WO2018123979A9 - ミエリンオリゴデンドロサイト糖タンパク質に結合する抗体 - Google Patents
ミエリンオリゴデンドロサイト糖タンパク質に結合する抗体 Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
- G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
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- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
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- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
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- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
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- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
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- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
- C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
- C07K2317/622—Single chain antibody (scFv)
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- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
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- C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
- C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
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- C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
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- C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
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- C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
- C07K2319/43—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a FLAG-tag
Definitions
- the present invention includes an antibody or antibody fragment that binds to myelin oligodendrocyte glycoprotein (MOG), a hybridoma that produces the antibody or the antibody fragment, and a nucleotide sequence encoding the antibody or the antibody fragment.
- the present invention relates to a method of detecting or measuring, a method of diagnosing or treating a brain disease, a method of improving brain retention of an antibody, and a method of increasing the amount of antibody in the brain.
- CDR complementarity determining region
- FR framework region
- Non-patent Document 1 a target antigen
- CD20, CD52, TNF ⁇ , HER2, EGFR etc. As a target antigen, antibody drugs targeting CD20, CD52, TNF ⁇ , HER2, EGFR etc. have already been approved for 60 or more items (Non-patent Document 1).
- antibodies have become a widely recognized drug format. Most of the antibody drugs approved to date are for cancer and immune diseases, and account for about 75% or more of the whole.
- Non-patent Document 2 glial-derived neurotrophic factor
- the delivery amount is lower compared to other organs, and the antibody transfer rate (ratio of cerebrospinal fluid (CSF) concentration to serum concentration) is 0. It is reported as 1-0.3% (non-patent documents 3-5).
- CSF cerebrospinal fluid
- BBB Blood Brain Barrier
- the blood-brain barrier has a physical / nonspecific control mechanism by intercellular junction of vascular endothelial cells and a substrate-specific efflux mechanism by efflux transporter, and protects the central nervous system from foreign substances or drugs. Plays an important role in maintaining homeostasis.
- Non-patent Document 11 Attempts have also been made to directly administer biologics intrathecally or intracerebrally to increase brain concentration. For example, a method of administering iduronate 2-sulfatase into the brain of a patient has been reported to prevent the progression of brain damage in a patient with Hunter syndrome (mucopolysaccharidosis type II) (Patent Document 1). However, direct intrathecal or intracerebral administration is highly invasive (Non-patent Document 11).
- RMT receptor-mediated transcytosis
- target cerebrovascular endothelial expression receptors include, for example, transferrin receptor, insulin There are receptors, insulin-like growth factor receptors, and low density lipoprotein receptor family (LDLRf).
- RMT receptor-mediated transcytosis
- LDLRf low density lipoprotein receptor family
- a transferrin receptor-mediated blood-brain barrier crossing technique has been reported by producing a fusion protein of an anti-transferrin receptor antibody and a nerve growth factor.
- an anti-transferrin receptor antibody bispecific antibodies of anti-transferrin receptor antibody and anti-beta-secretase (BACE1) antibody (patent documents 2 and 3 and non-patent documents 12 and 13), anti-amyloid ⁇ A fusion antibody (patent document 4 and non-patent document 14) in which a monovalent antibody of anti-transferrin receptor is fused to the carboxyl terminal side of the antibody has been reported.
- Non-Patent Document 13 It is reported that brain delivery by bispecific antibody of anti-transferrin receptor antibody and anti-BACE1 antibody increases the amount of antibody uptake in brain by about 4 times that of control when the antibody is administered at 20 mg / kg body weight in mouse.
- Non-patent Document 9 a technology has been reported in which a drug is allowed to pass through the blood-brain barrier by encapsulating the drug in a liposome having an anti-transferrin receptor antibody on the surface. It has been reported that the amount of uptake in rat brain is increased approximately 2 to 5 times by the anti-rat transferrin receptor antibody and the immunomicellar fusion (Non-patent Document 9).
- Fc5 is a variable domain of heavy chain antibody (VHH) antibody of a single-domain heavy chain antibody derived from llama, and Fc5 and human Fc fusions are compared with control IgG for brain delivery An increase is shown in in vitro BBB models and rat in vivo models.
- VHH variable domain of heavy chain antibody
- Non-patent Documents 24 and 25 fetal Fc receptor
- FcRn fetal Fc receptor
- MOG is a protein belonging to the immunoglobulin superfamily and constitutes myelin.
- the full-length human MOG consists of 218 amino acids and is expressed in the outermost layer of myelin in the central nervous system and plays a role in cell adhesion and cell surface interaction (Non-patent Document 26-28).
- MOG is considered to be a candidate for self antigen in inflammatory diseases in which glial cells in the central nerve are attacked by autoimmunity such as multiple sclerosis (MS) (Non-patent Documents 29 and 30) . Although the concentration of anti-MOG antibody in serum is low in MS patients, it has been reported that anti-MOG antibody is also detected in central nerves (Non-patent Document 29).
- Non-patent Documents 30, 32 and 33 The reason for this is that in disease states such as MS, it is reported that leakage of humoral factors and invasion of inflammatory cells cause breakdown of the blood-brain barrier, which makes it easier for antibodies to be transferred to the central nervous system. Furthermore, it has been reported that autoantibodies are produced locally in the central nervous system by B cells and plasma cells invading the central nervous system (Non-patent Documents 30, 32 and 33).
- EAE Experimental autoimmune encephalomyelitis
- Non-patent documents 29 and 35 there is a report that EAE score is exacerbated by administering anti-MOG antibody to EAE-induced animals (Non-patent documents 29 and 35), but 1-2 days after antibody administration (non-patent document 29) or 4 The day after (non-patent document 35) shows the peak of the EAE score, which is transient.
- non-patent Documents 36 and 37 it has been reported that EAE does not develop even when anti-MOG antibody alone is administered to normal animals.
- Boado RJ. Methods in Enzymology, 503, 269-292, 2012 Boado RJ., Et al., Drug Metab. Dispos., 37 (12), 2299-2304, 2009 Boado RJ., Et al., J. Pharmacol. Exp. Ther., 333 (3), 961-969, 2010 Boado RJ., Et al., Bioconjugate Chem., 1, 97-104, 2012 Yun Zhang. Et al., J. Pharmacol. Exp.
- Non-patent documents 29 and 35 disclose that when the anti-MOG antibody is administered to the EAE model, the antibody is detected in the brain, but when the anti-MOG antibody is administered to the periphery of a normal animal, the brain is There have been no reports of anti-MOG antibodies that can detect their internal presence.
- the present invention relates to MOG binding molecules that bind to myelin oligodendrocyte glycoprotein (MOG) and methods using such molecules.
- MOG myelin oligodendrocyte glycoprotein
- an antibody that binds to MOG or the antibody fragment a hybridoma that produces the antibody or the antibody fragment, a nucleic acid that includes the nucleotide sequence that encodes the antibody or the antibody fragment, and a transformation that includes the vector that includes the nucleic acid Cell
- method for producing the antibody or the antibody fragment composition containing the antibody or the antibody fragment, method for detecting or measuring an antigen present in the brain using the antibody or the antibody fragment, diagnosis or diagnosis of brain disease
- the present invention provides a MOG binding molecule that binds to MOG and a method using the molecule, specifically an antibody or an antibody fragment thereof.
- the present invention relates to the following (1) to (22).
- An antibody or antibody fragment that binds to myelin oligodendrocyte glycoprotein hereinafter referred to as MOG.
- MOG myelin oligodendrocyte glycoprotein
- the amino acid sequences of the complementarity determining regions (hereinafter referred to as "CDR") 1 to 3 of the heavy chain variable region hereinafter referred to as "VH" include the amino acid sequences described in SEQ ID NOs.
- the amino acid sequences of CDRs 1 to 3 of VH include the amino acid sequences described in SEQ ID NOs: 16, 17 and 18, respectively, and the amino acid sequences of CDRs 1 to 3 of VL correspond to SEQ ID NOs: 22, 23 and 24, respectively
- C) The amino acid sequences of CDRs 1 to 3 of VH include the amino acid sequences described in SEQ ID NOs: 28, 29 and 30, respectively, and the amino acid sequences of CDRs 1 to 3 of VL correspond to SEQ ID NOs: 34, 35 and 36, respectively
- (D) an antibody fragment wherein the amino acid sequences of CDRs 1 to 3 of the heavy chain variable region (hereinafter referred to as VHH) of the heavy chain antibody comprise the amino acid sequences set forth in S
- the amino acid sequences of CDRs 1 to 3 of VH include the amino acid sequences described in SEQ ID NOs: 163, 164 and 165, respectively, and the amino acid sequences of CDRs 1 to 3 of VL correspond to SEQ ID NOs: 168, 169 and 170, respectively
- (G) The amino acid sequences of CDRs 1 to 3 of VH include the amino acid sequences set forth in SEQ ID NOs: 173, 174 and 175, respectively, and the amino acid sequences of CDRs 1 to 3 of VL have SEQ ID NOs: 178, 179 and 180, respectively
- the amino acid sequences of CDRs 1 to 3 of VH include the amino acid sequences set forth in SEQ ID NOs: 183, 184 and 185, respectively, and the amino acid sequences of CDRs 1 to 3 of VL have SEQ ID NOs: 188, 189 and 190
- the amino acid sequences of CDRs 1 to 3 of VH include the amino acid sequences set forth in SEQ ID NOs: 213, 214 and 215, respectively, and the amino acid sequences of CDRs 1 to 3 of VL have SEQ ID NOs: 218, 219 and 220, respectively
- (L) The amino acid sequences of CDRs 1 to 3 of VH include the amino acid sequences described in SEQ ID NOs: 223, 224 and 225, respectively, and the amino acid sequences of CDRs 1 to 3 of VL correspond to SEQ ID NOs: 228, 229 and 230, respectively
- (M) The amino acid sequences of CDRs 1 to 3 of VH include the amino acid sequences set forth in SEQ ID NOs: 233, 234 and 235, respectively, and the amino acid sequences of CDRs 1 to 3 of VL have SEQ ID NOs: 238, 239 and 240, respectively
- an antibody comprising the amino acid sequence as set forth in SEQ ID NO: 272 and the amino acid sequence as set forth in SEQ ID NO: 274, wherein (O7) An antibody comprising an amino acid sequence as set forth in SEQ ID NO: 276 and an amino acid sequence as set forth in SEQ ID NO: 278 for an amino acid sequence of VH. (O8) an antibody comprising the amino acid sequence as set forth in SEQ ID NO: 280 and the amino acid sequence as set forth in SEQ ID NO: 282 in the amino acid sequence of VH (O9) An antibody comprising an amino acid sequence as set forth in SEQ ID NO: 284 and an amino acid sequence as set forth in SEQ ID NO: 286 for the amino acid sequence of VH.
- an antibody comprising an amino acid sequence as set forth in SEQ ID NO: 324 and an amino acid sequence as set forth in SEQ ID NO: 326 for an amino acid sequence of VH (O20) an antibody comprising the amino acid sequence as set forth in SEQ ID NO: 328 and the amino acid sequence as set forth in SEQ ID NO: 330, wherein the amino acid sequence of VH comprises (O21) an antibody comprising the amino acid sequence of which the amino acid sequence of VH is set forth in SEQ ID NO: 332, and wherein the amino acid sequence of VL comprises the amino acid sequence set forth in SEQ ID NO: 334; An antibody comprising the amino acid sequence set forth in 336, and wherein the amino acid sequence of VL comprises the amino acid sequence set forth in SEQ ID NO: 338.
- the antibody fragment contains Fab, Fab ′, F (ab ′) 2 , single chain antibody (scFv), dimerized V region (diabody), disulfide stabilized V region (dsFv), VHH and CDR
- the antibody and the antibody fragment according to any one.
- At least one member selected from the group consisting of the following (a) to (c) is bound to the antibody or antibody fragment that binds to the MOG according to any one of (1) to (10): Fusion antibody or fusion antibody fragment.
- a hybridoma that produces the antibody according to any one of (1) to (11).
- a nucleic acid comprising a nucleotide sequence encoding the antibody according to any one of (1) to (11).
- a transformed cell comprising a vector comprising the nucleic acid according to (13).
- culturing the hybridoma according to (12) or the transformed cell according to (14), and collecting the antibody according to any one of (1) to (11) or the antibody fragment thereof from the culture solution A method for producing the antibody or the antibody fragment according to any one of (1) to (11).
- (16) A composition comprising the antibody according to any one of (1) to (11) or the antibody fragment thereof.
- the composition according to (16) which is a composition for detection or measurement of an antigen present in the brain.
- (19) A method for detecting or measuring an antigen present in the brain, using the antibody according to any one of (1) to (11) or the antibody fragment thereof, or the composition according to (16).
- (20) A method for diagnosing or treating a brain disease, using the antibody according to any one of (1) to (11) or the antibody fragment thereof, or the composition according to (16).
- (21) The antibody, or the antibody fragment or fusion antibody or fusion antibody fragment according to any one of (1) to (11), or the composition according to (16) A method of improving brain retention of a fusion antibody or fusion antibody fragment.
- the MOG binding molecule of the present invention not only increases the brain retention of the binding molecule itself by specifically binding to MOG, but also transports into the brain and retention by modifying other molecules to the MOG binding molecule. Can be applied to the treatment of brain diseases.
- Specific MOG binding molecules of the invention include antibodies.
- the antibody or the antibody fragment of the present invention is an antibody having brain retention by binding to MOG in the brain. Therefore, the antibody of the present invention or the antibody fragment of the present invention is a composition for detection or measurement of an antigen present in the brain (MOG or MOG and other antigens present in the brain), a composition for diagnosis of a brain disease, And as a pharmaceutical composition for treating brain diseases.
- FIG. 1 shows the results of analysis by ELISA of the binding of scFv display phage clones that bind to MOG to rMOG-FLAG_Fc.
- the ordinate represents the absorbance for rMOG-FLAG_Fc, and the abscissa represents the name of scFv antibody displayed by each phage clone.
- FIG. 2 shows the results of analysis of the binding of each anti-MOG antibody to HEK cells, rat MOG / HEK cells, mouse MOG / HEK cells, cynomolgus monkey MOG / HEK cells or human MOG / HEK cells by a flow cytometer.
- FIG. 3 (A) and (B) show the results of rat brain migration evaluation of anti-MOG antibody.
- FIG. 3 (A) shows the antibody concentration in serum four days after administration of the antibody to rats.
- the vertical axis represents antibody concentration (ng / mL), and the horizontal axis represents administered antibody.
- FIG. 3 (B) shows the antibody concentration in brain tissue four days after administration of the antibody to rats.
- the ordinate represents the amount of antibody per brain weight (ng / g brain), and the abscissa represents the antibody administered.
- FIG. 4 (A) and (B) show the results of rat brain migration evaluation of anti-MOG antibody.
- FIG. 4 (A) shows antibody concentrations in serum four and ten days after administration of the antibody to rats.
- the vertical axis represents antibody concentration (ng / mL), and the horizontal axis represents the number of days since administration of antibody (days).
- FIG. 4 (B) shows the antibody concentration in brain tissue 4 and 10 days after administration of the antibody to rats.
- the vertical axis shows the amount of antibody per brain weight (ng / g brain), and the horizontal axis shows the number of days since administration of the antibody (days).
- FIG. 5 shows the results of analysis of the binding of various bispecific antibodies to HEK293F cells, rat MOG / HEK293F cells or human MOG / HEK293F cells using a flow cytometer.
- the ordinate represents the number of cells, and the abscissa represents the fluorescence intensity.
- the dotted histogram shows the binding of anti-AVM antibody used as a negative control, and the solid histogram shows the binding of each bispecific antibody.
- FIG. 6 shows the results of analysis of the binding of various bispecific antibodies to human breast cancer cell line SK-BR-3 using a flow cytometer.
- the ordinate represents the number of cells, and the abscissa represents the fluorescence intensity.
- the dotted histogram shows the binding of anti-AVM antibody used as a negative control, and the solid histogram shows the binding of each bispecific antibody.
- FIGS. 7 (A) and (B) show the results of rat brain transferability evaluation of bispecific antibodies binding to MOG.
- FIG. 7 (A) shows the antibody concentration in serum 10 days after administration of the antibody to rats.
- the ordinate represents the antibody concentration (ng / mL), and the abscissa represents the bispecific antibody used.
- FIG. 7 (B) shows the antibody concentration in brain tissue 10 days after administration of the antibody to rats.
- the ordinate represents the amount of antibody per brain weight (ng / g brain), and the abscissa represents the bispecific antibody used.
- FIG. 8 (A) and (B) show the results of mouse brain migration evaluation of anti-MOG01 antibody.
- FIG. 8 (A) shows antibody concentrations in serum at 3, 6, 10, 14, 21, 28 days after antibody administration to mice.
- the vertical axis represents antibody concentration (ng / mL), and the horizontal axis represents time (day).
- FIG. 8 (B) shows antibody concentrations in brain tissue 3, 6, 10, 14, 21, 28 days after administration of the antibody to mice.
- the vertical axis represents antibody concentration (ng / g brain), and the horizontal axis represents time (day).
- FIGS. 9 (A) to (C) show the results of mouse brain migration imaging evaluation of an anti-MOG01 antibody.
- Fig. 9 (A) shows fluorescence intensity measurement data of the brain six days after administering negative control Alexa Fluor R 488 labeled anti-AVM antibody and Alexa Fluor R 488 labeled anti MOG 01 antibody to mice, and
- FIG. 9C is a value obtained by correcting the amount of fluorescence in the brain after 6 days and 14 days with the fluorescence intensity of the administered antibody.
- FIGS. 11A and 11B show the structures of various bispecific antibodies that bind to AVM and MOG.
- FIG. 10 (A) shows the structure of an AVM-MOG01 IgG4PE (R409K) antibody
- FIG. 10 (B) shows an AVM IgG4PE (R409K) _MOG01 Fab antibody
- FIG. 10 (C) shows an AVM IgG4PE (R409K) _MOG01 sscFv antibody.
