blood, it is possible to predict the properties brought about by the structure (such as regulation of cell function, immune activation, and kinetics of exogenous immunoglobulins). Furthermore, since these properties affect the efficacy and / or safety of exogenous immunoglobulins, it is also possible to predict the efficacy and / or safety of the exogenous immunoglobulins.

[0034] Furthermore, analysis of N-linked glycans bound to endogenous proteins and / or prediction of glycan structures bound to exogenous immunoglobulins based on said analysis is preferably performed before administration of said immunoglobulin, but may also be performed simultaneously with or after said administration.

[0035] <Affinity chromatography analysis based on the affinity between antibodies and Fc-binding proteins immobilized on an insoluble carrier> More specifically, the above-mentioned affinity chromatography analysis is a method comprising the following steps [A] and [B]. [A] a step of applying a blood-derived sample to a column packed with an insoluble carrier onto which an Fc-binding protein has been immobilized, and separating endogenous proteins contained in the sample, thereby obtaining a separation pattern of the proteins; [B] A step of identifying peak regions in the separation pattern obtained in [A].

[0036] (Fc binding protein) The "Fc-binding protein" used to separate endogenous proteins from the blood-derived sample is not particularly limited, as long as it is a polypeptide that has the ability to bind to the Fc region of an endogenous protein contained in the sample and can recognize differences in the sugar chain structure of an antibody (e.g., the sugar chain structure of the Fc region). For example, when the endogenous protein is an antibody derived from a human, the Fc-binding protein can be a human Fc-binding protein. Preferred examples of human Fc-binding proteins include human Fc receptors. Examples of human Fc receptors include the human Fcγ receptor, which is a receptor for human immunoglobulin G (IgG), the human Fcα receptor, which is a receptor for human immunoglobulin A (IgA), the human Fcδ receptor, which is a receptor for human immunoglobulin D (IgD), and the human Fcε receptor, which is a receptor for human immunoglobulin E (IgE). Any of these receptors can be used as the human Fc-binding protein in the present invention.

[0037] Specific examples of human Fcγ receptors include polypeptides comprising at least a partial sequence of the extracellular region of human FcγRI (CD64), human FcγRIIa (CD32a), human FcγRIIb (CD32b), human FcγRIIc (CD32c), human FcγRIIIa (CD16a), or human FcγRIIIb (CD16b), as well as polypeptides in which some of the amino acid residues constituting these polypeptides have been substituted, deleted, inserted, and / or added. Among these, polypeptides comprising at least a partial sequence of the extracellular region of human FcγRIIIa and polypeptides in which some of the amino acid residues constituting these polypeptides have been substituted, deleted, inserted, and / or added are preferred as human Fcγ receptors used as human Fc-binding proteins in the present invention.

[0038] Specific examples of polypeptides comprising at least a partial sequence of the extracellular region of human FcγRIIIa, or polypeptides in which some of the amino acid residues constituting such polypeptides have been substituted, deleted, inserted, and / or added, include the polypeptides described in (1) to (3) below. (1) A polypeptide comprising amino acid residues 17 to 192 of the amino acid sequence set forth in SEQ ID NO: 1, in which at least valine at position 176 is substituted with phenylalanine; (2) A polypeptide comprising amino acid residues 17 to 192 of the amino acid sequence set forth in SEQ ID NO: 1, in which at least valine at position 176 is substituted with phenylalanine, and further having substitutions, deletions, insertions, and / or additions of one or several amino acid residues at one or several positions other than position 176, and having antibody-binding activity; (3) A polypeptide having an amino acid sequence having 70% or more homology with the sequence consisting of amino acid residues 17 to 192 of SEQ ID NO: 1, in which the amino acid residue corresponding to valine at position 176 of SEQ ID NO: 1 is substituted with phenylalanine, and having antibody binding activity.

[0039] Examples of the polypeptide described in (1) above include a polypeptide containing amino acid residues 24 to 199 of the amino acid sequence set forth in SEQ ID NO: 2, and the polypeptide (Fc-binding protein) disclosed in JP 2018-197224 A.

[0040] Examples of the substitution, deletion, insertion, or addition described in (2) above include the substitution of amino acid residues disclosed in JP-A-2015-086216, JP-A-2016-169167, and JP-A-2017-118871.

[0041] In (3) above, "homology" refers to similarity or identity, and can be determined using an alignment program such as BLAST (Basic Local Alignment Search Tool). For example, "amino acid sequence identity" may refer to the identity between amino acid sequences calculated using blastp, specifically, the identity between amino acid sequences calculated using blastp with default parameters. The homology may be 70% or more, and may also be 80% or more, 85% or more, 90% or more, or 95% or more (e.g., 96% or more, 97% or more, 98% or more, or 99% or more).