- FIGS. 11A and 11B show the structures of various bispecific antibodies that bind to AVM and MOG.
- FIG. 11A shows AVM IgG4PE (R409K) _MOG01 dscFv antibody, AVM IgG4PE (R409K) _MOG01 dscFv2 antibody and AVM IgG4PE (R409K) _MOG01 dscFv4 antibody.
- FIG. 11B shows AVM IgG4PE (R409K) _MOG01 dscFv3 antibody
- Antibody-AVM shows the structure of AVM IgG4PE (R409K) _MOG01 dscF11 antibody.
- 12 (A) to 12 (C) show the results of analysis of the binding of various bispecific antibodies to human MOG / L 929 cells using a flow cytometer.
- the ordinate represents the average fluorescence intensity
- the abscissa represents the antibody concentration.
- the white circles indicate the AVM IgG4PE (R409K) antibody (negative control)
- the black squares indicate the AVM-MOG01 IgG4PE (R409K) antibody.
- white circles indicate AVM IgG4PE (R409K) _AVMs scFv antibody (negative control)
- black squares indicate AVM IgG4PE (R409K) _MOG01 sscFv antibody.
- FIGS. 13 (A) and (B) show the results of analysis of the binding of various bispecific antibodies to human MOG / L 929 cells using a flow cytometer. The ordinate represents the average fluorescence intensity, and the abscissa represents the antibody concentration. In FIG.
- white squares indicate AVM IgG4PE (R409K) _MOG01 dscFv antibodies
- white circles indicate AVM IgG4PE (R409K) _MOG01 dscFv2 antibodies
- white triangles indicate AVM IgG4PE (R409K) _MOG01 dscFv4 antibodies.
- the white diamonds are AVM IgG4PE (R409K) _MOG01dscFv3 antibodies
- the black diamonds are AVM IgG4PE (R409K) _MOG01dscFv5 antibodies
- the white circles are AVM IgG4PE (R409K) _MOG01dscFv6 antibodies
- the black circles are AVM IgG4PE (R409K) _MOG01d
- the triangle indicates the AVM IgG4PE (R409K) _MOG01 dscFv8 antibody
- the black triangle indicates the AVM IgG4PE (R409K) _MOG01 dscFv9 antibody
- the white square indicates the AVM IgG4PE (R409K) _MOG01 dscFv10 antibody
- the black square indicates the AVM IgG4PE (R409K) _MOG01 dscFv11 antibody.
- FIGS. 14 (A) and (B) show the results of mouse brain migration evaluation of various bispecific antibodies.
- the ordinate represents the antibody concentration, and the abscissa represents the bispecific antibody used.
- FIGS. 14 (A) and (B) show antibody concentrations in serum and brain tissue 10 days after administration of AVM IgG4PE (R409K) antibody (negative control) and AVM-MOG01 IgG4PE (R409K) antibody, respectively.
- FIGS. 15 (A) and (B) show the results of mouse brain transferability evaluation of various bispecific antibodies. The ordinate represents the antibody concentration, and the abscissa represents the bispecific antibody used.
- FIGS. 16A and 16B show antibody concentrations in serum and brain tissue 10 days after administration of AVM IgG4 PE (R409K) _AVMs scFv antibody (negative control) and AVM IgG4 PE (R409K) _MOG01 scFv antibody, respectively.
- FIGS. 16A and 16B show the results of mouse brain migration evaluation of various bispecific antibodies. The ordinate represents the antibody concentration, and the abscissa represents the bispecific antibody used.
- FIGS. 17 (A) to (D) show the results of mouse brain migration evaluation of various bispecific antibodies. The ordinate represents the antibody concentration, and the abscissa represents the bispecific antibody used.
- Negative controls corresponding to the AVM IgG4PE (R409K) _MOG01 dscFv antibodies are AVM IgG4PE (R409K) _AVM dscFv antibodies
- AVM IgG4 PE (R409K) _MOG01 dscFv3 negative controls correspond to AVM IgG4PE (R409K) _AVM dscFv3 antibodies
- AVM dscFv3 The corresponding negative control is the AVM IgG4PE (R409K) _AVMdscFv5 antibody.
- FIG. 17 (A) shows the antibody concentration in serum 10 days after antibody administration.
- FIG. 17 (B) shows the antibody concentration in brain tissue 10 days after antibody administration.
- FIG. 17 (C) shows the antibody concentration in serum 28 days after antibody administration.
- FIG. 17 (D) shows the antibody concentration in brain tissue 28 days after antibody administration.
- FIG. 18 shows the amino acid sequences of scFv of similar clones of MOG antibody, and shows similar clones of MOG301 antibody.
- FIG. 19 shows the amino acid sequence of the scFv of a similar clone of the MOG antibody, and shows a similar clone of the MOG303 antibody.
- FIG. 20 shows the amino acid sequences of scFv of similar clones of MOG antibody, and shows similar clones of MOG 307 antibody.
- FIG. 21 shows the amino acid sequences of scFv of similar clones of MOG antibody, and shows similar clones of MOG310 antibody.
- FIG. 22 (A) and (B) shows the amino acid sequences of scFv of similar clones of MOG antibody.
- FIG. 22 (A) shows a similar clone of MOG 329 antibody, and
- FIG. 22 (B) shows a similar clone of MOG 456 antibody.
- FIG. 23 shows the results of analysis of the binding of anti-MOG antibodies to Expi293F cells using a flow cytometer. The ordinate represents the number of cells, and the abscissa represents the fluorescence intensity.
- FIG. 24 shows the results of analysis of the binding of anti-MOG antibodies to mouse MOG / Expi293F cells by a flow cytometer. The ordinate represents the number of cells, and the abscissa represents the fluorescence intensity.
- the dotted histogram shows the binding of anti-AVM antibody used as a negative control, and the solid histogram shows the binding of each MOG antibody.
- FIG. 25 shows the results of analysis of the binding of anti-MOG antibodies to human MOG / Expi293F cells by a flow cytometer.
- FIG. 26 shows the results of analysis of the binding of the enzyme-fused antibody MOG01 IgG4 PE (R409K) -ASM to human MOG / L 929 cells using a flow cytometer.
- the ordinate represents the average fluorescence intensity, and the abscissa represents the antibody concentration.
- FIG. 27 shows the results of ELISA analysis of the binding of anti-ASM antibody (manufactured by LSBio) to MOG01 IgG4PE (R409K) -ASM and AVM IgG4PE (R409K) -ASM.
- the ordinate represents the absorbance
- the abscissa represents the name of the immobilized antibody.
- MOG01 IgG4PE and AVM IgG4PE were used as negative controls.
- a thin hatched bar shows data of 5 ⁇ g / mL of anti-ASM antibody
- a thick hatched bar shows data of 1 ⁇ g / mL of anti-ASM antibody
- a white bar shows data of 0.2 ⁇ g / mL of anti-ASM antibody.
- FIGS. 28 (A) and (B) show the results of mouse brain migration evaluation of enzyme fusion antibodies MOG01 IgG4PE (R409K) -ASM and AVM IgG4PE (R409K) -ASM.
- the ordinate represents the antibody concentration
- the abscissa represents the enzyme-fused antibody used.
- FIG. 28 (A) shows the antibody concentration in serum 10 days after antibody administration.
- FIG. 28 (B) shows the antibody concentration in brain tissue 10 days after antibody administration.
- the present invention relates to an antigen binding molecule that binds to myelin oligodendrocyte glycoprotein (Myelin-oligodendrocyte glycoprotein, hereinafter referred to as MOG). More specifically, the present invention relates to an antibody or antibody fragment that binds to MOG.
- MOG myelin oligodendrocyte glycoprotein
- the MOG binding molecule of the present invention may be any molecular form as long as it is a molecule that specifically binds to MOG and the molecule is retained in the brain, and proteins, nucleic acids, organically synthesized low molecular weight compounds / high molecular weight compounds Etc. may be any molecule. Specifically, any of recombinant proteins, antibodies, aptamers, low molecular weight compounds obtained by small molecule screening and the like may be used, but preferably antibodies and antibody fragments thereof can be mentioned.
- the MOG binding molecule is preferably a molecule that binds to the extracellular domain of MOG.
- MOG is a protein belonging to the immunoglobulin superfamily and constitutes myelin.
- the full length human MOG consists of 218 amino acids and is expressed in the outermost layer of myelin in the central nervous system and plays a role in cell adhesion and cell surface interactions.
- Animal species of MOG to which the MOG binding molecule of the present invention binds include mouse, rat, cynomolgus monkey and / or human, etc., but it is not particularly limited to these species, and suitable depending on the use of the antibody. Animal species can be selected.
- the antibody of the present invention is used in human pharmaceutical applications, the antibody is preferably an antibody that binds to at least human MOG.
- a polypeptide comprising the amino acid sequence of SEQ ID NO: 78 or the amino acid sequence of NCBI accession number AAB08088 as human MOG, the amino acid sequence of SEQ ID NO: 78 or one of the amino acid sequences of NCBI accession number AAB08088 60% or more of the amino acid sequence described in SEQ ID NO: 78 or the amino acid sequence of NCBI Accession No. AAB08088, which comprises the amino acid sequence wherein the above amino acids are deleted, substituted or added, and which has the function of human MOG
- a polypeptide comprising an amino acid sequence having a homology of preferably 80% or more, more preferably 90% or more, most preferably 95% or more, and having a function of human MOG can be mentioned.
- a polypeptide having an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence set forth in SEQ ID NO: 78 or in the amino acid sequence shown in NCBI Accession No. AAB08088 is a site-directed mutagenesis [Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989), Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997), Nucleic acids Research, 10, 6487 (1982), Proc. Natl. Acad Sci. USA, 79, 6409 (1982), Gene, 34, 315 (1985), Nucleic Acids Research, 13, 4431 (1985), Proc. Natl. Acad. Sci. USA, 82, 488 (1985)], etc. Can be obtained, for example, by introducing a site-directed mutation into a DNA encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 78.
- the number of amino acids to be deleted, substituted or added is not particularly limited, but preferably 1 to several tens, for example, 1 to 20, more preferably 1 to several, for example 1 to 5 amino acids It is.
- the gene encoding human MOG includes the nucleotide sequence set forth in SEQ ID NO: 77, and the nucleotide sequence of NCBI Accession Number U64564.
- U64564 preferably a base sequence with 80% or more homology, more preferably 95 A gene comprising a nucleotide sequence having% homology or more and containing a DNA encoding a polypeptide having a MOG function, or a DNA comprising the nucleotide sequence of SEQ ID NO: 77, or the nucleotide sequence of NCBI accession number U64564 Hybrid under stringent conditions It consists size to DNA, and such a gene encoding a polypeptide having the function of MOG also included in the gene encoding MOG in the present invention.
- the colony hybridization method the plaque hybridization method using as a probe the DNA comprising the nucleotide sequence of SEQ ID NO: 77 or the nucleotide sequence of NCBI accession number U64564 as the DNA hybridizing under stringent conditions Or hybridizable DNA obtained by Southern blot hybridization or DNA microarray.
- the hybridizable DNA is a DNA having at least 60% or more homology, preferably 80% or more homology, with the nucleotide sequence of SEQ ID NO: 77, or the nucleotide sequence of NCBI accession number U64564.
- DNA having a homology of 95% or more can be mentioned.
- MOG The functions of MOG include involvement in cell adhesion and cell surface interaction on myelin and the like.
- genes in which small-scale mutations have been made in the nucleotide sequence due to such polymorphisms are also encompassed in the MOG-encoding gene in the present invention.
- the homology numerical value in the present invention may be a numerical value calculated using a homology search program known to those skilled in the art unless otherwise specified, but for nucleotide sequences, BLAST [J. Mol. Biol BLAST 2 [Nucleic Acids Res., 25, 3389 (1997), Genome Res., 7, 649 (1997), for amino acid sequences, such as those calculated using default parameters. , Http://www.ncbi.nlm.nih.gov/Education/BLASTinfo/information3.htmL], and numerical values calculated using default parameters.
- the default parameters are 5 if G (Cost to open gap) is a base sequence, 11 if an amino acid sequence, 2 if -E (Cost to extend gap) is a base sequence, and 1 if an amino acid sequence.
- -Q Pulalty for nucleotide mismatch
- -r forward for nucleotide match
- -e expect value
- -W wordsize
- amino acid sequence In the case of 3 residues, 20 if the -y [Dropoff (X) for blast extensions in bits] is blastn, 7 for programs other than blastn, -X (X dropoff value fo gapped alignment in bits 15 and 50 for final X dropoff values for gapped alignment in bits (blast) and 25 for programs other than blastn (http://www.ncbi.nlm.nih.gov /blast/htmL/blastcgihelp.htmL).
- Polypeptides containing partial sequences of the amino acid sequences of the various MOGs described above can be produced by methods known to those skilled in the art. Specifically, it can be prepared by deleting a part of the DNA encoding the amino acid sequences of the various MOGs described above and culturing a transformant into which an expression vector containing the same has been introduced. Further, a polypeptide having an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequences of various MOGs can be obtained by the same method as described above.
- a polypeptide comprising amino acid sequences of various MOGs, or a polypeptide having an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequences of various MOGs is the fluorenylmethyloxycarbonyl (Fmoc) method It can also be produced by chemical synthesis methods such as t-butyloxycarbonyl (tBoc) method.
- the extracellular region of human MOG refers to the amino acid sequence from 30th to 154th or 232th to 247th in the amino acid sequence set forth in SEQ ID NO: 78 or NCBI accession number AAB08088, and from 30th to 154th It is preferable that it is an amino acid sequence of
- the extracellular region of mouse MOG is the 30th to 157th or 232th to 247th amino acid sequence and the 30th to 157th amino acid sequence in the amino acid sequence described in SEQ ID NO: 74 or NCBI accession number NP_034944. Is preferred.
- the extracellular region of rat MOG refers to the 28th to 155th or 230th to 245th amino acid sequence and the 28th to 155th amino acid in the amino acid sequence described in SEQ ID NO: 68 or NCBI accession number AAA41628. Is preferred.
- the extracellular region of cynomolgus monkey MOG is the amino acid sequence from 30th to 154th or 232th to 247th in the amino acid sequence described in SEQ ID NO: 76 or NCBI accession number NP — 001271785, and the amino acid sequence from 30th to 154th Is preferred.
- the binding of the antibody of the present invention to the extracellular domain of MOG is determined by measuring the binding of the antibody of the present invention to MOG-expressing cells or a recombinant MOG protein using ELISA, flow cytometry, surface plasmon resonance, etc. It can be confirmed by In addition, known immunological detection methods [Monoclonal Antibodies-Principles and practice, Third edition, Academic Press (1996), Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory (1988), monoclonal antibody experiment manual, Kodansha Scientific (1987)] and the like can be combined and confirmed.
- the MOG binding molecule of the present invention is a molecule having brain retention by specifically binding to MOG in the brain.
- the antibody is an antibody having brain retention by binding to MOG in the brain.
- the antibody of the present invention when administered to the periphery of an animal, it penetrates from the periphery to the brain through the blood-brain barrier, translocates into the brain, and binds to MOG in the brain, thereby having an ability to retain brain It is.
- the antibody of the present invention is preferably an antibody excellent in brain retention or an antibody improved in brain retention.
- retention of brain means the property that the subject remains in the brain when the subject is administered to the subject animal. That is, at least selected from increased intracerebral transfer, increased intracerebral accumulation, reduced intracerebral to extracerebral transfer, reduced intracerebral to extracerebral drainage, and reduced intracerebral degradation
- increased intracerebral transfer at least selected from increased intracerebral transfer, increased intracerebral accumulation, reduced intracerebral to extracerebral transfer, reduced intracerebral to extracerebral drainage, and reduced intracerebral degradation
- excellent retention of brain, high retention of brain, or improved retention of brain means that when the subject is administered to a test animal, after the same day from the administration, as compared to the control, It means that the concentration (or amount in the brain) of the subject in the brain is increased, or the subject is present in the brain at a constant concentration (amount) to be detectable for a long time.
- excellent brain retention, high brain retention, or improved brain retention means, for example, when the subject is administered to a subject animal after administration compared to control 1
- the brain concentration (amount) of the subject is high, or preferably 2 to 10 days, preferably 3 to 10 days, and more preferably 4 to 10 days after administration;
- the peak of brain volume is 4 days after administration, preferably 5 days after administration, 6 days after, 7 days after, 8 days after, 9 days after, more preferably 10 days after administration And the like.
- An antibody with excellent brain retention, an antibody with high brain retention, or an antibody with improved brain retention has an antibody concentration (amount of antibody) in the brain higher than that of the control antibody, or in the brain for a long time Any antibody may be used as long as it has an optional feature.
- the feature of high transferability into the brain and / or accumulation in the brain For example, the feature of high transferability into the brain and / or accumulation in the brain, the feature of transfer from the inside of the brain to the outside of the brain, the feature of low excretory ability and / or degradation in brain compared to the control antibody, and the brain
- the antibody include an antibody having features such as high migration into the brain and / or accumulation in the brain as compared to migration from the inside to the brain, excretion, and / or degradability in the brain.
- the antibody or antibody fragment of the present invention when the antibody or antibody fragment is administered to an animal, an antibody having a high antibody concentration (or amount of antibody) in the brain after the same day after administration compared to the control antibody. Or the antibody fragment or an antibody or antibody fragment that can be present in the brain for a long time.
- the change in antibody concentration (or antibody amount) in the brain may be any, for example, if the antibody concentration in the brain gradually decreases after reaching a peak during the measurement period, After the antibody concentration reaches a peak, the antibody concentration may be maintained or may increase after administration of the antibody.
- the antibody of the present invention or the antibody fragment thereof is, for example, an antibody having a higher concentration or amount of antibody in the brain than the control antibody on day 4 or 10 after administration to rat, 4 days after administration to rat Antibody whose concentration or amount of antibody in the brain is maintained or increased from day 10 to day 10 or antibody whose presence in the brain can be clearly confirmed even after day 10 after administration to rats Etc. but are not limited to these.
- any antibody of the same species or subclass as the test antibody may be used.
- an anti-avermectin (AVM) antibody can be used.
- examples of the brain include, but are not limited to, brain parenchyma, intracerebroventricular, cerebrospinal fluid and the like.
- methods of administering antibodies to animals include, for example, intravenous administration or intracerebroventricular administration, intraperitoneal administration, subcutaneous administration, intradermal administration, nasal administration, intrathecal administration, etc. It is not limited to the method.
- a method for measuring the brain retention of an antibody for example, after administering an antibody to an animal and collecting brain tissue after several days, homogenizing and measuring the antibody concentration in the supernatant after centrifugation
- a method of calculating the amount of antibody per unit brain weight, a method of detecting the presence of antibody using known immunological techniques using collected brain tissue, or administering a labeled antibody to an animal examples include a method of detecting the presence of the antibody over time with an in vivo imaging system.
- Examples of the antibody of the present invention include one antibody selected from the group consisting of the following (a) to (q).
- (A) The amino acid sequences of CDRs 1 to 3 of VH are the amino acid sequences described in SEQ ID NOs: 4, 5 and 6, respectively, and the amino acid sequences of CDRs 1 to 3 of VL are SEQ ID NOs: 10, 11 and 12 respectively
- the amino acid sequences of CDRs 1 to 3 of VH are the amino acid sequences described in SEQ ID NOs: 16, 17 and 18 respectively, and the amino acid sequences of CDRs 1 to 3 of VL are SEQ ID NOs: 22, 23 and 24 respectively
- the amino acid sequences of CDRs 1 to 3 of VH are the amino acid sequences described in SEQ ID NOs: 28, 29 and 30, respectively, and the amino acid sequences of CDRs 1 to 3 of VL are SEQ ID NOs: 34, 35 and 36,
- the amino acid sequences of CDRs 1 to 3 of VH include the amino acid sequences described in SEQ ID NOs: 163, 164 and 165, respectively, and the amino acid sequences of CDRs 1 to 3 of VL correspond to SEQ ID NOs: 168, 169 and 170, respectively
- (G) The amino acid sequences of CDRs 1 to 3 of VH include the amino acid sequences set forth in SEQ ID NOs: 173, 174 and 175, respectively, and the amino acid sequences of CDRs 1 to 3 of VL have SEQ ID NOs: 178, 179 and 180, respectively
- the amino acid sequences of CDRs 1 to 3 of VH include the amino acid sequences set forth in SEQ ID NOs: 183, 184 and 185, respectively, and the amino acid sequences of CDRs 1 to 3 of VL have SEQ ID NOs: 188, 189 and 190
- the amino acid sequences of CDRs 1 to 3 of VH include the amino acid sequences set forth in SEQ ID NOs: 213, 214 and 215, respectively, and the amino acid sequences of CDRs 1 to 3 of VL have SEQ ID NOs: 218, 219 and 220, respectively
- (L) The amino acid sequences of CDRs 1 to 3 of VH include the amino acid sequences described in SEQ ID NOs: 223, 224 and 225, respectively, and the amino acid sequences of CDRs 1 to 3 of VL correspond to SEQ ID NOs: 228, 229 and 230, respectively
- (M) The amino acid sequences of CDRs 1 to 3 of VH include the amino acid sequences set forth in SEQ ID NOs: 233, 234 and 235, respectively, and the amino acid sequences of CDRs 1 to 3 of VL have SEQ ID NOs: 238, 239 and 240, respectively
- the antibodies of the present invention include the amino acid sequences of CDRs 1 to 3 of VH and CDRs 1 to 3 of VL of any one of the antibodies described in (a) to (n) above, each 85% or more, preferably 90% It includes antibodies having the amino acid sequences of CDRs 1 to 3 of VH and CDRs 1 to 3 of VL, which exhibit the above homology. More preferably, 90% or more homology includes 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% homology.
- human anti-MOG monoclonal antibody MOG01 antibody, MOG09 antibody, MOG14 antibody and alpaca anti-MOG monoclonal VHH antibody iMOG-3Rim1-S32 antibody Can be mentioned.
- Other examples include human chimeric antibodies of iMOG-3Rim1-S32, and humanized antibodies of iMOG-3 Rim1-S32.
- the antibody of (o) above refers to a second antibody that inhibits the binding between the first antibody and MOG when the antibodies described in (a) to (n) above are used as the first antibody.
- the antibody described in (a) to (n) above is the first antibody, and the epitope to which the first antibody binds is the first epitope
- the antibody of (p) above is the first epitope. It refers to a second antibody that binds to a second epitope.
- the antibody of the above (q) of the present invention means the antibody described in the above (a) to (n) as the first antibody, and the epitope to which the first antibody binds is the first epitope. Refers to a second antibody that binds to one epitope.
- an antibody comprising the amino acid sequence of SEQ ID NO: 3 and the amino acid sequence of VL of SEQ ID NO: 3;
- B an antibody comprising the amino acid sequence of SEQ ID NO: 15 and the amino acid sequence of VL of SEQ ID NO: 21;
- C an antibody comprising the amino acid sequence of SEQ ID NO: 27 and the amino acid sequence of VL of SEQ ID NO: 33;
- D an antibody comprising the amino acid sequence of which the amino acid sequence of VHH is set forth in SEQ ID NO: 39,
- E An antibody comprising an amino acid sequence as set forth in SEQ ID NO: 152 and an amino acid sequence as set forth in SEQ ID NO: 157 for the amino acid sequence of VH.
- an antibody comprising the amino acid sequence as set forth in SEQ ID NO: 272 and the amino acid sequence as set forth in SEQ ID NO: 274, wherein (O7) An antibody comprising an amino acid sequence as set forth in SEQ ID NO: 276 and an amino acid sequence as set forth in SEQ ID NO: 278 for an amino acid sequence of VH. (O8) an antibody comprising the amino acid sequence as set forth in SEQ ID NO: 280 and the amino acid sequence as set forth in SEQ ID NO: 282 in the amino acid sequence of VH (O9) An antibody comprising an amino acid sequence as set forth in SEQ ID NO: 284 and an amino acid sequence as set forth in SEQ ID NO: 286 for the amino acid sequence of VH.
- an antibody comprising an amino acid sequence as set forth in SEQ ID NO: 324 and an amino acid sequence as set forth in SEQ ID NO: 326 for an amino acid sequence of VH (O20) an antibody comprising the amino acid sequence as set forth in SEQ ID NO: 328 and the amino acid sequence as set forth in SEQ ID NO: 330, wherein the amino acid sequence of VH comprises (O21) an antibody comprising the amino acid sequence of which the amino acid sequence of VH is set forth in SEQ ID NO: 332, and wherein the amino acid sequence of VL comprises the amino acid sequence set forth in SEQ ID NO: 334; An antibody comprising the amino acid sequence set forth in 336, and wherein the amino acid sequence of VL comprises the amino acid sequence set forth in SEQ ID NO: 338.
- the antibody of the present invention has an amino acid sequence of at least 85%, preferably 90%, of each of the VH and VL amino acid sequences of any one of the antibodies described in (a) to (n) and (o1) to (o22) above. It includes antibodies having the VH and VL amino acid sequences of antibodies showing the above homology. More preferably, 90% or more homology includes 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% homology.
- human anti-MOG monoclonal antibody MOG01 antibody, MOG09 antibody, MOG14 antibody, and alpaca anti-MOG monoclonal antibody, respectively.
- VHH antibodies include iMOG-3 Rim1-S32 antibody.
- Other examples include iMOG-3 Rim1-S32 human chimeric antibody, and iMOG-3 Rim1-S32 humanized antibody.
- the EU index refers to the position of the amino acid residue shown in Sequence of Proteins of Immunological Interest 5th Edition (1991).
- the positions of amino acid residues shown below all indicate the positions of amino acid residues described in the EU index, unless otherwise specified.
- Antibody molecules are also referred to as immunoglobulins (hereinafter referred to as Ig) and their basic structures are referred to as heavy chains (hereinafter referred to as heavy chains) and light chains (hereinafter referred to as light chains). It is a tetramer having two polypeptides each.
- the H chain is also referred to as the H chain variable region (also referred to as VH) from the N-terminal side, the H chain constant region (also referred to as CH), and the L chain as the L chain variable region (VL as the N terminal side) And L chain constant region (also denoted as CL).
- VH H chain variable region
- CH H chain constant region
- CL L chain constant region
- CH As for CH, ⁇ , ⁇ , ⁇ , ⁇ and ⁇ chains are known for each subclass.
- the CH is further composed of each domain of CH1 domain, hinge domain, CH2 domain, and CH3 domain from the N-terminal side.
- a domain refers to a functional structural unit that constitutes each polypeptide of an antibody molecule.
- the CH2 domain and the CH3 domain are collectively referred to as Fc (Fragment, crystallizable) region or simply Fc.
- Fc Frament, crystallizable region
- CL C ⁇ and C ⁇ chains are known.
- the subclasses of antibodies in which CH is ⁇ , ⁇ , ⁇ , ⁇ and ⁇ chains are referred to as IgA, IgD, IgE, IgG and IgM, respectively.
- the subclass of each antibody has an isotype depending on the animal, and in humans, IgA1 and IgA2 for IgA, and IgG1, IgG2, IgG3 and IgG4 for IgG.
- the CH1 domain, hinge domain, CH2 domain, CH3 domain and Fc region in the present invention can be identified by the number of amino acid residues from the N-terminus by EU index.
- CH1 is the amino acid sequence of EU index 118-215
- hinge is the amino acid sequence of EU index 216-230
- CH2 is the amino acid sequence of EU index 231-340
- CH3 is EU index 341-447
- the amino acid sequence and the Fc region are specified as the amino acid sequence of EU index 231 to 447, respectively.
- the antibodies of the present invention include any of polyclonal antibodies, monoclonal antibodies and oligoclonal antibodies.
- Polyclonal antibody refers to a population of antibody molecules secreted by antibody producing cells of different clones.
- a monoclonal antibody is an antibody secreted by a single clone of antibody-producing cells, and is an antibody that recognizes a single epitope (also referred to as an antigenic determinant) and is uniform in amino acid sequence (primary sequence) constituting the monoclonal antibody.
- Oligoclonal antibody refers to a population of antibody molecules in which a plurality of different monoclonal antibodies are mixed.
- Examples of monoclonal antibodies in the present invention include antibodies produced by hybridomas, or recombinant antibodies produced by transformants transformed with an expression vector containing an antibody gene.
- the epitope includes a single amino acid sequence that a monoclonal antibody recognizes and binds, a three-dimensional structure consisting of an amino acid sequence, a three-dimensional structure consisting of a post-translationally modified amino acid sequence, and a post-translationally modified amino acid sequence.
- the post-translationally modified amino acid sequence includes an O-linked sugar chain in which the sugar chain is linked to Tyr and Ser having an OH substituent, an N-linked sugar chain linked to Gln and Asn having an NH 2 substituent, and a sulfate molecule Is linked to Tyr having an OH substituent and includes a tyrosine sulfated amino acid sequence.
- the epitope of MOG to which the antibody of the present invention binds is a deletion from which a partial domain of MOG is deleted, a variant substituted with a domain derived from another protein, a partial peptide fragment of MOG, etc. It can be determined by conducting binding experiments. Alternatively, antibody binding experiments can also be performed using cells expressing the above-mentioned deficient body or mutant.
- the epitope of MOG to which the antibody of the present invention binds can also be determined by adding the antibody of the present invention to a peptide fragment of MOG digested with a proteolytic enzyme and performing epitope mapping using known mass spectrometry. can do.
- CDR Combination Determining Region
- a chimeric antibody refers to an antibody derived from an animal species in which VH and VL and CH and CL are different.
- An antibody consisting of non-human animal (non-human animal) antibodies VH and VL and human antibody CH and CL comprises human chimeric antibody, non-mouse animal antibody VH and VL, and mouse antibody CH and CH
- An antibody consisting of CL is referred to as a mouse-type chimeric antibody, and other chimeric antibodies are named in the same manner.
- any animal can be used as long as it is an animal capable of producing a hybridoma or producing an antibody phage library, such as a mouse, a rat, a hamster, a rabbit, a llama, a camel, an alpaca and the like.
- a hybridoma is a cell producing a monoclonal antibody having a desired antigen specificity, obtained by fusing a B cell obtained by immunizing a non-human animal with an antigen and a myeloma cell derived from a mouse or the like.
- An antibody phage library refers to a library prepared by cloning an immunoglobulin variable region gene into phage and expressing an antigen-binding molecule on its surface.
- Phages to be used include M13 phage and the like, but are not particularly limited.
- the antigen-binding molecule displayed on the phage may be in any form, but is preferably an antibody fragment such as scFv, Fab or VHH.
- the antibody phage library may be any library among an immune library, a naive library and a synthetic library.
- the immune library refers to an antibody-immunized library constructed from an antibody-immunized animal or an antibody gene derived from a patient's lymphocyte.
- the naive library refers to an antibody phage library constructed based on antibody genes derived from normal animal or healthy human lymphocytes.
- a synthetic library is a library in which V genes in genomic DNA and CDRs of reconstructed functional V genes are replaced with oligonucleotides encoding random amino acid sequences of appropriate length.
- a method for producing a chimeric antibody the method for producing a human chimeric antibody is described below.
- the other chimeric antibodies can also be produced by the same method.
- Human chimeric antibodies are obtained by obtaining cDNAs encoding VH and VL from non-human animal cell-derived hybridomas producing monoclonal antibodies, and using them as animal cell expression vectors having DNAs encoding human antibodies CH and CL, respectively.
- the vector can be inserted to construct a human chimeric antibody expression vector, which can be expressed and produced by introduction into animal cells.
- genes encoding VH and VL are cloned from antibody phage libraries derived from non-human animals, and inserted into animal cell expression vectors having DNAs encoding human antibodies CH and CL, respectively. Can be expressed and produced by introducing into animal cells.
- a humanized antibody refers to an antibody in which the amino acid sequences of the CDRs of VH and VL of a non-human animal antibody are grafted to the corresponding CDRs of VH and VL of a human antibody. Regions other than CDRs of VH and VL are referred to as framework regions (hereinafter referred to as FR).
- the humanized antibody comprises a cDNA encoding a VH amino acid sequence consisting of the amino acid sequence of the CDR of the VH of the non-human animal antibody and the amino acid sequence of the FR of any human antibody, and the amino acids of the CDR of the VL of the non-human animal antibody
- a cDNA encoding a VL amino acid sequence consisting of the sequence and the amino acid sequence of the FR of any human antibody is constructed, and inserted into an animal cell expression vector having DNAs encoding CH and CL of the human antibody, respectively.
- An antibody expression vector can be constructed and expressed by introducing it into animal cells for production.
- the human antibody originally refers to an antibody naturally present in the human body, but also includes a human antibody phage library and an antibody obtained from a human antibody-producing transgenic animal.
- a human antibody is obtained by immunizing a mouse carrying a human immunoglobulin gene (Tomizuka K. et al., Proc Natl Acad Sci USA 97, 722-7, 2000.) with a desired antigen. Can.
- human antibodies can be obtained without immunization by selecting human antibodies having a desired binding activity using a phage display library obtained by amplifying antibody genes from human-derived B cells (Winter G. et al. et al., Annu Rev Immunol. 12: 433-55. 1994).
- the human antibody phage library is a library of phage in which antibody fragments such as Fab, scFv and VHH are expressed on the surface by inserting antibody genes prepared from human (healthy people or patients) lymphocytes into phage genes. . From the library, phage expressing an antibody fragment having a desired antigen binding activity can be recovered using the binding activity to a substrate on which the antigen is immobilized as an index. The antibody fragment can be further converted into a human antibody molecule consisting of two complete H chains and two complete L chains by genetic engineering techniques.
- a human antibody-producing transgenic animal refers to an animal in which a human antibody gene has been integrated into the host animal chromosome. Specifically, a human antibody-producing transgenic animal can be produced by introducing a human antibody gene into mouse ES cells, and transplanting the ES cells into early embryos of other mice to generate them.
- Production of human antibodies from human antibody-producing transgenic animals is carried out by culturing human antibody-producing hybridomas obtained by the hybridoma production method carried out in normal non-human mammals and producing and accumulating human antibodies in the culture. This can be done by purifying the antibody from the culture.
- the antibodies of the present invention include heavy chain antibodies consisting of only heavy chains.
- the heavy chain antibody refers to an antibody obtained from a camelid animal such as llama, camel and alpaca, and a recombinant antibody produced based on the antibody.
- an antibody fragment refers to a fragment of an antibody that has antigen binding activity.
- Fab, Fab ′, F (ab ′) 2 , single chain Fv (scFv), diabody, dsFv, a peptide containing a plurality of CDRs, and VHH and the like can be mentioned.
- antibody fragments of the present invention include partial fragments of antibodies such as antibody fragments in which the antibody constant region or full length or a part of Fc is fused to the antibody fragment, antibody regions containing constant region or Fc, etc. Any antibody fragment is also included as long as it has binding activity.
- the disulfide bond between the N-terminal half of the H chain and the entire L chain is an antibody fragment having a molecular weight of about 50,000 and antigen binding activity.
- F (ab ') 2 is a fragment obtained by treating IgG with the proteolytic enzyme pepsin (cleaved at the 234th amino acid residue of the H chain), Fab is via the S-S bond of the hinge region An antibody fragment having an antigen binding activity and a molecular weight of about 100,000, which is slightly larger than that bound.
- Fab ′ is an antibody fragment having a molecular weight of about 50,000 and having an antigen binding activity, which is obtained by cleaving the S—S bond of the hinge region of F (ab ′) 2 described above.