[0042] Furthermore, in the present invention, the "position" of each amino acid residue refers to the order in which the first methionine is placed as position 1 in the amino acid sequence set forth in each SEQ ID NO. Therefore, "position 176" according to the present invention refers to position 176 in the amino acid sequence set forth in SEQ ID NO: 1. Furthermore, "the amino acid residue corresponding to valine at position 176 in SEQ ID NO: 1" refers to an amino acid residue in the amino acid sequence having 70% or more homology, which is arranged at the same position as valine at position 176 in the amino acid sequence set forth in SEQ ID NO: 1, when the amino acid sequence is aligned with the sequence consisting of amino acid residues 17 to 192 in SEQ ID NO: 1.

[0043] (Insoluble carrier) In the present invention, the Fc-binding protein is immobilized on an insoluble carrier before use to separate endogenous proteins from the blood-derived sample. The term "insoluble carrier" refers to a carrier that is insoluble in a liquid (e.g., a liquid used for antibody adsorption or elution, such as an equilibration liquid or elution liquid) passed through a column packed with the carrier. The insoluble carrier may have a functional group (e.g., a hydroxy group) for covalently immobilizing the Fc-binding protein. Examples of insoluble carriers include carriers derived from inorganic substances such as zirconia, zeolite, silica, and coated silica; carriers derived from natural organic polymers such as cellulose, agarose, and dextran; and carriers derived from synthetic organic polymers such as polyacrylic acid, polystyrene, polyacrylamide, polymethacrylamide, polymethacrylate, and vinyl polymers.

[0044] For example, immobilization of an Fc-binding protein to an insoluble carrier can be achieved by utilizing functional groups present on the carrier surface that can covalently immobilize the Fc-binding protein. More specifically, when an insoluble carrier has hydroxyl groups on its surface, an activator can be used to convert the hydroxyl groups to activated groups capable of covalently binding to the Fc-binding protein, thereby covalently binding the activated groups to the Fc-binding protein. Specific examples of activators for hydroxyl groups include epichlorohydrin (which forms an epoxy group as the activated group), 1,4-butanediol diglycidyl ether (which forms an epoxy group as the activated group), tresyl chloride (which forms a tresyl group as the activated group), and vinyl bromide (which forms a vinyl group as the activated group). Alternatively, the hydroxyl groups can be converted to amino groups, carboxyl groups, or the like, and then activated with an activator. Specific examples of activators for amino groups, carboxy groups, etc. include N-succinimidyl 3-maleimidopropionate (which forms a maleimide group as the activating group), 1,1'-carbonyldiimidazole (which forms a carbonylimidazole group as the activating group), and halogenated acetic acids (which form a halogenated acetyl group as the activating group).

[0045] (Method for analyzing endogenous proteins) In the above step (A), the separated pattern can be obtained by carrying out the following steps (a) and (b). (a) adding a sample to a column packed with an insoluble carrier onto which an Fc-binding protein has been immobilized, and allowing endogenous proteins contained in the sample to be adsorbed onto the carrier (hereinafter also referred to as the "adsorption step"); (b) A step of eluting the endogenous proteins adsorbed to the carrier with an eluent to obtain a separation pattern of the proteins (hereinafter also referred to as the "separation step").

[0046] Each step will be described in detail below.

[0047] (a) Adsorption process In this step, a sample containing an endogenous protein is added to a column packed with an insoluble carrier onto which an Fc-binding protein has been immobilized, and the protein is adsorbed onto the carrier.

[0048] The sample used in this step is the blood-derived sample described above. The sample may be added to the column after being dissolved, suspended, dispersed, or solvent-exchanged in a suitable liquid medium. Examples of the liquid medium include the equilibration solution described below.

[0049] A sample can be added to a column packed with an insoluble carrier on which an Fc-binding protein has been immobilized, for example, using a liquid delivery device such as a pump (hereinafter, in this specification, adding a liquid to a column is also referred to as "delivering a liquid to the column"). The conditions for the adsorption step, such as the amount of sample added (delivered), the type of liquid phase, the liquid phase delivery rate, and the column temperature, are not particularly limited, as long as the endogenous protein is adsorbed to the carrier. The conditions for the adsorption step can be appropriately set depending on various conditions, such as the type of endogenous protein, the type of Fc-binding protein, the type of insoluble carrier, and the scale of the column. An example of the liquid phase is the equilibration liquid described below. For example, when the inner diameter of the column is 4.6 mm, the delivery rate may be 0.1 mL / min to 2.0 mL / min, 0.2 mL / min to 1.5 mL / min, or 0.4 mL / min to 1.2 mL / min. The delivery rate may be set, for example, so that it is proportional to the square of the inner diameter of the column. The column temperature may be set appropriately within the range of 0°C to 50°C.