- the scFv is prepared by using an appropriate peptide linker (P) such as a linker peptide consisting of a linker (G4S) consisting of 4 Gly and 1 Ser residue (G4S) consisting of 1 VH and 1 VL
- P an appropriate peptide linker
- G4S linker peptide consisting of a linker (G4S) consisting of 4 Gly and 1 Ser residue (G4S) consisting of 1 VH and 1 VL
- G4S linker peptide consisting of a linker (G4S) consisting of 4 Gly and 1 Ser residue (G4S) consisting of 1 VH and 1 VL
- G4S linker peptide consisting of a linker (G4S) consisting of 4 Gly and 1 Ser residue (G4S) consisting of 1 VH and 1 VL
- G4S Ser residue
- a diabody is an antibody fragment in which scFvs of the same or different antigen binding specificities form a dimer and are antibody fragments having a bivalent antigen binding activity to the same antigen or a specific antigen binding activity to different antigens.
- DsFv refers to a polypeptide in which one amino acid residue in each of VH and VL is substituted with a cysteine residue, which is linked via an S-S bond between the cysteine residues.
- the peptide containing CDR is comprised including at least one or more regions of CDR of VH or VL.
- Peptides containing a plurality of CDRs can be linked together directly or via an appropriate peptide linker.
- a DNA encoding the VH and VL CDRs of the antibody of the present invention is constructed, the DNA is inserted into a prokaryotic expression vector or a eukaryotic expression vector, and the expression vector is introduced into a prokaryotic or eukaryotic organism. Expression and production.
- peptides containing CDRs can also be produced by chemical synthesis methods such as Fmoc method or tBoc method.
- VHHs are variable regions of heavy chain antibodies, also referred to as nanobodies.
- the antibody fragment of the present invention includes any antibody fragment or antibody fragment as described above as long as it is an antibody fragment having MOG binding activity.
- an antibody having one antigen binding site or the antibody fragment is referred to as a monovalent antibody.
- a monovalent antibody antigen binding sites described in WO 2014/054804, WO 2011/090754, WO 2007/048037, and WO 2012/116927 etc. can be used.
- the format etc. of the antibody which has one or the antibody fragment are mentioned.
- one molecule of antibody or antibody fragment that binds to three or more different antigens or epitopes is referred to as multispecific antibody.
- one molecule of antibody or antibody fragment that binds to two different antigens or epitopes is referred to as a bispecific antibody.
- bispecific antibody examples include bispecific antibodies described below.
- the bispecific antibody described in (1) above is a bispecific antibody in which the antigen binding site containing VH of heavy chain A binds to MOG and the antigen binding site containing VH of heavy chain B binds to an antigen present in the brain It may be or vice versa.
- the bispecific antibody described in the above (2) is a bispecific antibody in which an antibody fragment is bound to the C terminus of one of the two heavy chains constituting the antibody and the antibody fragment binds to both of the two heavy chains.
- the bispecific antibody may be any bispecific antibody.
- a suitable linker may be present between the C-terminus of the antibody heavy chain and the antibody fragment.
- the antibody fragment possessed by the bispecific antibody described in the above (2) is preferably, but not limited to, scFv, Fab and VHH.
- the bispecific antibody described in the above (2) may be a bispecific antibody in which the N-terminal antigen binding site binds to MOG and the C-terminal antigen binding site binds to an antigen present in the brain, or vice versa .
- the bispecific antibody described in the above (3) refers to a bispecific antibody in which an antibody fragment is bound to the N terminus of at least one of two heavy chains or light chains constituting the antibody. Also, a suitable linker may be present between the N-terminus of the antibody heavy and / or light chain and the antibody fragment.
- the antibody fragment possessed by the bispecific antibody described in the above (3) is preferably, but not limited to, scFv, Fab, VHH and the like.
- bispecific antibody described in the above (3), a bispecific antibody having a structure of VH 1 -CH 1 -VH 2 -CH 1 -hinge-CH 2 -CH 3 from the N-terminus of heavy chain, and the above heavy chain structure
- bispecific antibodies that have an antigen-binding site with each of VH 1 and VH 2 and VL.
- the VH in which VH 1 and VH 2 form an antigen binding site may be the same amino acid sequence or may be different amino acid sequences.
- the multispecific antibody or bispecific antibody may be any antibody that is multispecific antibody and bispecific antibody that binds to MOG.
- a multispecific antibody or bispecific antibody that binds to MOG and an antigen present in the brain is preferable, and a multispecific antibody or an antigen binding site that binds to MOG and an antigen binding site that binds to an antigen present in the brain More preferred are bispecific antibodies.
- antigens present in the brain include proteins, sugar chains, lipids and the like, and among them, proteins are preferable.
- sugar chains present in the brain include, but are not limited to, Lewis-x, Lewis-y, CD15 and the like.
- lipids present in the brain include, but are not limited to, GD1a, GD2, GD3, GM1, GM2, GM3, or Phosphatidylserine, but are not limited to these lipids.
- the antibody or antibody fragment of the present invention also includes an antibody containing any post-translationally modified amino acid.
- Post-translational modifications include, for example, deletion of a lysine residue at the C-terminus of the heavy chain [lysine clipping], conversion of glutamine residue at the N-terminus of the polypeptide to pyroglutamine (puroGlu), etc. [Beck et al, Analytical Chemistry, 85, 715-736 (2013)].
- the antibody of the present invention or the antibody fragment may carry out amino acid modification of the Fc region.
- Amino acid modifications of the Fc region include, for example, amino acid modifications for stabilizing the antibody or controlling the half life in blood.
- Specific examples of amino acid modifications of the Fc region include, for example, WO 2006/033386, WO 2006/075668, WO 2011/122011, and WO 2009/125825. .
- the antibody or the antibody fragment of the present invention also encompasses a fusion antibody or a fusion antibody fragment, in which the antibody or the antibody fragment is modified.
- the method for modifying the antibody is not particularly limited, and any method can be used as long as it can modify the desired amino acid residue and sugar chain.
- examples of the molecule that modifies the antibody or the antibody fragment include hydrophilic polymers, amphiphilic polymers, and functional molecules.
- examples of the hydrophilic polymer and amphiphilic polymer include molecules containing polyoxyalkylene, polyol or polysaccharide.
- polyoxyalkylenes examples include linear or branched polyethylene glycol (hereinafter referred to as PEG), polypropylene glycol, polypropylene ethylene glycol and the like.
- molecules containing polyols or polysaccharides include amylose consisting of linear or branched polyglycerol, homo- or hetero-polysaccharides such as dextran, pullulan or glycogen, and the like.
- the molecular weight of the hydrophilic polymer or the molecule containing an amphiphilic polymer is not particularly limited, but is preferably 100 Da or more, and for example, preferably 100 Da to 100 kDa.
- Examples of functional molecules include antigen-binding molecules and fragments thereof, drugs, bioactive peptides, bioactive proteins, nucleic acids, radiolabeled compounds, sugar chains, lipids or fluorescent compounds.
- a functional molecule such as an antigen-binding molecule
- the molecule having bispecificity is a bispecific antibody.
- Antigen binding molecules include, for example, antibodies, receptors, and ligands.
- the fragment of the antigen binding molecule may be any fragment of the antigen binding molecule as long as it has antigen binding activity.
- alkylating agents for example, alkylating agents, nitrosoureas, antimetabolites, antivirals, antibiotics, plant alkaloids, topoisomerase inhibitors, tubulin polymerization inhibitors, hormone therapeutic agents, hormone antagonists, aromatase inhibitors, Anticancer drugs such as P-glycoprotein inhibitors, platinum complex derivatives, M phase inhibitors or kinase inhibitors [Clinical Oncology, Cancer and Chemotherapy (1996)], steroids such as hydrocortisone and prednisone, aspirin, indomethacin, etc.
- steroids such as hydrocortisone and prednisone, aspirin, indomethacin, etc.
- Non-steroidal agents such as gold thiomalate, penicillamine, immunosuppressants such as cyclophosphamide, azathioprine, or anti-inflammatory agents such as antihistamines such as chlorpheniramine maleate or cremathinin [inflammatory and anti-inflammatory therapies, Medical and dental drug publishing corporation (19 2)] and the like.
- anticancer agents include mertansine, emtansine, amifostin (Ethiol), cisplatin, dacarbazine (DTIC), dactinomycin, mechlorethamine (nitrogen mustard), streptozocin, cyclophosphamide, ifosfamide, carmustine (BCNU), lomustine (BCNU) CCNU), doxorubicin (adriamycin), epirubicin, gemcitabine (gemzar), daunorubicin, procarbazine, mitomycin, cytarabine, etoposide, 5-fluorouracil, fluorouracil, vinblastine, vincristine, bleomycin, daepomycin, pepromycin, estramustine, paclitaxel (taxol), Docetaxel (taxotere), aldesleukin, asparagine , Busulfan, carboplatin, oxaliplatin, ned
- physiologically active peptide or physiologically active protein for example, interferon (hereinafter referred to as IFN) - ⁇ , IFN- ⁇ , IFN- ⁇ , interleukin (hereinafter referred to as IL) -2, IL-12, IL-15 , IL-18, IL-21, IL-23, granulocyte colony stimulating factor (G-CSF), granulocyte / macrophage colony stimulating factor (GM-CSF), or macrophage colony stimulating factor (M-CSF), etc.
- IFN interferon
- IL-15 interleukin
- IL-18 interleukin
- IL-21 interleukin-23
- G-CSF granulocyte colony stimulating factor
- GM-CSF granulocyte / macrophage colony stimulating factor
- M-CSF macrophage colony stimulating factor
- NK Cytokines or growth factors that activate immunocompetent cells such as cells, macrophages or neutrophils, proteolytic enzymes such as hydrolases, lyases and isomerases, enzymes such as acid sphingomyelinase, such as lysine, diphtheria toxin, or ONTAK Bacterial toxins and toxins such as plant toxins, cell membrane injury Antimicrobial peptides having sex, peptides having cell membrane bound or cell membrane permeability, and derivatives thereof.
- the nucleic acid may be any molecule obtained by polymerizing a nucleotide or a molecule having a function equivalent to that of the nucleotide, such as siRNA, microRNA, antisense RNA, DNA aptamer and the like.
- the radiolabeled compound may be any nuclide used for diagnostic or therapeutic applications, for example, 3 H, 14 C, 32 P, 33 P, 35 S, 51 Cr, 57 CO, 18 F, 153 Gd , 159 Gd, 64 Cu, 68 Ge, 166 Ho, 115 In, 113 In, 112 In, 111 In, 111 In, 131 I, 125 I, 123 I, 121 I, 140 La, 177 Lu, 54 Mn, 99 Mo, 103 Pd, 142 Pr, 149 Pm, 186 Re, 188 Re, 211 At, 105 At, Rh, 97 Ru, 153 Sm, 47 Sc, 75 Se, 85 Sr, 99 Tc, 201 Ti, 113 Sn, 117 Sn, 133 Xe, 169 Yb, 175 Yb, 90 Y and 65 Zn etc., or containing the above mentioned nuclides A compound is mentioned.
- Radioactively labeled compounds can be attached directly to the antibody, such as by the chloramine T method.
- a substance that chelates the radiolabeled compound may be bound to the antibody.
- the chelating agent includes, for example, DOTA, PA-DOTA, TRITA and DTPA, etc.
- An antibody modified by a chelating agent, a modified antibody in which a radiolabeled compound is labeled via a chelating agent is also included in the antibody of the present invention included.
- sugar chains include fucose, mannose, glucose, allose, gulose, idose, galactose, talose, ribose, arabinose, xylose, lyxose, erythose, erythrose, threose, cellobiose, maltose, isomaltose, lactose, lipoarabi
- sugar chains include fucose, mannose, glucose, allose, gulose, idose, galactose, talose, ribose, arabinose, xylose, lyxose, erythose, erythrose, threose, cellobiose, maltose, isomaltose, lactose, lipoarabi
- it may be a natural product containing a sugar chain known as an immunoadjuvant, such as ⁇ (1 ⁇ 3) glucan (lentinan, schizophyllan) or ⁇ galactosyl ceramide (KRN 7000).
- an immunoadjuvant such as ⁇ (1 ⁇ 3) glucan (lentinan, schizophyllan) or ⁇ galactosyl ceramide (KRN 7000).
- lipids examples include simple lipids (neutral lipids) which are esters of fatty acids and various alcohols and their analogs.
- simple lipids neutral lipids
- esters of fatty acids and various alcohols and their analogs For example, fats and oils (eg, triacylglycerol), waxes (eg, fatty acid esters of higher alcohols), sterol esters, cholesterol esters, fatty acid esters of vitamins, etc., fatty acids and alcohols, as well as phosphoric acids, sugars, sulfuric acids, amines etc.
- Group-containing complex lipids such as phospholipids (eg, glycerophospholipids and sphingophospholipids) and glycolipids (eg, glyceroglycolipids and glycosphingolipids), compounds produced by hydrolysis of simple lipids and complex lipids
- derivatized lipids such as fatty acids, higher alcohols, fat-soluble vitamins, steroids, hydrocarbons and the like refer to fat-soluble ones.
- Fluorescent compounds include, for example, fluorescein series such as fluorescein isothiocyanate (FITC), rhodamine series, Cy3, Cy5, eosin series, fluorescent dyes such as alexafluoro series and NBD series, luminescent substances such as acridinium ester, or rophine And fluorescent proteins such as green fluorescent protein (GFP).
- fluorescein series such as fluorescein isothiocyanate (FITC), rhodamine series, Cy3, Cy5, eosin series, fluorescent dyes such as alexafluoro series and NBD series, luminescent substances such as acridinium ester, or rophine And fluorescent proteins such as green fluorescent protein (GFP).
- the above-mentioned hydrophilic polymer or amphiphilic polymer, and the functional molecule can be linked directly or via an appropriate linker.
- the linker for example, ester, disulfide, hydrazone, dipeptide and the like can be mentioned.
- the cDNA encoding the antibody is ligated to the cDNA encoding the protein, and the fusion antibody or fusion is performed. Constructing a DNA encoding the antibody fragment, inserting the DNA into a prokaryotic or eukaryotic expression vector, and introducing the expression vector into the prokaryotic or eukaryotic organism to express a fusion antibody or a fusion antibody fragment
- fusion antibodies or fusion antibody fragments can be produced.
- composition of the present invention may be any composition containing the antibody of the present invention or the antibody fragment.
- Such composition may contain, in addition to the antibody or the antibody fragment, an additive such as a suitable carrier and a stabilizer.
- the composition of the present invention includes, for example, a composition for detection or measurement comprising the antibody of the present invention or the antibody fragment thereof.
- the composition of the present invention includes, for example, a pharmaceutical composition (therapeutic agent) containing the antibody of the present invention or the antibody fragment as an active ingredient, etc., and is formulated into a desired dosage form with a pharmacologically acceptable carrier. It is formulated.
- the composition for detection or measurement includes the antibody of the present invention or the antibody fragment thereof, which can detect or measure the antigen to which the antibody of the present invention or the antibody fragment specifically binds. Any composition may be used.
- the antigen to which the antibody of the present invention or the antibody fragment specifically binds includes MOG or an antigen present in MOG and brain.
- the antibody or antibody fragment of the present invention binds to MOG in the brain when administered to an animal and has the property of staying in the brain. Therefore, by using the composition for detection or measurement containing the antibody or the antibody fragment, it is possible to maintain the antibody in the brain or to increase the antibody concentration in the brain, and MOG or MOG over a long period of time And detecting or measuring an antigen present in the brain, and / or detecting or measuring MOG or an antigen present in MOG and the brain.
- the composition for detection or measurement is a composition containing a bispecific antibody that binds to MOG and an antigen present in the brain
- the MOG to which the bispecific antibody binds and the antigen present in the brain are detected for a long time
- the composition for detection or measurement is a composition comprising a fusion antibody or a fusion antibody fragment that binds to a MOG labeled with a radioactive labeling compound or a fluorescent dye
- the MOG is detected or measured for a long time
- / or MOG can be detected or measured with high sensitivity.
- the pharmaceutical composition (therapeutic agent) containing the antibody of the present invention may be a therapeutic agent for any disease as long as the disease expresses an antigen to which the antibody of the present invention or the antibody fragment specifically binds.
- Therapeutic agents for brain diseases are preferred.
- Brain diseases include, for example, Alzheimer's disease, prodromal Alzheimer's disease, Huntington's disease, Parkinson's disease, brain tumor, multiple sclerosis, muscular dystrophy, amyotrophic lateral sclerosis, multiple system atrophy, progressive supranuclear palsy, Nigrostriatal degeneration, Olivopon cerebellar atrophy, bulbo-spinal muscular atrophy, spinocerebellar degeneration, cerebrovascular disorder, epilepsy, migraine headache, hyperactivity disorder, Creutzfeldt-Jakob disease, cerebral cortex basal ganglia degeneration Disease, lysosome disease, depression, dystonia and the like.
- the antibody of the present invention has the property of binding to MOG in the brain when administered to an animal and staying in the brain. Therefore, by using the therapeutic agent containing the antibody or the antibody fragment, it is possible to maintain the antibody or the antibody fragment in the brain for a long period of time and to increase the antibody concentration in the brain. It can show therapeutic effects.
- the therapeutic agent is a therapeutic agent comprising a bispecific antibody that binds to MOG and an antigen present in the brain
- the therapeutic effect on a brain disease associated with an antigen present in the brain to which the bispecific antibody binds Can be shown.
- the therapeutic agent is a fusion antibody or a fusion antibody fragment that binds to MOG modified with a small molecule drug
- a therapeutic effect can be shown against a brain disease targeted by the small molecule drug.
- the therapeutic effect is higher when the therapeutic agent of the present invention is used than when the low molecular weight drug is used alone.