[0050] An equilibration step in which an equilibration solution is added (pumped) to the column may be performed before and / or after this step. Performing an equilibration step after this step is particularly preferable because it allows for the removal from the column of endogenous proteins that do not bind to the Fc-binding protein and endogenous proteins that bind to the Fc-binding protein in the sample atmosphere but not in the equilibration buffer atmosphere (the removed endogenous protein-containing fraction is also referred to as the "unadsorbed fraction" herein). The unadsorbed fraction refers to the fraction in the region from which the peak detected after the sample is added (pumped) to the column reaches its minimum value during the equilibration step in the separation pattern described below. Separating the unadsorbed fraction from the peak detected after the addition of the eluent in the separation step described below is preferable, as this increases separation accuracy. A constant detection value between the unadsorbed fraction and the peak region detected after the addition of the eluent is particularly preferable, as it indicates that the unadsorbed fraction has been sufficiently removed from the column by the equilibration step. The term "constant value" as used here refers not only to a constant value but also to a state in which the detection value changes with a constant slope.

[0051] Examples of equilibration solutions include aqueous buffer solutions. Specific examples include weakly acidic to weakly alkaline buffer solutions with a pH of 5.0 to 8.0. The components of the buffer solution can be selected appropriately depending on various conditions, such as the pH of the buffer solution. Examples of buffer solution components include phosphoric acid, acetic acid, formic acid, MES (2-Morpholinoethanesulfonic acid), MOPS (3-Morpholinopropanesulfonic acid), citric acid, succinic acid, glycine, and piperazine. A salt such as sodium chloride or potassium chloride may also be added to the buffer solution. The salt is not particularly limited as long as it is a salt that can be easily imagined by a person skilled in the art.

[0052] (b) Separation process This step involves eluting the endogenous proteins adsorbed to the insoluble carrier in step (a) using an elution solution to obtain a separation pattern for the endogenous proteins. That is, the endogenous proteins adsorbed to the carrier can be eluted by adding (delivering) the elution solution to the column. The conditions for the elution step, such as the type of elution solution, the delivery format of the elution solution, the liquid phase delivery rate, and the column temperature, are not particularly limited as long as the antibodies are separated in the desired manner, e.g., as long as the desired separation pattern is obtained. The conditions for the elution step can be appropriately set depending on various conditions, such as the type of endogenous protein, the type of Fc-binding protein, the type of insoluble carrier, and the scale of the column. The elution solution used may be one that weakens the affinity between the endogenous protein and the Fc-binding protein, and examples thereof include aqueous buffer solutions with a more acidic pH than the liquid phase prior to elution (more specifically, the equilibration solution used in the equilibration step, if any). As a specific example, if the liquid phase before elution (e.g., the equilibration solution) is a weakly acidic to weakly alkaline buffer solution with a pH of 5.0 to 8.0, an acidic buffer solution with a pH of 2.5 to 4.5 can be used as the eluent. The components of the buffer solution can be selected appropriately depending on various conditions, such as the pH of the buffer solution. Examples of buffer solution components include phosphoric acid, acetic acid, formic acid, MES (2-Morpholinoethanesulfonic acid), MOPS (3-Morpholinopropanesulfonic acid), citric acid, succinic acid, glycine, and piperazine. The eluent delivery method may be linear gradient elution, in which the ratio of the eluent in the liquid phase is continuously changed, or stepwise elution, in which the ratio is gradually changed, or a combination thereof. The gradient may be set, for example, so that the ratio of the eluent in the liquid phase increases from 0% (v / v) to 100% (v / v) over 10 to 60 minutes, 15 to 50 minutes, or 20 to 40 minutes. The flow rate may be, for example, 0.1 mL / min to 2.0 mL / min, 0.2 mL / min to 1.5 mL / min, or 0.4 mL / min to 1.2 mL / min when the column inner diameter is 4.6 mm. The flow rate may be set, for example, so that it is proportional to the square of the column inner diameter.The column temperature may be set appropriately within the range of, for example, 0°C to 50°C.

[0053] The endogenous proteins eluted from the column are detected using a detector to obtain a separation pattern of the endogenous proteins. Examples of the detector include a UV detector and a mass detector. Examples of the antibody separation pattern include a chromatogram obtained during the elution of the endogenous proteins. The intervals at which measurement data is acquired by the detector may be any time interval. However, as the time interval increases, the accuracy of reflecting the original characteristics of the separation pattern of the endogenous proteins in the chromatogram decreases. Therefore, the time interval is preferably 1 minute or less, more preferably 10 seconds or less, and even more preferably 2 seconds or less.