- the therapeutic agent containing the antibody of the present invention or the antibody fragment may contain only the antibody or the antibody fragment as an active ingredient, but usually one or more pharmacologically acceptable carriers and It is desirable that they be mixed together and provided as a pharmaceutical preparation made by any method known in the pharmaceutical arts.
- the administration route is preferably that which is most effective in treatment, for example, oral administration, or intraoral, intratracheal, intrarectal, subcutaneous, intradermal, intramuscular, intracerebroventricular, intrathecal, intranasal And parenteral administration such as intraperitoneal or intravenous injection, particularly preferably intravenous or intracerebroventricular administration and the like.
- the dosage form includes, for example, sprays, capsules, tablets, powders, granules, syrups, emulsions, suppositories, injections, ointments, tapes and the like.
- the dose or the number of administrations vary depending on the intended therapeutic effect, administration method, treatment period, age, body weight and the like, it is usually 10 ⁇ g / kg to 20 mg / kg per adult per day.
- the present invention also provides a method of retaining an antibody in the brain using the antibody of the present invention or the antibody fragment thereof, a method of improving the brain retention of the antibody, and a method of increasing the antibody concentration (or amount of antibody) in the brain. Also includes
- the present invention relates to a peptide that binds to MOG, a nucleic acid containing a nucleotide sequence encoding the peptide, a transformed cell containing a vector containing the nucleic acid, culturing the transformed cell, and collecting the peptide from the culture solution
- a method of producing the peptide, a composition containing the peptide, or a method of detecting or measuring an antigen present in the brain using the peptide or the composition a method of diagnosing or treating a brain disease, a peptide Or a method of increasing the amount of peptide in the brain.
- the peptides of the present invention include fusion peptides in which the peptide is modified.
- Preparation of Antigen MOG or MOG-expressing cell serving as an antigen is to introduce an expression vector containing cDNA encoding MOG full length or partial length thereof into E. coli, yeast, insect cells or animal cells, etc.
- Can be obtained by MOG can also be obtained by purifying MOG from various animal cell lines, animal cells, animal tissues, etc. expressing MOG in large amounts.
- these animal cell lines, animal cells and animal tissues can be used as antigens as they are.
- a synthetic peptide having a partial sequence of MOG can be prepared by a chemical synthesis method such as Fmoc method or tBoc method and used as an antigen.
- a known tag such as FLAG or His may be added to the C-terminal or N-terminal of a synthetic peptide having a MOG or a partial sequence of MOG.
- MOG used in the present invention may be any of the methods described in Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989), Current Protocols In Molecular Biology, John Wiley & Sons (1987-1997), etc.
- MOG-encoding DNA can be expressed and produced in host cells by the following method.
- a recombinant vector is prepared by inserting a full-length cDNA containing a portion encoding MOG downstream of the promoter of an appropriate expression vector.
- a DNA fragment of an appropriate length which is prepared based on the full-length cDNA and contains a polypeptide-encoding portion, may be used.
- the resulting recombinant vector can be introduced into a host cell compatible with the expression vector to obtain a transformant producing a polypeptide.
- any vector capable of autonomous replication or integration into a chromosome in the host cell to be used and containing an appropriate promoter at a position capable of transcribing DNA encoding the polypeptide may be used.
- any microorganism can be used as long as it can express a target gene, such as a microorganism belonging to the genus Escherichia such as E. coli, yeast, insect cells or animal cells.
- the expression vector is capable of autonomous replication in prokaryote, and at the same time, contains a promoter, a ribosome binding sequence, a DNA encoding a human MOG-encoding portion, and a transcription termination sequence It is preferable that it is a vector containing. Although a transcription termination sequence is not necessarily required for the expression vector, it is preferable to place the transcription termination sequence immediately below the structural gene. Furthermore, the recombinant vector may contain a gene that controls a promoter.
- the expression vector it is preferable to use a plasmid in which the distance between the Shine-Dalgarno sequence (also referred to as SD sequence), which is a ribosome binding sequence, and the initiation codon is adjusted to an appropriate distance (eg 6 to 18 bases).
- SD sequence also referred to as SD sequence
- initiation codon is adjusted to an appropriate distance (eg 6 to 18 bases).
- bases can be substituted so as to be optimal codons for expression in a host, whereby the target MOG production rate can be improved.
- any expression vector can be used as long as it can exert its function in the host cell to be used, and for example, pBTrp2, pBTac1, pBTac2 (all manufactured by Roche Diagnostics), pKK233-2 (preferable) Manufactured by Pharmacia, pSE280 (manufactured by Invitrogen), pGEMEX-1 (manufactured by Promega), pQE-8 (manufactured by Qiagen), pKYP10 (Japanese Unexamined Patent Publication No. 58-110600), pKYP200 [Agricultural Biological Chemistry, 48, 669 (1984)], pLSA1 [Agric. Biol.
- any promoter may be used as long as it can function in the host cell used.
- promoters derived from E. coli, phage or the like such as trp promoter (Ptrp), lac promoter, PL promoter, PR promoter or T7 promoter can be mentioned.
- trp promoter Ptrp
- lac promoter PL promoter
- PR promoter PR promoter
- T7 promoter T7 promoter
- a tandem promoter in which two Ptrps are in tandem, and an artificially designed and modified promoter such as a tac promoter, lacT7 promoter or let I promoter can be mentioned.
- Examples of host cells include E. coli XL1-Blue, E. coli XL2-Blue, E. coli DH1, E. coli MC1000, E. coli KY3276, E. coli W1485, E. coli JM109, E. coli HB101, E. coli No. 49, E. coli W3110, E. coli NY49 or E. coli DH5 ⁇ and the like.
- any method for introducing a recombinant vector into a host cell any method can be used as long as it is a method for introducing DNA into a host cell to be used, for example, a method using calcium ion [Proc. Natl. Acad. Sci. USA , 69, 2110 (1972), Gene, 17, 107 (1982), and Molecular & General Genetics, 168, 111 (1979)].
- any expression vector can be used as long as it can exhibit its function in animal cells.
- pCDM8 [Nature, 329, 840 (1987)], pcDNAI / Amp (manufactured by Invitrogen Corporation) , PcDNA3.1 (manufactured by Invitrogen), pREP4 (manufactured by Invitrogen), pAGE 103 [J. Biochemistry, 101, 1307 (1987)], pAGE 210, pME18 SFL3, pKANTEX 93 (WO 97/10354), N5KG1 val (US Patent No. 6,001,358), INPEP 4 (Bi Made gen-IDEC, Inc.), pCI (Promega Corp.) and transposon vector (WO 2010/143698), and the like.
- Any promoter can be used as long as it can exert a function in animal cells, for example, a cytomegalovirus (CMV) immediate early (IE) gene promoter, SV40 early promoter, retrovirus promoter And metallothionein promoter, heat shock promoter, SR ⁇ promoter or promoter or enhancer of Moloney murine leukemia virus.
- CMV cytomegalovirus
- IE immediate early gene promoter
- SV40 early promoter SV40 early promoter
- retrovirus promoter And metallothionein promoter metallothionein promoter
- heat shock promoter SR ⁇ promoter or promoter or enhancer of Moloney murine leukemia virus.
- SR ⁇ promoter promoter or enhancer of Moloney murine leukemia virus.
- the enhancer of IE gene of human CMV may be used together with the promoter.
- host cells examples include human leukemia cells Namalwa cells, monkey cells COS cells, Chinese hamster ovary cells CHO cells [Journal of Experimental Medicine, 108, 945 (1958); Proc. Natl. Acad. Sci. USA, 60, 1275 (1968); Genetics, 55, 513 (1968); Chromosoma, 41, 129 (1973); Methods in Cell Science, 18, 115 (1996); Radiation Research, 148, 260 (1997); Proc. Natl. Acad Sci. USA, 77, 4216 (1980); Proc. Natl. Acad. Sci., 60, 1275 (1968); Cell, 6, 121 (1975); Molecular Cell Genetics, Appendix I, II (pp. 883-).
- CHO cells deficient in the dihydrofolate reductase gene (hereinafter referred to as dhfr) (CHO / DG44 cells) [Proc. Natl. Acad. Sci. USA, 77, 4216 (1980)], CHO-K1 (ATCC CCL-61), DUkXB11 (ATCC CCL-9096), Pro-5 (ATCC) CCL-1781), CHO-S (Life Technologies, Cat # 11619), Pro-3, rat myeloma cells YB2 / 3HL. P2. G11.16 Ag.
- dhfr dihydrofolate reductase gene
- mice 20 also referred to as YB2 / 0
- mouse myeloma cells NS0 mouse myeloma cells SP2 / 0-Ag14
- Syrian hamster cells BHK or HBT5637 Japanese Unexamined Patent Publication No. 63-000299
- any method for introducing an expression vector into a host cell any method can be used as long as it is a method for introducing DNA into an animal cell, and, for example, electroporation [Cytotechnology, 3, 133 (1990)], calcium phosphate ( JP-A-2-227075) or lipofection [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)], and the like.
- a transformant derived from a microorganism or animal cell carrying an expression vector into which the MOG-encoding DNA obtained as described above has been incorporated is cultured in a medium, and the MOG is produced and accumulated in a culture solution,
- the MOG can be produced by collecting it from the culture solution.
- the method of culturing the transformant in a medium can be carried out according to a conventional method used for culturing a host.
- MOGs When expressed in cells derived from eukaryotes, it is possible to obtain MOGs to which sugars or sugar chains have been added.
- an inducer may be added to the medium as needed.
- an inducer may be added to the medium as needed.
- a lac promoter culturing a microorganism transformed with an expression vector using an trp promoter such as isopropyl- ⁇ -D-thiogalactopyranoside
- trp promoter such as isopropyl- ⁇ -D-thiogalactopyranoside
- indole acrylic acid may be added to the medium.
- a culture medium for culturing transformants obtained using animal cells as a host for example, commonly used RPMI 1640 medium [The Journal of the American Medical Association, 199, 519 (1967)], Eagle's MEM medium [Science] , 122, 501 (1952)], Dulbecco's modified MEM medium [Virology, 8, 396 (1959)], 199 medium [Proc. Soc. Exp. Biol. Med., 73, 1 (1950)] or Iscove's Modified Examples include Dulbecco's Medium (IMDM) medium or a medium obtained by adding fetal bovine serum (FBS) or the like to these medium.
- IMDM Dulbecco's Medium
- FBS fetal bovine serum
- Culturing is usually carried out under conditions such as pH 6-8, 30-40 ° C., 5% CO 2 presence, etc. for 1-7 days.
- antibiotics such as kanamycin or penicillin may be added to the medium during the culture, if necessary.
- Examples of the method of expressing the MOG-encoding gene include, in addition to direct expression, methods such as secretory production or fusion protein expression [Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989)]. .
- MOG As a method of producing MOG, for example, a method of producing it in a host cell, a method of secreting it outside a host cell, or a method of producing it on a host extracellular membrane can be mentioned. By changing, an appropriate method can be selected.
- MOG When MOG is produced in the host cell or on the host extracellular membrane, the method of Paulson et al. [J. Biol. Chem., 264, 17619 (1989)], the method of Lowe et al. [Proc. Natl. Acad. Sci. , USA, 86, 8227 (1989), Genes Develop., 4, 1288 (1990)], Japanese Patent Application Laid-Open No. 05-336963, WO 94/23021 etc. Can be actively secreted out of host cells. Moreover, the production amount of MOG can also be increased using a gene amplification system (Japanese Patent Application Laid-Open No. 2-227075) using a dihydrofolate reductase gene or the like.
- the obtained MOG can be isolated and purified, for example, as follows.
- MOG When MOG is expressed in a dissolved state in cells, cells are collected by centrifugation after completion of culture, suspended in an aqueous buffer, and then disrupted with an ultrasonic crusher, French press, Manton Gaulin homogenizer, Dinomill, etc. The cells are disrupted to obtain a cell-free extract.
- a conventional protein isolation and purification method ie, solvent extraction method, salting out method with ammonium sulfate, desalting method, precipitation method with organic solvent, diethylamino Anion exchange chromatography method using a resin such as ethyl (DEAE) -sepharose, DIAION HPA-75 (manufactured by Mitsubishi Chemical Corporation), cation exchange chromatography using a resin such as S-Sepharose FF (manufactured by Pharmacia) Method, hydrophobic chromatography method using resin such as butyl sepharose, phenyl sepharose, gel filtration method using molecular sieve, affinity chromatography method, chromatofocusing method, electrophoresis method such as isoelectric focusing, etc. Is used alone or in combination to obtain a purified preparation It is possible.
- a resin such as ethyl (DEAE) -sepharose, DIAION HPA-75 (manufactured by Mitsubishi Chemical Corporation),
- MOG forms an insoluble matter in cells and is expressed
- the cells are recovered and disrupted in the same manner as described above, and centrifuged to separate the MOG insoluble matter as a precipitate fraction.
- the recovered insoluble form of the MOG is solubilized with a protein denaturant.
- a purified preparation of the polypeptide can be obtained by the same isolation and purification method as described above.
- MOG or a derivative such as a glycosylated product When MOG or a derivative such as a glycosylated product is secreted extracellularly, the MOG or a derivative such as a glycosylated product can be recovered from the culture supernatant.
- a soluble fraction is obtained by treating the culture as described above by a technique such as centrifugation, and a purified preparation is obtained from the soluble fraction by using the same isolation and purification method as described above. it can.
- the MOG used in the present invention can also be produced by a chemical synthesis method such as the Fmoc method or tBoc method.
- chemical synthesis using a peptide synthesizer such as Advanced Chemtech, Perkin-Elmer, Pharmacia, Protein Technology Instrument, Synthesel-Vega, Perceptib or Shimadzu Corporation You can also be produced by a chemical synthesis method such as the Fmoc method or tBoc method.
- a peptide synthesizer such as Advanced Chemtech, Perkin-Elmer, Pharmacia, Protein Technology Instrument, Synthesel-Vega, Perceptib or Shimadzu Corporation You can also
- mice, rats, rabbits or hamsters are immunized with the antigen obtained in (1) Collect antibody-producing cells in the peripheral blood.
- animals such as llama, alpaca and camels can be used as the animals to be immunized.
- Immunization is performed by administering the antigen subcutaneously, intravenously or intraperitoneally to the animal, for example, with Freund's complete adjuvant, or with an appropriate adjuvant such as aluminum hydroxide gel and B. pertussis vaccine.
- a conjugate is prepared with a carrier protein such as BSA (bovine serum albumin) or KLH (Keyhole Limpet hemocyanin), which is used as an immunogen.
- BSA bovine serum albumin
- KLH Keyhole Limpet hemocyanin
- tissues containing antibody-producing cells such as spleen are excised from the immunized animals, and antibody-producing cells are collected.
- spleen cells When spleen cells are used, the spleen is shredded and loosened, and then centrifuged to remove red blood cells to obtain fusion antibody-producing cells.
- Immunization can be performed on other immunized animals in the same manner to obtain antibody-producing cells.
- An appropriate interval can be selected according to the species of the animal to be immunized, for the interval between immunizations and the period from the last immunization to the removal of the tissue.
- myeloma cell for example, 8-azaguanine resistant mouse (derived from BALB / c) myeloma cell line P3-X63Ag8-U1 (P3-) is used using a cell line obtained from mouse.
- U1 Current Topics in Microbiology and Immunology, 18, 1 (1978)
- P3-NS1 / 1-Ag41 NS-1
- SP2 / 0-Ag14 SP-2
- SP-2 / 0-Ag14 SP-2
- P3-X63-Ag8653 6
- J. Immunology, 123, 1548 (1979)] or P3-X63-Ag8 (X63) [Nature, 256, 495] (1975)] and the like.
- the myeloma cells are passaged in normal medium [RPMI 1640 medium supplemented with glutamine, 2-mercaptoethanol, gentamycin, FBS, and 8-azaguanine], and passaged to normal medium 3-4 days before cell fusion. , To secure the cell number of 2 ⁇ 10 7 or more on the day of fusion.
- a mixture of polyethylene glycol-1000 (PEG-1000), MEM medium and dimethyl sulfoxide is added at 37 ° C. with stirring. Further, 1 to 2 mL of MEM medium is added several times every 1 to 2 minutes, and then MEM medium is added to a total volume of 50 mL.
- HAT medium normal medium supplemented with hypoxanthine, thymidine, and aminopterin. This suspension is cultured for 7-14 days at 37 ° C. in a 5% CO 2 incubator.
- a portion of the culture supernatant is withdrawn, and a cell group which reacts with MOG but does not react with an antigen other than MOG is selected by a hybridoma selection method such as the binding assay described later.
- cloning is performed by limiting dilution, and those which show stable and strong antibody titers are selected as monoclonal antibody-producing hybridomas.
- Ascites fluid is collected from this mouse, centrifuged to remove solids, salted out with 40 to 50% ammonium sulfate, and purified by caprylic acid precipitation, DEAE-Sepharose column, protein A-column or gel filtration column. , IgG or IgM fractions are collected and made into purified monoclonal antibodies.
- the monoclonal antibody-producing hybridoma obtained in (4) is cultured in RPMI 1640 medium or the like to which 10% FBS is added, the supernatant is removed by centrifugation, suspended in Hybridoma SFM medium, and cultured for 3 to 7 days.
- the obtained cell suspension can be centrifuged, and the obtained supernatant can be purified with a protein A-column or a protein G-column to collect the IgG fraction to obtain a purified monoclonal antibody.
- 5% Digo GF21 can also be added to Hybridoma SFM culture medium.
- Determination of antibody subclasses is performed by enzyme immunoassay using a subclass typing kit.
- the quantification of the amount of protein is calculated from the Lowry method or the absorbance at 280 nm.
- the MOG-expressing cell may be any cell as long as MOG is expressed on the cell surface, and examples thereof include animal cells, animal cell lines and MOG forced expression cell lines obtained in (1).
- MOG-expressing cells are aliquoted into a plate such as a 96-well plate, a test substance such as serum, culture supernatant of hybridoma or purified antibody is aliquoted as a first antibody and reacted.