[0054] This step allows the endogenous protein contained in the sample to be obtained in a separated form. The separated endogenous protein may be obtained, for example, as an elution fraction containing the endogenous protein. That is, the separated endogenous protein can be obtained by collecting the elution fraction containing the separated endogenous protein. The elution fraction can be collected, for example, by a conventional method. Specifically, the elution fraction can be collected, for example, by an automatic fraction collector such as an autosampler. Furthermore, the separated endogenous protein may be recovered from the elution fraction. The separated endogenous protein can be collected, for example, by a conventional method. Specifically, the separated endogenous protein can be collected from the elution fraction by, for example, a known method used for protein separation and purification.

[0055] Then, in the above step [B], the peak region of the separation pattern of the endogenous proteins contained in the blood-derived sample is identified.

[0056] Specific examples of peaks in the separation pattern in the present invention include peak area, peak elution time, peak width, number of detected peaks, and peak height. In particular, examples of peaks identified in the present invention include peak elution time, peak width, and number of detected peaks. The separation pattern of endogenous proteins may be used for peak extraction either directly or after appropriate correction, such as baseline correction. Peaks may be expressed as absolute values ​​or relative values. As relative values, any elution time may be used as an arbitrary value; for example, the time at which elution begins may be set to 0, or the relative value may be a ratio or difference relative to the total time during which the eluent was introduced.

[0057] In the present invention, unless otherwise specified, the "first to third peaks" may refer to the peaks that elute first to third after the start of elution (for example, after the start of the gradient), respectively. The peaks may particularly have a peak area% of 1% or more. In other words, the "first to third peaks" may particularly refer to the peaks that elute first to third after the start of elution, respectively, and have a peak area% of 1% or more. The "peak area%" refers to the value (percentage) obtained by dividing the area of ​​each peak (peak region) by the area of ​​all peaks.

[0058] In the present invention, the term "peak region" refers to a range of detection values ​​that includes the aforementioned peaks and is sandwiched between specific elution times. For example, the first peak region refers to the region of detection values ​​between the elution time that marks the boundary between the first and second peaks (hereinafter also referred to as the boundary time) and any elution time value before the boundary time. The arbitrary elution time value may be a reference point in baseline correction or the start time of elution, and is not particularly limited. However, it is preferably set between the peak of the non-adsorbed fraction that does not bind to the endogenous protein immobilized on the column containing the Fc-binding protein, and the peak derived from the endogenous protein bound to the carrier. The second peak region refers to the region of detection values ​​between the boundary time between the first and second peaks and the boundary time between the second and third peaks, and the third peak region refers to the region of detection values ​​between the boundary time between the second and third peaks and any elution time value after the boundary time. The arbitrary value of the elution time is not particularly limited and may be a reference point when baseline correction is performed or the end time of elution. However, it is preferable to set the arbitrary value of the elution time within the time period from when a separation peak derived from an endogenous protein bound to an insoluble carrier onto which an Fc-binding protein packed in the column is immobilized is obtained to when, after washing the column with the elution solution, the column is switched to an equilibration solution to equilibrate the column, a change in the detection value that occurs as a result of the switch is observed.

[0059] In the present invention, the peak region of an endogenous protein may be determined by any conventional method, for example, by visually determining the boundary time of the peak region. However, a method in which the boundary time of each peak is determined as the elution time at which the detected value is at a minimum in the region between each peak top, or the elution time at which the sign of the derivative obtained by differentiating a chromatogram showing the detected value in the region between each peak top, is changed, which allows the definition of the peak region to be automated and also reduces differences between operators, is particularly preferred.

[0060] Furthermore, the peak regions of endogenous proteins can be determined by applying the separation pattern of a standard substance, as shown in the Examples below. There are no particular limitations on the type of standard substance, but it is preferable that the standard substance exhibits a clear separation pattern that allows easy identification of peak regions, and that the peak regions match those of the endogenous protein. Furthermore, the separation pattern of the standard substance may be measured before or after the measurement of the endogenous protein, and it is preferable to identify the peaks based on values ​​measured immediately before or immediately after the measurement of the endogenous protein, as this allows for highly accurate identification of the peaks of the protein.

[0061] The peak regions identified in this way can be used to distinguish between differences in the glycan structures of N-linked glycans bound to endogenous proteins contained in blood-derived samples (WO 2019 / 244901). Examples of distinguishable glycan structures include sialic acid, galactose, mannose, N-acetylglucosamine, and fucose, which constitute N-linked glycans, and examples of glycan structures include G0, G0F, G1, G0F+GN, G1Fa, G1Fb, G1F+GN, G2, G2F, G1F+SA, G2F+SA, G2F+2SA, G2F+GN, G2+SA, G2+2SA, S1, S2, and S3.