- a test substance such as serum, culture supernatant of hybridoma or purified antibody is aliquoted as a first antibody and reacted.
- the cells are thoroughly washed with PBS containing 1-10% bovine serum albumin (BSA) (hereinafter referred to as BSA-PBS) or the like, and then an anti-immunoglobulin antibody labeled with a fluorescent reagent is used as a second antibody. Dispense and react.
- BSA-PBS bovine serum albumin
- the amount of fluorescence of the labeled antibody is measured using a flow cytometer to select an antibody that specifically reacts with MOG-expressing cells.
- selection of antibodies can also be performed by measuring the binding of monoclonal antibodies to MOG-expressing cells or MOG proteins using ELISA or surface plasmon resonance described below.
- the MOG protein may be a protein consisting of a partial domain of MOG or a protein to which a tag such as GST is attached.
- MOG-expressing cells or MOG protein are dispensed into a plate such as a 96-well plate, and then blocked with BSA-PBS, and a test substance such as serum, hybridoma culture supernatant or purified antibody is dispensed as a first antibody. And react. Next, after thoroughly washing with PBS or the like, an anti-immunoglobulin antibody labeled with a fluorescent reagent or the like is dispensed as a second antibody and reacted.
- the affinity of an antibody binding to MOG can be measured by immobilizing the antibody on an appropriate sensor chip and converting the MOG protein into an analyte using a known protocol.
- An antibody having a desired affinity for the MOG protein can be selected based on the affinity of the obtained antibody.
- the MOG protein may be immobilized on a sensor chip, and the antibody may be used as an analyte to measure the affinity of the antibody that binds to the MOG.
- an antibody that competes with the antibody of the present invention and binds to MOG can be obtained by adding a test antibody to a measurement system using the above-mentioned flow cytometry or ELISA and reacting it. That is, an antibody that competes with the antibody of the present invention for binding to the amino acid sequence of MOG or its steric structure by screening an antibody that inhibits the binding of the antibody of the present invention to MOG when the test antibody is added. You can get
- the antibody that binds to the epitope containing the epitope to which the antibody of the present invention binds is identified by the known method, the epitope of the antibody obtained by the above-mentioned screening method, and the synthetic peptide containing the identified epitope, or the steric of the epitope It can be obtained by preparing and immunizing a synthetic peptide or the like mimicking a structure.
- the epitope to which the antibody of the present invention binds and an antibody that binds to the same epitope identify the epitope of the antibody obtained by the screening method described above, and a partially synthetic peptide of the identified epitope or the conformation of the epitope Can be obtained by preparing and immunizing a synthetic peptide or the like mimicked in
- the immune library is obtained by lymphocyte from animal or patient immunized by the same method as the above (1), naive library is taken lymphocyte from normal animal or healthy person, RNA is extracted and reverse transcription reaction is performed CDNA is synthesized by reaction.
- an antibody gene fragment amplified by PCR is inserted into a phagemid vector, and E. coli is transformed with the phagemid vector.
- E. coli is transformed with the phagemid vector.
- an antibody phage library in which an antibody gene is library can be obtained.
- a synthetic library is a phagemid vector in which V genes in the genomic DNA and CDRs of reconstructed functional V genes are replaced with oligonucleotides encoding random amino acid sequences of appropriate length, and the V genes are inserted.
- Transform E. coli with An antibody phage library can be obtained by infecting the obtained transformant with a helper phage.
- Lymphocyte-derived cDNA and antibody phage libraries can also be used commercially.
- Phagemid vectors include pCANTAB 5E (Amersham Pharmacia), pUC118 / pUC119 vector (TaKaRa), pBlueScript II Phagemid Vector (Agilent Technologies), and pKSTV-02 (Miyazaki et al, J. Biochem. 2015; 1). be able to.
- helper phage M13KO7 helper phage (Invitrogen), VCSM13 Interference Resistant Helper Phage (Agilent Technologies), R408 Interference Resistant Helper Phage (Agilent Technologies) and the like can be used.
- Phage vectors can also be used for phage display. There are a peptide phage library (manufactured by New England Biolabs, etc.) using g3p of filamentous phage as a display molecule and a method using g7p, g8p, g9p as a display molecule.
- phage display using T7 phage can also be used.
- Display systems for T7 phage include T7Select vector (Novagen).
- Immobilize MOG on the immunotube and block the tube with blocking buffer The antibody phage library prepared in (7-1) above is added to each well of the tube and reacted. Next, the wells are washed, and fluorescently labeled anti-phage antibody is added and reacted, and then the wells are washed again and a coloring solution is added. The color reaction is then stopped with a reaction stop solution, and the absorbance in each well is measured with a microplate reader. Thereby, antibody phage clones that bind to MOG are selected.
- Recombinant Antibody As a production example of a recombinant antibody, methods of producing a human chimeric antibody and a humanized antibody are shown below. Recombinant mouse antibody, rat antibody, rabbit antibody, hamster antibody, camel antibody, llama antibody, alpaca antibody, human antibody, various chimeric antibodies, heavy chain antibody and the like can be produced by the same method.
- the vector for recombinant antibody expression is an expression vector for animal cells into which DNA encoding human antibody CH and CL has been incorporated, and the expression vector for animal cells is human It can be constructed by cloning DNAs encoding antibody CH and CL, respectively.
- CH and CL of any human antibody can be used.
- CH and ⁇ class CL of ⁇ 1 subclass of human antibody are used.
- cDNA is used for DNA encoding CH and CL of a human antibody, chromosomal DNA consisting of exons and introns can also be used.
- Any expression vector for animal cells can be used as long as it can integrate and express the gene encoding the C region of human antibody.
- pAGE 107 [Cytotechnol., 3, 133 (1990)]
- pAGE 103 [J. Biochem., 101, 1307 (1987)]
- pHSG274 [Gene, 27, 223 (1984)]
- pKCR Proc. Natl. Acad. Sci. USA, 78, 1527 (1981)]
- pSG1bd2-4 [Cytotechnol., 4, 173 (1990)]
- pSE1UK1Sed1-3 [Cytotechnol., 13, 79 (1993)], etc.
- the promoter and enhancer of the SV40 early promoter [J. Biochem., 101, 1307 (1987)], Moloney murine leukemia virus LTR [Biochem. Biophys. Res. Commun., 149, 960 ( 1987)] or an immunoglobulin heavy chain promoter [Cell, 41, 479 (1985)] and an enhancer [Cell, 33, 717 (1983)], and the like.
- the recombinant antibody expression vector balances the ease of construction of the recombinant antibody expression vector, the ease of introduction into animal cells, and the balance of the expression amounts of antibody H chain and L chain in animal cells
- a vector for expressing a recombinant antibody of a type (tandem type) in which the antibody H chain and L chain are present on the same vector [J. Immunol. Methods, 167, 271 (1994)]
- pKANTEX 93 WO 97/10354
- pEE 18 [Hybridoma, 17, 559 (1998)] or the like is used.
- DNA encoding the C region or V region of the non-human antibody is used as a probe to isolate a recombinant phage or recombinant plasmid having a cDNA encoding VH or VL, respectively.
- the entire base sequence of VH or VL of the non-human antibody of interest on the recombinant phage or recombinant plasmid is determined, and the entire amino acid sequence of VH or VL is deduced from the base sequence.
- mice, rats, hamsters, rabbits, rabbits, llamas, camels or alpacas are used, but any hybridoma cells can be produced. Animals can also be used.
- RNA easy kit for preparation of total RNA from hybridoma cells.
- Oligo (dT) -immobilized cellulose column method [Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989)], or Oligo-dT30 ⁇ Super> mRNA Purification (for mRNA preparation from total RNA)
- a kit such as a registered trademark Kit (manufactured by Takara Bio Inc.) is used.
- mRNA can be prepared from hybridoma cells using a kit such as Fast Track mRNA Isolation (registered trademark) Kit (manufactured by Invitrogen Corporation) or QuickPrep mRNA Purification (registered trademark) Kit (manufactured by Pharmacia).
- any vector can be used as a vector into which cDNA synthesized using mRNA extracted from hybridoma cells as a template can be incorporated, as long as the vector can incorporate the cDNA.
- ZAP Express [Strategies, 5, 58 (1992)]
- pBluescript II SK (+) [Nucleic Acids Research, 17, 9494 (1989)]
- ⁇ ZAPII (Stratagene)
- ⁇ gt10, ⁇ gt11 [DNA Cloning: A Practical] Approach, I, 49 (1985)]
- Lambda BlueMid (Clontech), ⁇ ExCell, pT7T3-18U (Pharmacia)
- pCD2 [Mol. Cell. Biol., 3, 280 (1983)] or pUC18 [Gene , 33, 103 (1985)] and the like.
- any vector capable of introducing, expressing and maintaining the cDNA library can be used.
- Selection of cDNA clones encoding VH or VL of a non-human antibody from a cDNA library can be performed by colony hybridization using a isotope or fluorescently labeled probe, or plaque hybridization [Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989)] or the like.
- primers are prepared, and a cDNA or cDNA library synthesized from mRNA is used as a template, Polymerase Chain Reaction method [hereinafter referred to as PCR method, Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989) CDNA encoding VH or VL can also be prepared by performing Current Protocols in Molecular Biology, Supplement 1, John Wiley & Sons (1987-1997)].
- PCR method Polymerase Chain Reaction method
- the selected cDNA is cleaved with an appropriate restriction enzyme and then cloned into a plasmid such as pBluescript SK (-) (Stratagene) to determine the base sequence of the cDNA by a commonly used base sequence analysis method or the like.
- a sequence analysis method for example, a reaction such as dideoxy method [Proc. Natl. Acad. Sci. USA, 74, 5463 (1977)] is performed, and then ABI PRISM 3700 (manufactured by PE Biosystems) or A.P. L. F.
- a base sequence automatic analyzer such as a DNA sequencer (manufactured by Pharmacia) or the like is used.
- the entire amino acid sequences of VH and VL are respectively deduced from the determined base sequences, and the entire amino acid sequences of VH and VL of known antibodies [Sequences of Proteins of Immunological Interest, US By comparing with Dept. Health and Human Services (1991)], it is confirmed whether the obtained cDNA encodes the complete amino acid sequences of VH and VL of an antibody containing a secretory signal sequence, respectively.
- the complete amino acid sequences of the VH and VL of the antibody containing the secretory signal sequence are compared with the complete amino acid sequences of the known antibody VH and VL [Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)]. By doing this, the length of the secretory signal sequence and the N-terminal amino acid sequence can be deduced, and furthermore, the subgroup to which they belong can be known.
- amino acid sequences of VH and VL CDRs are also found by comparison with the amino acid sequences of the known antibodies VH and VL [Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)]. Can.
- a human chimeric antibody expression vector can be constructed by cloning each of the encoding cDNAs.
- the base sequence of the linking part encodes an appropriate amino acid in order to link the 3 'end of the cDNA encoding the non-human antibody VH or VL with the end of the CH or CL of the human antibody, and CDNAs of VH and VL designed to have appropriate restriction enzyme recognition sequences are prepared.
- the prepared VH and VL cDNAs are respectively expressed upstream of the respective genes encoding CH or CL of the human antibody of the vector for recombinant antibody expression obtained in (1) so that they can be appropriately expressed. Clone and construct a human chimeric antibody expression vector.
- a cDNA encoding non-human antibody VH or VL is amplified by PCR using synthetic DNAs having recognition sequences of appropriate restriction enzymes at both ends, and the vector for expressing recombinant antibody obtained in (1) Can also be cloned into
- amino acid sequences of the human antibody VH or VL FR are selected, respectively, for grafting the amino acid sequences of the non-human antibody VH or VL CDRs. Any amino acid sequence of FR to be selected can be used as long as it is derived from a human antibody.
- the amino acid sequence of the human antibody FR registered in a database such as Protein Data Bank or the common amino acid sequence of each subgroup of the human antibody FR [Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services ( 1991)] and the like.
- the amino acid sequence of FR having as high homology (at least 60% or more) as possible with that of the FR of VH or VL of the original antibody is selected.
- the amino acid sequence of the CDR of the original antibody is grafted to the amino acid sequence of FR of VH or VL of the selected human antibody, and the amino acid sequence of VH or VL of the humanized antibody is designed respectively.
- the designed amino acid sequence is converted into a DNA sequence in consideration of usage frequency of codons found in the nucleotide sequence of antibody gene [Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)], and a humanized antibody
- synthetic DNAs each having a length of about 100 bases are synthesized and PCR reaction is performed using them.
- six synthetic DNAs are designed for each of VH and VL, from the reaction efficiency in the PCR reaction and the length of the synthesizable DNA.
- a cDNA encoding a VH or VL of a humanized antibody can be obtained by combining (1) the gene pair obtained by introducing a recognition sequence of an appropriate restriction enzyme into the 5 'or 3' end of the synthetic DNA located at both ends. It can be easily cloned into a vector for expressing a replacement antibody.
- the amplified product is cloned into a plasmid such as pBluescript SK (-) (Stratagene) and the base sequence is determined by the same method as described in (2), and the desired humanized antibody is obtained.
- a plasmid such as pBluescript SK (-) (Stratagene) and the base sequence is determined by the same method as described in (2), and the desired humanized antibody is obtained.
- Humanized antibody can be obtained from its original antigen-binding activity only by grafting only the CDRs of VH and VL of non-human antibody to VH and VL of human antibody. It is reduced compared to non-human antibody of [BIO / TECHNOLOGY, 9, 266 (1991)].
- an amino acid residue directly involved in antigen binding an amino acid residue interacting with an amino acid residue of CDR, and an antibody Reduced antigen binding by identifying amino acid residues that maintain conformation and indirectly participate in antigen binding, and replacing those amino acid residues with amino acid residues of the original non-human antibody
- the activity can be increased.
- the amino acid residues of the human antibody VH and VL FR can be modified by carrying out the PCR reaction described in (4) using the synthetic DNA for modification.
- the nucleotide sequence of the amplification product after the PCR reaction is determined by the method described in (2) to confirm that the desired modification has been performed.
- Transient expression of genetically modified antibodies is performed using the genetically modified antibody expression vectors obtained in (3) and (6), or expression vectors obtained by modifying them.
- the antigen binding activity of various types of human chimeric antibodies and humanized antibodies produced can be efficiently evaluated.
- any host cell capable of expressing a recombinant antibody can be used.
- COS-7 cells American Type Culture Collection (ATCC) No .: CRL1651] [Methods in Nucleic Acids Res., CRC press, 283 (1991)].
- the expression amount and antigen binding activity of the recombinant antibody in the culture supernatant are determined by enzyme-linked immunosorbent assay [Monoclonal Antibodies-Principles and practice, Third edition, Academic Press (1996), Antibodies-A Laboratory Manual, It measures using Cold Spring Harbor Laboratory (1988), a monoclonal antibody experiment manual, Kodansha Scientific (1987)], and the like.
- Any host cell into which the recombinant antibody expression vector has been introduced may be any host cell capable of expressing the recombinant antibody.
- CHO-K1 ATCC CCL-61
- DUKXB11 ATCC CCL 9096
- Pro-5 ATCC CCL-1781
- CHO-S Life Technologies, Cat # 11619
- mice 20 also referred to as ATCC No .: CRL1662 or YB2 / 0
- mouse myeloma cell NS0 mouse myeloma cell SP2 / 0-Ag14
- mouse P3 X 63-Ag 8.653 cell ATCC No: CRL 1580
- dihydro A CHO cell CHO / DG44 cell
- dhfr folate reductase gene
- a protein such as an enzyme involved in the synthesis of intracellular sugar nucleotide GDP-fucose, a sugar chain modification in which the 1 position of fucose is ⁇ -linked to the 6th position of N-acetylglucosamine at the reducing end of N-glycosidic bond complex type sugar chain
- Host cells whose activity has been reduced or deleted, such as proteins involved in the transport of proteins involved in the transport of the intracellular glyconucleotide GDP-fucose to the Golgi apparatus, eg, deletion of the ⁇ 1,6-fucosyltransferase gene
- CHO cells WO 2005/035586, WO 02/31140
- Lec13 Somatic Cell and Molecular genetics, 12, 55 (1986)] which has acquired lectin resistance, and the like.
- transformant strains which stably express the recombinant antibody are selected by culturing in a culture medium for animal cell culture containing a drug such as G418 sulfate (hereinafter referred to as G418) (Japan Japanese Patent Laid-Open Publication No. 2-257891).
- the culture medium for animal cell culture includes RPMI 1640 medium (manufactured by Invitrogen), GIT medium (manufactured by Japan Pharmaceutical Co., Ltd.), EX-CELL 301 medium (manufactured by JAH), IMDM medium (manufactured by Invitrogen) or Hybridoma-SFM medium (manufactured by Invitrogen) In Vitro Invitrogen) or media obtained by adding various additives such as FBS to these media are used.
- the recombinant antibody is expressed and accumulated in the culture supernatant by culturing the obtained transformant in a medium.
- the expression amount of the recombinant antibody in the culture supernatant and the antigen binding activity can be measured by ELISA or the like.
- the expression amount of the recombinant antibody produced by the transformed strain can be improved by using a dhfr gene amplification system (Japanese Patent Application Laid-Open No. 2-257891) or the like.
- Recombinant antibodies are purified from culture supernatants of transformants using protein A-column [Monoclonal Antibodies-Principles and practice, Third edition, Academic Press (1996), Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory (1988)]. Also, methods used in protein purification such as gel filtration, ion exchange chromatography and ultrafiltration can be combined.
- the molecular weight of the H chain, L chain or whole antibody molecule of the purified recombinant antibody is determined by polyacrylamide gel electrophoresis [Nature, 227, 680 (1970)], or Western blotting [Monoclonal Antibodies-Principles and practice, Third] measurement, Academic Press (1996), Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory (1988)], and the like.
- the antibody fragment of the present invention can be produced according to a known method.
- the antibody fragment of the present invention may be prepared by cleaving the antibody prepared according to the method described in the above (1) to (8) with an enzyme or the like, or the nucleotide sequence encoding the desired antibody fragment It may be prepared and produced by genetic engineering techniques.
- the bispecific antibody or multispecific antibody of the present invention can be produced according to the above-described method for producing an antibody.
- a bispecific antibody expression vector in which a scFv binding to MOG is fused to the C terminus of an IgG antibody binding to an antigen present in the brain can be prepared by the method described below, and the expression of the antibody described above
- the bispecific antibody can be produced according to the method and the purification method of the antibody.
- bispecific antibodies in which an antibody fragment is fused to the C terminus of the antibody can also be produced by the same method.
- the gene fragment of the CH1-Hinge-CH2-CH3-linker region is amplified by PCR using the synthetic gene of the heavy chain constant region of the IgG antibody that binds to an antigen present in the brain as a template.
- the nucleotide sequence of the antibody that binds to MOG is prepared using PCR or the like.
- the above two regions are linked by PCR or the like, and the resulting gene fragment is inserted into a suitable vector such as a pCI vector.
- gene fragments of the light chain region (VL and CL) of the IgG antibody that binds to an antigen present in the brain, and a gene fragment of the VH of the antibody are amplified by PCR using an appropriate template, respectively, Insert in the appropriate position of.
- the bispecific antibody of the present invention can also be prepared by binding an antigen binding site containing an antibody fragment to an IgG antibody by a chemical method.
- activity of the antibody or antibody fragment can be evaluated as follows.
- Binding Activity to MOG The binding activity to MOG of the antibody of the present invention or the antibody fragment thereof is measured using flow cytometry described in 1- (6) above, ELISA, surface plasmon resonance detection and the like. It can also be measured using a fluorescent antibody method [Cancer Immunol. Immunother., 36, 373 (1993)].
- the binding activity of the monovalent antibody to MOG can be measured by the same method. Even when the antibody of the present invention or the antibody fragment is a bispecific antibody or multispecific antibody that binds to MOG and an antigen present in the brain, the bispecific antibody or multispecific antibody is present in the MOG or brain in a similar manner. The binding activity to the antigen can be measured.
- brain tissue is collected, homogenized and centrifuged to measure the concentration of the antibody or the antibody fragment in the supernatant, and the antibody per unit brain weight or Examples include a method of calculating the amount of the antibody fragment, and a method of detecting the presence of the antibody or the antibody fragment by a known immunological method using the collected brain tissue.
- a pharmacologically acceptable labeled antibody or antibody fragment is administered to an animal, and a method of detecting the presence of the antibody or antibody fragment over time with an in vivo imaging system, and the like can be mentioned.
- the animal to be used can select the appropriate animal according to the use of the antibody of this invention, or this antibody fragment.
- Method for controlling the effector activity of the antibody or the antibody fragment of the present invention binds to asparagine (Asn) at position 297 of the Fc region of the antibody or antibody fragment containing Fc.
- Method for controlling the amount of fucose also referred to as core fucose which is ⁇ 1,6 bound to N-acetylglucosamine (GlcNAc) present at the reducing end of the N-linked complex type sugar chain (WO 2005/035586, WO 2002/31140, WO 00/61739), or a method of control by modifying amino acid residues of the Fc region of the antibody or the antibody fragment is known.
- the effector activity can be controlled using any method for the antibody of the present invention or the antibody fragment thereof.
- the effector activity refers to antibody-dependent activity caused via the Fc region of the antibody or antibody fragment thereof, and antibody-dependent phagocytosis by ADCC, CDC, or phagocytes such as macrophages or dendritic cells (Antibody- Dependent phagocytosis (ADP) etc. are known.
- effector activity for example, target cells, human peripheral blood mononuclear cells (PBMC) as effectors, and target cell-specific antibodies or antibody fragments thereof are mixed, and after incubation for about 4 hours, an indicator of cell damage Lactate dehydrogenase (LDH) that has been released can be measured.
- PBMC peripheral blood mononuclear cells
- LDH Lactate dehydrogenase
- effector activity can also be measured by the free 51 Cr method or flow cytometry.
- the effector activity of the antibody or antibody fragment containing Fc can be increased or decreased.
- an antibody or the above is used using CHO cells deficient in the ⁇ 1,6-fucosyltransferase gene. By expressing the antibody fragment, it is possible to obtain an antibody to which fucose is not bound or the antibody fragment.
- the antibody or antibody fragment to which fucose is not bound has high ADCC.
- the antibody fragment can be obtained by expressing fucose by expressing the antibody fragment.
- the antibody to which fucose is bound or the antibody fragment has lower ADCC than the antibody or antibody fragment to which fucose is not bound.
- ADCC or CDC can be increased or decreased by modifying amino acid residues in the Fc region of the antibody or the antibody fragment. For example, by using the amino acid sequence of the Fc region described in US Patent Application Publication No. 2007/0148165, CDC of the antibody or the antibody fragment can be increased.
- ADCC or CDC can be obtained by performing the amino acid modification described in US Pat. No. 6,737,056, US Pat. No. 7,297,775 or US Pat. No. 7,317,091. Can be increased or decreased.
- the antibody or antibody fragment of the present invention may be, for example, disclosed in JP-A-2013-165716 or JP-A-2013-165716 in accordance with amino acid modification or sugar chain modification in the constant region contained in the antibody or antibody fragment described above. Also included are antibodies or antibody fragments whose blood half life has been controlled by controlling the reactivity to the Fc receptor by performing the amino acid modification described in JP-A-2012-021004.
- Method of treating a disease using the antibody of the present invention or the antibody fragment thereof can be used for the treatment of a brain disease of an animal expressing MOG in the brain.
- Brain diseases include, for example, Alzheimer's disease, prodromal Alzheimer's disease, Huntington's disease, Parkinson's disease, brain tumor, multiple sclerosis, muscular dystrophy, amyotrophic lateral sclerosis, multiple system atrophy, progressive supranuclear palsy, Nigrostriatal degeneration, Olivopon cerebellar atrophy, bulbo-spinal muscular atrophy, spinocerebellar degeneration, cerebrovascular disorder, epilepsy, migraine headache, hyperactivity disorder, Creutzfeldt-Jakob disease, cerebral cortex basal ganglia degeneration Disease, lysosome disease, depression, dystonia and the like.
- Brain diseases that can be treated by the antibody or the antibody fragment of the present invention include an antigen to which the antibody or the antibody fragment of the present invention binds, and a molecule that modifies the antibody or the antibody fragment with the fusion antibody or fusion antibody fragment of the present invention. It depends on the type etc.
- the therapeutic agent containing the antibody of the present invention or the antibody fragment may contain only the antibody or the antibody fragment as an active ingredient, but usually one or more pharmacologically acceptable carriers and They are mixed together and provided as a pharmaceutical preparation produced by methods known in the pharmaceutical arts.
- the administration route is, for example, oral administration, or non-oral administration such as oral cavity, airway, rectum, subcutaneous, intramuscular, intracerebroventricular, intraperitoneal administration, intradermal administration, nasal administration, intrathecal administration or intravenous administration. Oral administration is mentioned.
- the dosage form includes, for example, sprays, capsules, tablets, powders, granules, syrups, emulsions, suppositories, injections, ointments or tapes.
- the formulations suitable for oral administration include emulsions, syrups, capsules, tablets, powders or granules.
- Liquid preparations such as emulsions or syrups may be water, sugars such as sucrose, sorbitol or fructose, glycols such as polyethylene glycol or propylene glycol, oils such as sesame oil, olive oil or soybean oil, p-hydroxybenzoic acid It is manufactured using preservatives such as esters or flavors such as strawberry flavor or peppermint as additives.
- Formulations suitable for parenteral administration include injections, suppositories or sprays.
- the injection is produced using a carrier or the like consisting of a salt solution or a glucose solution, or a mixture of both.
- Suppositories are prepared with carriers such as cocoa butter, hydrogenated fats or carboxylic acids.
- the spray is produced using a carrier or the like which does not irritate the recipient's oral and respiratory tract mucosa and disperses the antibody or antibody fragment of the present invention as fine particles to facilitate absorption.
- a carrier for example, lactose or glycerin is used. It can also be manufactured as an aerosol or dry powder.
- components exemplified as additives in a formulation suitable for oral administration can be added.
- a method of detecting or measuring an antigen present in the brain using the antibody of the present invention or the antibody fragment thereof, or a method of diagnosing a disease using MOG or MOG and an antigen present in the brain using the antibody or the antibody fragment of the present invention Can be detected or measured.
- detecting or measuring MOG or MOG and an antigen present in the brain it is possible to diagnose a brain disease of an animal expressing MOG in the brain.
- Brain diseases include, for example, Alzheimer's disease, prodromal Alzheimer's disease, Huntington's disease, Parkinson's disease, brain tumor, multiple sclerosis, muscular dystrophy, amyotrophic lateral sclerosis, multiple system atrophy, progressive supranuclear palsy, Nigrostriatal degeneration, Olivopon cerebellar atrophy, bulbo-spinal muscular atrophy, spinocerebellar degeneration, cerebrovascular disorder, epilepsy, migraine headache, hyperactivity disorder, Creutzfeldt-Jakob disease, cerebral cortex basal ganglia degeneration Disease, lysosome disease, depression, dystonia etc., but the brain disease which can be diagnosed by the antibody or the antibody fragment of the present invention is an antigen to which the antibody or the antibody fragment of the present invention binds, and the fusion antibody or the fusion antibody of the present invention The fusion antibody fragment differs depending on the type of antibody or molecule that modifies the antibody fragment.
- the diagnosis of a brain disease of an animal in which MOG is expressed in the brain can be performed, for example, by detecting or measuring MOG present in the brain of a patient or a patient animal by an immunological method.
- diagnosis can be performed by detecting MOG expressed or present in cells in the brain of the patient or patient animal using an immunological technique such as flow cytometry.
- MOG in the brain can be measured by the same method as described above.
- a bispecific antibody or multispecific antibody which binds to MOG and an antigen present in the brain as the antibody of the present invention or the antibody fragment, the same method as above is used to detect or measure the MOG present in the brain or the antigen present in the brain can do.
- the immunological technique is a method of detecting or measuring the amount of antibody or the amount of antigen using a labeled antigen or antibody or the like.
- a radioactive substance labeled immunoantibody assay an enzyme immunoassay, a fluorescence immunoassay, a luminescence immunoassay, a Western blotting method, a physicochemical method or the like is used.
- radioactive substance labeled immunoantibody method for example, an antigen or a cell expressing an antigen or the like is reacted with the antibody or the antibody fragment of the present invention, and further radiolabeled anti-immunoglobulin antibody or the antibody fragment is reacted Then, measure with a scintillation counter or the like.
- the enzyme-linked immunosorbent assay is, for example, an antigen or a cell expressing an antigen or the like, reacted with the antibody or the antibody fragment of the present invention, and further reacted with an anti-immunoglobulin antibody or the antibody fragment labeled with an enzyme or the like. After that, the substrate is added and the absorbance of the reaction solution is measured with a spectrophotometer. For example, a sandwich ELISA method is used.
- an enzyme label of known [enzyme immunoassay, Medical Shoin (1987)] can be used.
- an alkaline phosphatase label, a peroxidase label, a luciferase label or a biotin label is used.
- the sandwich ELISA method is a method of binding an antibody to a solid phase, trapping an antigen to be detected or measured, and reacting a second antibody with the trapped antigen.
- the first antibody is adsorbed to a plate (for example, 96 well plate) in advance.
- the second antibody is labeled with a fluorescent substance such as FITC, an enzyme such as peroxidase, or biotin.
- the second antibody After reacting the cells or their cell-free fluid, tissue or cell-free fluid, cell culture supernatant, serum, pleural fluid, ascites fluid, eye fluid, etc. isolated from in vivo on the plate to which the first antibody mentioned above has been adsorbed The second antibody is reacted, and a detection reaction corresponding to the labeling substance is performed.
- the antigen concentration in the test sample is calculated from a calibration curve prepared by serially diluting the antigen of known concentration.
- an antibody used for the sandwich ELISA method either a polyclonal antibody or a monoclonal antibody may be used.
- antibody fragments such as Fab, Fab ′ or F (ab) 2 may be used instead of antibodies.
- the combination of two types of antibodies used in sandwich ELISA may be a combination of monoclonal antibodies or antibody fragments that recognize different epitopes, or a combination of polyclonal antibodies and monoclonal antibodies or their antibody fragments.
- the fluorescent immunoassay is measured by the method described in the literature [Monoclonal Antibodies-Principles and practice, Third edition, Academic Press (1996), monoclonal antibody experimental manual, Kodansha Scientific (1987)], and the like.
- a label used in the fluorescence immunoassay a fluorescent label of known [fluorescent antibody method, soft science company (1983)] can be used.
- FITC or RITC is used.
- the luminescence immunoassay is measured by the method described in the literature [Bioluminescence and chemiluminescence clinical examination 42, Kamogawa Shoten (1998)].
- Examples of the label used in the luminescence immunoassay include known luminescent labels, and acridinium ester or rophine is used.
- an antigen or cells expressing an antigen or the like are fractionated by SDS (sodium dodecyl sulfate) -PAGE (polyacrylamide gel) [Antibodies-A Laboratory Manual Cold Spring Harbor Laboratory (1988)], and then the gel is subjected to
- the membrane was blotted to a polyvinylidene fluoride (PVDF) membrane or nitrocellulose membrane, and the membrane was reacted with an antibody that recognizes an antigen or an antibody fragment thereof, and further provided with a fluorescent substance such as FITC, an enzyme label such as peroxidase, or a biotin label
- FITC fluorescent substance
- an enzyme label such as peroxidase
- biotin label an enzyme label such as peroxidase
- An example is shown below.
- BSA-PBS PBS containing 1-10% BSA
- the antibody of the present invention or the antibody fragment thereof is reacted, washed with PBS containing 0.05 to 0.1% Tween-20 (hereinafter referred to as Tween-PBS), and peroxidase-labeled goat anti-mouse IgG Is allowed to react for 2 hours at room temperature.
- Tween-PBS PBS containing 0.05 to 0.1% Tween-20
- peroxidase-labeled goat anti-mouse IgG Is allowed to react for 2 hours at room temperature.
- the polypeptide having the amino acid sequence of MOG is detected by washing with Tween-PBS and detecting a band bound to the antibody of the present invention or the antibody fragment using ECL Western Blotting Detection Reagents (manufactured by Amersham) or the like. .
- an antibody or antibody fragment capable of binding to a polypeptide which does not retain a natural three-dimensional structure is used.
- the physicochemical method is performed, for example, by forming an aggregate by binding MOG which is an antigen to the antibody of the present invention or the antibody fragment of the present invention, and detecting the aggregate.
- MOG an antigen to the antibody of the present invention or the antibody fragment of the present invention
- capillary method one-dimensional immunodiffusion method, immunoturbidimetric method or latex immunoturbidimetric method [clinical examination method provision, Kinbara Publishing (1998)] can be used.
- Latex turbidimetric immunoassay uses a carrier such as polystyrene latex having a particle diameter of about 0.1 to 1 ⁇ m sensitized with an antibody or antigen, and when the antigen-antibody reaction is caused by the corresponding antigen or antibody, the reaction in the reaction solution Scattered light increases and transmitted light decreases. By detecting this change as absorbance or integral sphere turbidity, the antigen concentration etc. in the test sample is measured.
- immunological detection methods For detection or measurement of MOG-expressing cells, known immunological detection methods can be used. Among them, immunoprecipitation, immunocytostaining, immunohistological staining, fluorescent antibody staining, etc. are used. Is preferred.
- a cell or the like expressing MOG is reacted with the antibody of the present invention or the antibody fragment of the present invention, and then a carrier having specific binding ability to an immunoglobulin such as protein G-Sepharose is added to form an antigen-antibody complex. Settle. Or it can also carry out by the following methods.
- the antibody of the present invention or the antibody fragment described above is immobilized on a 96-well plate for ELISA and then blocked with BSA-PBS.
- the antibody is in an unpurified state such as hybridoma culture supernatant, for example, anti-mouse immunoglobulin, anti-rat immunoglobulin, protein-A or protein-G etc. are previously immobilized on a 96-well plate for ELISA.
- hybridoma culture supernatants are aliquoted and allowed to bind.
- cells or tissues expressing an antigen are treated with a surfactant, methanol or the like in some cases to improve antibody passage, and then reacted with the antibody of the present invention. Furthermore, after reacting with a fluorescent label such as FITC, an enzyme label such as peroxidase, or an anti-immunoglobulin antibody or a binding fragment thereof to which a biotin label or the like has been applied, the label is visualized and observed with a microscope.
- a fluorescent label such as FITC
- an enzyme label such as peroxidase, or an anti-immunoglobulin antibody or a binding fragment thereof to which a biotin label or the like has been applied
- fluorescent antibody and antibody are reacted with each other and analyzed with a flow cytometer [Fluorescent antibody staining method [Monoclonal Antibodies-Principles and practice, Third edition, Academic Press (1996), Monoclonal antibody experiment manual, Kodansha Scientific Fick (1987)] can perform detection.
- the antibody of the present invention or the antibody fragment of the present invention can detect cells expressing and retaining a natural three-dimensional structure by fluorescent antibody staining.
- the formed antibody-antigen complex and a free antibody not involved in the formation of the antibody-antigen complex in the case of using the FMAT 8100 HTS system (manufactured by Applied Biosystems) among fluorescent antibody staining methods, the formed antibody-antigen complex and a free antibody not involved in the formation of the antibody-antigen complex.
- the amount of antigen or antibody can be measured without separation from the antibody or antigen.