[0062] The N-linked glycan structures thus obtained correlate with the structures of glycans bound to exogenous immunoglobulins in the blood after administration. Therefore, as described above, it is possible to determine the structures of glycans bound to exogenous immunoglobulins based on the N-linked glycans bound to endogenous proteins.

[0063] More specifically, as shown in the Examples below, when the area % of the first peak region is high and / or the area % of the third peak region is low, the blood retention of the exogenous immunoglobulin can be determined to be high. On the other hand, when the area % of the first peak region is low and / or the area % of the third peak region is high, the blood retention of the exogenous immunoglobulin can be determined to be low.

[0064] It has also been reported that core fucose-deficient glycan structures in N-linked glycans enhance ADCC activity (J. Biol. Chem., 2010, Vol. 285, pp. 16012-16022). Furthermore, affinity chromatography analysis using Fc-binding proteins has revealed that immunoglobulins with such core fucose-deficient N-linked glycans and high ADCC activity have slower elution times (Tosoh Research and Technical Report, Vol. 61, 2017, pp. 33-41). It has also been reported that affinity chromatography analysis using Fc-binding proteins demonstrates that the higher the area percentage of the third peak region of an antibody drug, the higher its ADCC activity (Bio Drugs, 2020, Vol. 34, pp. 363-379). Furthermore, affinity chromatography analysis using the Fc-binding protein has also been shown to increase ADCC activity and CDC activity as the affinity for the Fc-binding protein increases in the order of the first, second, and third peaks (SCIENTIFIC REPORTS, 2018, Vol. 8, Article No. 3955). Therefore, if the area percentage of the first peak region is high and / or the area percentage of the third peak region is low, the ADCC activity of the exogenous immunoglobulin can be determined to be low. On the other hand, if the area percentage of the first peak region is low and / or the area percentage of the third peak region is high, the ADCC activity of the exogenous immunoglobulin can be determined to be high.

[0065] Furthermore, the strength of the affinity of an immunoglobulin for an Fc-binding protein affects the ADCC activity and / or ADCP activity of immune cells such as natural killer cells, monocytes, and macrophages, which have the ability to damage or phagocytose the binding substance bound by the immunoglobulin, and therefore, the activities can be determined by detecting the difference in affinity.

[0066] Those skilled in the art can distinguish between "high" and "low" by setting a cutoff value corresponding to the area percentage of each peak region. [Example]

[0067] The present invention will be described in more detail below with reference to examples and comparative examples, but the present invention is not limited to these examples.

[0068] <Preparation of affinity chromatography column (FcR9_F column)> The Fc-binding protein FcR9_F_Cys obtained by the method of JP 2018-197224 A was immobilized on a gel by the method shown below to prepare an FcR9_F column. In FcR9_F_Cys (amino acid sequence set forth in SEQ ID NO: 2), the portion from the 1st methionine (Met) to the 22nd alanine (Ala) is the improved PelB signal peptide, the portion from the 24th glycine (Gly) to the 199th glutamine (Gln) is the amino acid sequence of the Fc-binding protein FcR9_F (JP 2018-197224 A) (corresponding to the region from the 17th to the 192nd of SEQ ID NO: 1), and the portion from the 200th glycine (Gly) to the 207th glycine (Gly) is the cysteine ​​tag sequence. Furthermore, the FcR9_F is a polypeptide consisting of the 17th to 192nd amino acid residues of native FcγRIIIa shown in SEQ ID NO: 1, with the following amino acid substitutions (I) to (X): (I) Substitution of valine (Val) at position 27 of SEQ ID NO: 1 (position 34 of SEQ ID NO: 2) with glutamic acid (Glu) (II) Substitution of phenylalanine (Phe) at position 29 of SEQ ID NO: 1 (position 36 of SEQ ID NO: 2) with isoleucine (Ile) (III) Substitution of tyrosine (Tyr) at position 35 of SEQ ID NO: 1 (position 42 of SEQ ID NO: 2) with asparagine (Asn) (IV) Substitution of glutamine (Gln) at position 48 of SEQ ID NO: 1 (position 55 of SEQ ID NO: 2) with arginine (Arg) (V) Substitution of phenylalanine (Phe) at position 75 of SEQ ID NO: 1 (position 82 of SEQ ID NO: 2) with leucine (Leu) (VI) Substitution of asparagine (Asn) at position 92 of SEQ ID NO: 1 (position 99 of SEQ ID NO: 2) with serine (Ser) (VII) Substitution of valine (Val) at position 117 of SEQ ID NO: 1 (position 124 of SEQ ID NO: 2) with glutamic acid (Glu) (VIII) Substitution of glutamic acid (Glu) at position 121 of SEQ ID NO: 1 (position 128 of SEQ ID NO: 2) with glycine (Gly) (IX) Substitution of phenylalanine (Phe) at position 171 of SEQ ID NO: 1 (position 178 of SEQ ID NO: 2) with serine (Ser) (X) Valine (Val) at position 176 of SEQ ID NO: 1 (position 183 of SEQ ID NO: 2) is replaced with phenylalanine (Phe).

[0069] (1) The hydroxyl groups on the surface of 2 mL of hydrophilic vinyl polymer for separation agent (manufactured by Tosoh Corporation: packing material for liquid chromatography) were activated with iodoacetyl groups, and then 4 mg of FcR9_F_Cys obtained by the method of JP 2018-197224 A was reacted to obtain an FcR9_F immobilized gel. (2) 1.2 mL of the FcR9_F-immobilized gel prepared in (1) was packed into a φ4.6 mm × 50 mm stainless steel column to prepare an FcR9_F column.

[0070] <Purification of rituximab contained in blood> As shown in FIG. 1, rituximab was isolated and purified from the serum of a patient after administration of rituximab by the following method. (1) Magnetic particles 10 (Dynabeads M-280 Tosylactivated; Thermo Fisher Scientific) were modified with anti-rituximab antibody 20 (Bio-Rad Laboratories), and the remaining unreacted tosyl groups on the magnetic particle surface were blocked with BSA. The resulting magnetic particles were designated as anti-rituximab antibody-modified magnetic particles. (2) Blood samples were collected from 13 patients with hematological malignancies who had received rituximab and given informed consent. Serum was obtained by centrifugation. 2 mg of the anti-rituximab antibody-modified magnetic particles prepared in (1) was added to 500 μL of the serum, and the mixture was mixed by inversion at room temperature for 3 hours to bind rituximab 30 to the magnetic particles. (3) By approaching a magnet to the mixed solution of the serum obtained in (2) and the magnetic particles, the magnetic particles 10 were magnetically collected, the supernatant was removed, and washing was performed by adding a phosphate buffer at pH 7.4. By performing the washing operation four times in total, the serum component 40 was removed. (4) By approaching a magnet to the suspension of the magnetic particles 10 obtained in (3), the magnetic particles 10 were magnetically collected, the supernatant was removed, and 60 μL of a glycine-HCl buffer at pH 2.7 was added. By performing amplitude stirring for 5 minutes, the rituximab 30 bound to the magnetic particles was dissociated. After stirring, by approaching a magnet, the magnetic particles 10 were magnetically collected, the supernatant was recovered, and the recovered supernatant was neutralized by adding 30 μL of a Tris-HCl buffer at pH 7.5. The obtained solution was used as a purified rituximab solution.

[0071] (Example 1) <Analysis of Blood Antibodies Using an FcR9_F Column> (1) Among the blood tumor patients, blood obtained by blood collection before rituximab administration from the same 12 specimens was centrifuged to obtain serum. The serum was diluted 20-fold with PBS (Phosphate Buffered Saline) (pH 7.4), and then passed through a 0.2-μm diameter filter (manufactured by Merck Millipore) to prepare a serum sample. (2) The FcR9_F column prepared above was connected to a high-performance liquid chromatograph (manufactured by Tosoh Corporation), and after equilibration being equilibrating with a 10 mM citrate buffer (pH 6.5) containing 100 mM sodium chloride (hereinafter also referred to as "equilibrating solution"), a rituximab antibody solution prepared by adjusting rituximab (manufactured by Zenyaku Kogyo Co., Ltd., trade name: Rituxan intravenous drip injection 100 mg) to a rituximab concentration of 1 mg / mL with the equilibrating solution was added at a flow rate of 1.2 mL / min at 10 μL. The measurement interval by the detector was performed by acquiring data every 1 / 5 second. (3) After washing with the equilibrating solution for 7 minutes at a flow rate of 1.2 mL / min, the adsorbed immunoglobulin (antibody) was eluted with a pH gradient (a gradient in which the eluent becomes 100% in 11 minutes) using a 10 mM citrate buffer (pH 4.5) containing 500 mM sodium chloride (hereinafter also referred to as "eluent") to obtain a separation pattern. (4) After analyzing the reference substance, 10 μL of the serum sample prepared in (1) was added as a measurement sample and analyzed in the same procedure as in (2) and (3) to obtain a separation pattern of immunoglobulins. The analysis was performed by measuring the reference substance and the measurement sample alternately. (5) The separation patterns obtained in (3) and (4) were baseline-corrected so that the detected values both became 0 at the time when the pH gradient started (7 minutes after the start of elution) and at the time when the pH gradient ended (i.e., when the eluate became 100%) (18 minutes after the start of elution). (6) From the separation pattern of the baseline-corrected reference substance (Figure 2), the regions divided by two elution times at which the derivative takes 0 in the valley region between the three peaks detected between 7 minutes and 18 minutes after the start of elution (the 1st, 2nd, and 3rd peaks from the shorter elution time) were defined as peak regions. The 1st peak region was defined as the range from 7 minutes after the start of elution to the elution time at which the derivative of the valley region between the 1st and 2nd peaks takes 0, the 2nd peak region was defined as the range from the elution time at which the derivative of the valley region between the 1st and 2nd peaks takes 0 to the elution time at which the derivative of the valley region between the 2nd and 3rd peaks takes 0, and the 3rd peak region was defined as the range from the elution time at which the derivative of the valley region between the 2nd and 3rd peaks takes 0 to 18 minutes after the start of elution. By applying the peak regions defined with the reference substance to the measurement sample measured immediately after analyzing the reference substance, the peak regions of the measurement sample were defined using the reference substance. (7) The peak area of each peak region of the measurement sample defined in (6) was calculated, and each peak area % was calculated from the value (percentage) obtained by dividing the peak area by the total value of the peak areas between 7 minutes and 18 minutes after the start of elution.

[0072] (Example 2) <Purification rituximab analysis using FcR9_F column> (1) Blood collected from a subject who gave informed consent was centrifuged to obtain serum. Rituximab was added to the serum to a concentration of 100 μg / mL, and rituximab was purified in the same manner as in <Purification of rituximab contained in blood>. The obtained rituximab solution was used as a standard rituximab solution. (2) The area percentage of each peak was calculated from the separation pattern in the same manner as in Example 1(2) to (7), except that 60 μL of the standard rituximab solution prepared in (1) was used as the standard substance and 60 μL of the purified rituximab solution prepared in <Purification of rituximab contained in blood> was used as the measurement sample.

[0073] FIG. 3 shows the correlation between the first peak area % of gamma globulin (serum gamma globulin) isolated from the serum of the specimen obtained in Example 1 and the first peak area % of rituximab (purified rituximab) isolated from the serum of the same specimen obtained in Example 2.

[0074] As shown in Figure 3, as the first peak area% of serum gamma globulins before administration of the antibody drug increased, the first peak area% value of purified rituximab also increased in a significant correlation (correlation coefficient: 0.714, p-value: 0.0091), and as the third peak area% of the serum gamma globulins increased, the third peak area% value of purified rituximab also increased in a significant correlation (correlation coefficient: 0.697, p-value: 0.0117). These results revealed that the separation pattern of purified rituximab correlates with the separation pattern of serum gamma globulins before administration of the antibody drug.

[0075] It is known from patent document (WO2019 / 244901) that the separation pattern of gamma globulin obtained using an FcR9_F column varies depending on the type of glycan bound to the gamma globulin. This indicates that the separation pattern of rituximab correlates with the glycan structure estimated from the separation pattern of serum gamma globulin before administration, since the glycan structure of the administered rituximab changed in the patient's body to take a similar structure.

[0076] The separation patterns of rituximab as the standard substance obtained in Examples 1 and 2 (FIG. 4a) and the separation patterns of serum gamma globulins and purified rituximab for each patient with disease, samples A to D, listed in FIG. 3 (FIG. 4b) are shown in FIG. 4.

[0077] As shown in Figure 4, it can be seen that the separation pattern of rituximab after administration changes significantly from the separation pattern of rituximab before administration (Figure 4a), particularly for samples A and D (Figure 4b). Furthermore, in the separation pattern of serum gamma globulin, for example, if the first peak area % is high and the third peak area % is low when comparing samples, the separation pattern of purified rituximab will show a similar trend. These results also show that the separation patterns of serum gamma globulin and purified rituximab are correlated.

[0078] (Example 3) <Evaluation of correlation with rituximab blood concentration after administration> (1) The amount of purified rituximab in the blood was measured using a NanoDrop ultra-microspectrophotometer (Thermo Scientific). The amount of purified rituximab depends on the concentration of rituximab in the patient's serum, so blood retention can be determined. Therefore, samples with purified rituximab levels of less than 2 μg were defined as having low blood retention, and samples with levels of 2 μg or more were defined as having high blood retention. (2) The correlation between the third peak area % of the purified rituximab solution calculated in Example 1 and the blood retention defined in (1) was evaluated. The results of Example 3 are summarized in Figures 5 and 6.

[0079] As is clear from the results shown in Figure 5, the amount of purified rituximab differed between samples. Using a purification amount of 2 μg as the standard, samples were divided into two groups: those with a high purification amount (high blood retention) and those with a low purification amount (low blood retention), and evaluated based on the third peak area % of the purified rituximab solution. As a result, as shown in Figure 6, it can be seen that the third peak area % is significantly higher in samples with low blood retention as a separation pattern of purified rituximab. This result indicates that the separation pattern of purified rituximab predicted from the separation pattern of serum gamma globulin before administration of an antibody drug is effective for predicting blood retention.

[0080] (Example 4) <Evaluation of correlation with rituximab blood concentration after administration> (1) As the blood in Example 1, blood obtained by drawing blood from the same 12 specimens among the above-mentioned hematological malignant patients before administration of rituximab, and blood obtained by drawing blood from the same specimen among the above-mentioned hematological malignant patients excluding the above-mentioned 12 specimens after administration of rituximab, were used. Except for this, each peak area % was calculated in the same manner as in Example 1. (2) After rituximab administration, blood samples were collected from the 13 patients with hematological malignancies and centrifuged to obtain serum. The rituximab concentration in the serum was measured using a rituximab ELISA (Enzyme-Linked Immunosorbent Assay) kit (Eagle Biosciences). (3) The number of days from when the patient with a blood tumor was administered rituximab (administration immediately before blood collection, as described below) until blood collection was performed was plotted against the rituximab concentration in the serum evaluated in (2), and the correlation with the third peak area % calculated in (1) was evaluated.

[0081] The results of Example 4 are summarized in Table 1 and Figure 7. Table 1 shows the third peak area % obtained from (1), the blood rituximab concentration obtained from (2), and the number of days from the most recent administration to blood collection for the 13 hematological malignant patient samples. Based on the data in Table 1, the blood rituximab concentration and the number of days from the most recent administration to blood collection were plotted and the results are shown in Figure 7. In Figure 7, the group in which the blood concentration decreased early and the group in which the blood concentration was maintained are each surrounded by a dotted line, and the distribution of the third peak area % values ​​was evaluated. As a result, in the group in which the blood concentration was maintained, the third peak area % was 40.5% to 59.6%, whereas in the group in which the blood concentration decreased early, the third peak area % was 68.1% to 68.4%, indicating a tendency for the blood concentration of rituximab to decrease as the third peak area % value increases. These results indicate that the serum gamma globulin separation pattern before or after administration of an antibody drug (here, rituximab) is effective in predicting the blood retention of the antibody drug.

[0082] [Table 1] [Industrial Applicability]

[0083] As described above, according to the present invention, when immunoglobulin is administered to a subject, it is possible to predict the structure of sugar chains bound to the immunoglobulin in the subject's blood after the administration. Based on the sugar chain structure predicted in this way, it is also possible to predict the properties brought about by the structure (such as regulation of cell function, immune activation, and immunoglobulin dynamics). Since these properties affect the efficacy and / or safety of immunoglobulins, the present invention is useful in the fields of pharmaceuticals and medicine through prediction of the efficacy or safety of immunoglobulins such as antibody drugs in the body. [Explanation of symbols]

[0084] 10...magnetic particles, 20...anti-rituximab antibody, 30...rituximab, 40...serum component

Claims

1. A method for predicting the structure of a sugar chain bound to an exogenous immunoglobulin in the blood of a subject after administration of the exogenous immunoglobulin, the method comprising the following steps (1) and (2): (1) analyzing the structure of N-linked glycans bound to endogenous proteins in a blood-derived sample collected from the subject; (2) A method for determining the structure of the sugar chain bound to the exogenous immunoglobulin based on the structure of the N-linked sugar chain obtained in (1).

2. The method according to claim 1, wherein the sugar chain bound to the exogenous immunoglobulin is a sugar chain involved in regulating cell function or immune activation.

3. The method of claim 1 or 2, wherein the endogenous protein is an endogenous immunoglobulin.

4. The method according to any one of claims 1 to 3, wherein the structure of the N-linked sugar chain is analyzed using a column packed with an insoluble carrier on which an Fc-binding protein has been immobilized.

5. The method described in claim 4, wherein the Fc binding protein is FcγRIIIa.

6. The method of claim 4, wherein the Fc-binding protein is a polypeptide according to any one of (1) and (3) below: (1) A polypeptide comprising a sequence consisting of amino acid residues 17 to 192 set forth in SEQ ID NO: 1, in which at least valine at position 176 set forth in SEQ ID NO: 1 is substituted with phenylalanine; (3) A polypeptide having an amino acid sequence that is 90% or more identical to the sequence consisting of amino acid residues 17 to 192 of SEQ ID NO: 1, in which the amino acid residue corresponding to valine at position 176 of SEQ ID NO: 1 is substituted with phenylalanine, and which has antibody binding activity.

7. A step of predicting the structure of a glycan by the method according to any one of claims 1 to 6; and a step of presenting the structure of the glycan, wherein the glycan structure is present.

8. The method according to any one of claims 1 to 7, wherein the exogenous immunoglobulin is an antibody pharmaceutical.

9. A step of predicting the structure of a glycan by the method according to claim 8; and presenting the structure of the glycan.

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