- Example 1 Acquisition of Anti-MOG Antibody (1) Acquisition of Antibody in Human Antibody Phage Library From cDNA of human PBMC, VH gene fragment and VL gene fragment were amplified by PCR. The VH gene fragment and the VL gene fragment were respectively inserted into a phagemid vector pCANTAB 5E (manufactured by Amersham Pharmacia), and E. coli TG1 (manufactured by Lucigen) was transformed to obtain a plasmid.
- pCANTAB 5E manufactured by Amersham Pharmacia
- E. coli TG1 manufactured by Lucigen
- the obtained plasmid was infected with M13KO7 Helper Phage (manufactured by Invitrogen) to obtain a human antibody M13 phage library in which the VH gene and the VL gene were library-formed.
- an anti-rat MOG (rMOG) monoclonal antibody was obtained using the following phage display method.
- the rMOG-FLAG_Fc of Example 4 to be described later was immobilized on MAXISORP STARTUBE (manufactured by NUNC), and a portion to which rMOG-FLAG_Fc was not bound was blocked using Super Block Blockig Buffer (manufactured by Thermo).
- the tube is reacted with human antibody M13 phage library at room temperature for 1 hour, washed with PBS or PBS containing 0.1% Tween 20 (hereinafter referred to as PBS-T), and washed with 0.1 M Gly-HCl (pH 2.2)
- PBS-T PBS or PBS containing 0.1% Tween 20
- the phage was eluted with The eluate was neutralized by adding Tris-HCl (pH 8.5).
- the eluted phage was infected into TG1 competent cells to amplify the phage.
- rMOG-FLAG_Fc was immobilized on MAXISORP (manufactured by NUNC), and SuperBlock Blockig Buffer (manufactured by Thermo) was used to block the site where rMOG-FLAG_Fc is not bound.
- MAXISORP manufactured by NUNC
- SuperBlock Blockig Buffer manufactured by Thermo
- a plate on which FLAG_Fc was immobilized was also prepared as a negative control.
- Each phage clone was added to each well, reacted at room temperature for 30 minutes, and then each well was washed with PBS-T. Then, a solution of horseradish peroxidase-labeled anti-M13 antibody (manufactured by GE Healthcare) diluted with 10% Block Ace (manufactured by Dainippon Pharmaceutical Co., Ltd.) in PBS-T is added to each well, and the solution is Incubate for a minute.
- TMB chromogenic substrate solution manufactured by DAKO
- a microplate reader manufactured by Molecular Device
- Lymphocytes (2 ⁇ 10 7 cells) were collected from the blood (50 mL) of immunized alpaca blood, and RNA was extracted from the resulting cells using RNA IsoPlus (manufactured by TAKARA). Furthermore, after cDNA is synthesized by reverse transcription using SuperScript (registered trademark) III First-Strand Synthesis System for RT-PC (manufactured by Invitrogen), Alpaca IgG2 (Short hinge-heavy chain antibody), IgG3 (Long hinge- Amplification of the VHH gene was performed using a primer specific for heavy chain antibody).
- VHH gene fragment was inserted into the phagemid vector pKSTV-02 (described in Miyazaki et al, J. Biochem. 2015; 1), and E. coli TG1 was transformed by electroporation using a MicroPulser electroporator (BioRad).
- the transformant had a titer of 2.6 ⁇ 10 7 for IgG2 and 3.2 ⁇ 10 7 for IgG3).
- the resulting transformant was infected with M13KO7 Helper Phage (manufactured by Invitrogen) to obtain an alpaca antibody M13 phage library in which the VHH gene was library-formed.
- an anti-MOG antibody was obtained using the following biopanning method.
- the immunotube was immobilized with rMOG-GST (4 ⁇ g / 2 mL), and 0.5% BSA was used to block the site where rMOG-GST was not bound.
- the tube was reacted with an alpaca antibody M13 phage library at room temperature for 1 hour, and after washing with PBS-T, the phage was eluted with 0.1 M Gly-HCl (pH 2.7). The eluate was neutralized by adding Tris-HCl (pH 9.1). The eluted phages amplified phages after infection with E. coli TG1. After that, it was made to react again with immobilized rMOG-GST in an immunotube, and washing and elution were carried out.
- This operation was repeated three times for IgG2 and twice for IgG3 to concentrate phage that displayed VHH that specifically bind to rMOG-GST. From the concentrated phage, 96 phage clones displaying IgG2 and IgG3 VHH were respectively cloned, and clones having binding to rMOG-GST were selected by ELISA.
- rMOG-GST was immobilized (50 ng / 50 ⁇ L) on MAXISORP (manufactured by NUNC), and 0.5% BSA was used to block the site where rMOG-GST is not bound.
- MAXISORP manufactured by NUNC
- BSA 0.5% BSA was used to block the site where rMOG-GST is not bound.
- Each phage clone was added to each well and reacted at room temperature for 1 hour, and then each well was washed 5 times with PBS-T.
- TMB chromogenic substrate solution (CALBIOCHEM) was added to each well and incubated at room temperature. The color reaction was stopped by adding 1 M hydrochloric acid to each well, and the absorbance at a wavelength of 450 nm (reference wavelength 570 nm) was measured with a microplate reader (Model 680 XR, manufactured by BioRad).
- Example 2 Construction of antibody expression vector (1) Construction of expression vector of anti-MOG antibody In order to produce an anti-MOG antibody of human IgG type, the anti-MOG scFv antibody derived from human antibody phage library obtained in Example 1 An expression vector of various anti-MOG antibodies in which a DNA sequence encoding the amino acid sequence of each variable region was incorporated into an amino acid sequence encoding the amino acid sequence of human IgG antibody constant region was prepared by the method described below.
- the nucleotide sequence encoding the lambda chain constant region of human IgG was synthesized and inserted into the BglII-EcoRI site of N5KG4PE vector (described in WO 2002/088186) to construct N5LG4PE vector.
- N5LG4PE_MOG01 and N5LG4PE_MOG09 expression vectors in which the nucleotide sequences encoding the amino acid sequences of the VH and VL of the MOG01 antibody and the MOG09 antibody were inserted into N5LG4PE were named N5LG4PE_MOG01 and N5LG4PE_MOG09, respectively.
- N5KG4PE_MOG14 an expression vector in which the nucleotide sequence encoding the amino acid sequence of VH and VL of the MOG14 antibody was inserted into the N5KG4PE vector was designated as N5KG4PE_MOG14.
- MOG01 antibody expression vector N5LG4PE_MOG01 The gene fragment of the VL region was amplified by PCR using Primer 1 (SEQ ID NO: 43) and Primer 2 (SEQ ID NO: 44) and KOD plus DNA Polymerase (manufactured by Toyobo Co., Ltd.) with phagemid vector pCANTAB_MOG01 as a template. The PCR was carried out for 30 cycles of a reaction consisting of 94 ° C. for 30 seconds, 58 ° C. for 30 seconds, and 68 ° C. for 45 seconds. The PCR described in Example 2 was performed under the conditions described above unless otherwise stated.
- a signal sequence was added to the gene fragment of the VL region by PCR using PCR product as a template and using Primer 3 (SEQ ID NO: 45) and Primer 2 (SEQ ID NO: 44) and KOD plus DNA Polymerase (Toyobo Co., Ltd.).
- the obtained gene fragment was inserted into the BglII-BlpI site of the N5LG4PE vector to obtain N5LG4PE_MOG01VL.
- a gene fragment of the VH region was amplified by PCR using pCANTAB_MOG01 as a template and primers 4 (SEQ ID NO: 46) and 5 (SEQ ID NO: 47) and KOD plus DNA Polymerase (manufactured by Toyobo Co., Ltd.).
- a signal sequence was added to the gene fragment of the VH region by PCR using PCR product as a template and Primer 6 (SEQ ID NO: 48) and Primer 5 (SEQ ID NO: 47), and KOD plus DNA Polymerase (Toyobo Co., Ltd.).
- the obtained gene fragment was inserted into the SalI-NheI site of the N5LG4PE_MOG01VL vector to obtain N5LG4PE_MOG01.
- MOG09 antibody expression vector N5LG4PE_MOG09 N5LG4PE_MOG09 was produced by the same method as (1-1) above.
- MOG14 antibody expression vector N5KG4PE_MOG14 N5KG4PE_MOG14 was produced by the same method as (1-1) above.
- Primer 11 (SEQ ID NO: 53) and Primer 12 (SEQ ID NO: 54) for amplification of a gene fragment of the VL region using the phagemid vector pCANTAB_MOG14 as a template, primer 3 (sequence when adding a signal sequence to the gene fragment of the VL region No. 45) and primer 12 (SEQ ID No. 54) were used.
- the gene fragment of the VL region to which the obtained signal sequence was added was inserted into the BglII-BsiWI site of the N5KG4PE vector to obtain N5KG4PE_MOG14VL.
- primers 13 (SEQ ID NO: 55) and 14 (SEQ ID NO: 56) are used to amplify a gene fragment of the VH region using pCANTAB_MOG14 as a template, and primer 6 (SEQ ID NO: 48) when amplifying a gene fragment of the VH region. And primer 14 (SEQ ID NO: 56) were used.
- the gene fragment of the VH region to which the obtained signal sequence was added was inserted into the SalI-NheI site of N5KG4PE_MOG14VL to obtain N5KG4PE_MOG14.
- iMOG-3Rim1-S32 antibody expression vector N5G4PEFc_iMOG-3Rim1-S32 A sequence in which a signal sequence is added to the gene encoding the Fc region of human IgG4 PE is synthesized, and Primer 25 (SEQ ID NO: 79) and Primer 26 (SEQ ID NO: 80) and KOD plus DNA Polymerase (Toyobo Co., Ltd.) are used. The gene fragment of human Fc region was amplified by PCR.
- the PCR was carried out 30 cycles of a reaction consisting of 94 ° C. for 30 seconds, 58 ° C. for 30 seconds, and 68 ° C. for 60 seconds.
- the obtained Fc gene fragment was inserted into the BglII-BamHI site of the N5KG4PE vector to prepare an N5G4PEFc vector.
- the expression vector in which the nucleotide sequence encoding the VHH amino acid sequence of iMOG-3 Riml-S32 was inserted into N5G4 PEFc was designated N5 G4 PE Fc_iMOG-3 Riml-S32.
- the VHH-Fc expression vector was constructed as described below.
- the nucleotide sequence of VHH of iMOG-3Rim1-S32 was synthesized, and the gene of the VHH region was PCR by using Primer 15 (SEQ ID NO: 57) and Primer 16 (SEQ ID NO: 58), and KOD plus DNA Polymerase (Toyobo Co., Ltd.) The fragment was amplified. The PCR was carried out 30 cycles of a reaction consisting of 94 ° C. for 30 seconds, 58 ° C. for 30 seconds, and 68 ° C. for 60 seconds. The obtained VHH gene fragment was inserted into the EcoRI-BglII site of the N5G4 PEFc vector to obtain N5G4 PEFc_iMOG-3 Rim1-S32.
- N5LG4PE_AVM Anti-Avermectin antibody expression vector
- N5LG4PE_AVM a chimeric anti-Avermectin (AVM) antibody was produced by the same method as the above (1-1).
- the expression vector in which the nucleotide sequence encoding the amino acid sequence of VH and VL of the AVM antibody was inserted into N5LG4PE was named N5LG4PE_AVM.
- SD rats were immunized with AVM, and anti-AVM antibody-producing hybridomas were established by a conventional method.
- amplification of the gene fragment of the VL region by adding a signal sequence to the primers 29 (SEQ ID NO: 83) and 30 (SEQ ID NO: 84), and the gene fragment of the VL region
- primer 3 SEQ ID NO: 45
- primer 30 SEQ ID NO: 84
- primers 31 SEQ ID NO: 85
- 32 SEQ ID NO: 86
- primer 6 SEQ ID NO: 48
- primer 32 SEQ ID NO: 86
- Anti-rat transferrin receptor antibody OX26 antibody expression vector N5KG4PE (R409K) OX26 The anti-rat transferrin receptor antibody OX26 antibody described in [Protein Engineering, 12, 787-796, 1999] was prepared as a positive control antibody of anti-rat transferrin receptor antibody.
- An expression vector in which the nucleotide sequence encoding the amino acid sequence of VH and VL of the OX26 antibody was inserted into N5KG4PE (R409K) (described in WO 2002/088186) was prepared in the same manner as in (1-1) above. , N5KG4PE (R409K) _OX26.
- the gene encoding the amino acid sequence of the VL of the OX26 antibody is synthesized and used as a template, and for amplification of the gene fragment of the VL region, the primers 40 (SEQ ID NO: 94) and 41 (SEQ ID NO: 95) and the gene fragment of the VH region Primer 42 (SEQ ID NO: 96) and Primer 43 (SEQ ID NO: 97) were used for the amplification of.
- the PCR was carried out 30 cycles of a reaction consisting of 94 ° C. for 30 seconds, 58 ° C. for 30 seconds, and 68 ° C. for 2 minutes.
- Gene fragment of scFv region (hereinafter referred to as MOG01 scFv) by PCR using phagemid vector pCANTAB_MOG01 as a template, primers 19 (SEQ ID NO: 61) and primer 20 (SEQ ID NO: 62), and KOD plus DNA Polymerase (made by Toyobo Co., Ltd.) was amplified.
- PCR was carried out for 30 cycles of a reaction consisting of 94 ° C. for 30 seconds, 58 ° C. for 30 seconds, and 68 ° C. for 90 seconds.
- a reaction consisting of 94 ° C. for 30 seconds, 58 ° C. for 30 seconds, and 68 ° C. for 90 seconds.
- Primer 17 SEQ ID NO: 59
- Primer 20 SEQ ID NO: 62
- KOD plus DNA Polymerase Toyobo Co., Ltd.
- the PCR was carried out 30 cycles of a reaction consisting of 94 ° C. for 30 seconds, 58 ° C. for 30 seconds, and 68 ° C. for 2 minutes.
- the obtained gene fragment was inserted into pCI vector (Promega) to prepare pCI-hG4PE (R409K) _MOG01 scFv vector.
- a gene encoding the amino acid sequence of the VL of an anti-HER2 antibody (Trastuzumab) (described in WO 1999/57134) is synthesized and used as a template, and primers 21 (SEQ ID NO: 63) and 22 (SEQ ID NO: 64) The gene fragment of the VL region was amplified by PCR using KOD plus DNA Polymerase (Toyobo Co., Ltd.).
- the PCR was carried out for 30 cycles of a reaction consisting of 94 ° C. for 30 seconds, 58 ° C. for 30 seconds, and 68 ° C. for 45 seconds.
- CL25 SEQ ID NO: 81
- Primer 28 SEQ ID NO: 82
- KOD plus DNA Polymerase Toyobo Co., Ltd.
- N5KG4 PE vector described in WO 2002/088186
- the PCR was carried out for 30 cycles of a reaction consisting of 94 ° C. for 30 seconds, 58 ° C. for 30 seconds, and 68 ° C. for 45 seconds.
- the gene fragments were amplified by PCR using Primers 21 (SEQ ID NO: 63) and Primer 28 (SEQ ID NO: 82) and KOD plus DNA Polymerase (manufactured by Toyobo Co., Ltd.) with the obtained gene fragments VL and CL as templates.
- PCR was carried out for 30 cycles of a reaction consisting of 94 ° C. for 30 seconds, 58 ° C. for 30 seconds, and 68 ° C. for 90 seconds.
- the obtained gene fragment was inserted into pCI-hG4PE (R409K) _MOG01 scFv to obtain pCI-Trastuzumab VL-hKG4 PE (R409K) _MOG01 scFv.
- a gene encoding the amino acid sequence of Trastuzumab VH is synthesized and used as a template, and PCR is performed using Primer 23 (SEQ ID NO: 65) and Primer 24 (SEQ ID NO: 66), and KOD plus DNA Polymerase (Toyobo Co., Ltd.).
- the gene fragment of VH region was amplified by The PCR was carried out for 30 cycles of a reaction consisting of 94 ° C. for 30 seconds, 58 ° C. for 30 seconds, and 68 ° C. for 45 seconds.
- the obtained gene fragment was inserted into pCI-Trastuzumab VL-hKG4PE (R409K) _MOG01 scFv, to obtain pCI-Trastuzumab-hKG4PE (R409K) _MOG01 scFv.
- expression vector for bispecific antibody binding to AVM and MOG was prepared by the method described below for pCI-AVM-hLG4PE (R409K) _MOG01 scFv .
- the bispecific antibody is obtained by fusing the scFv of anti-MOG antibody to the C-terminus of IgG of anti-AVM antibody.
- the gene fragment of the AVM light chain region was amplified by PCR using N5LG4PE_AVM as a template, primer 33 (SEQ ID NO: 87) and primer 34 (SEQ ID NO: 88), and KOD plus DNA Polymerase (manufactured by Toyobo Co., Ltd.).
- the PCR was carried out 30 cycles of a reaction consisting of 94 ° C. for 30 seconds, 58 ° C. for 30 seconds, and 68 ° C. for 60 seconds.
- the gene fragment of the AVM VH region was amplified by PCR using N5LG4PE_AVM as a template, primer 35 (SEQ ID NO: 89) and primer 32 (SEQ ID NO: 86), and KOD plus DNA Polymerase (manufactured by Toyobo Co., Ltd.).
- the PCR was carried out for 30 cycles of a reaction consisting of 94 ° C. for 30 seconds, 58 ° C. for 30 seconds, and 68 ° C. for 45 seconds.
- the obtained gene fragment was inserted into pCI-hG4PE (R409K) _MOG01scFv prepared above to obtain pCI-AVM-hLG4PE (R409K) _MOG01scFv.
- the PCR was carried out 30 cycles of a reaction consisting of 94 ° C. for 30 seconds, 58 ° C. for 30 seconds, and 68 ° C. for 2 minutes.
- the gene fragment of the scFv region was amplified by PCR using the synthetic gene of AVM scFv as a template and the primers 38 (SEQ ID NO: 92) and 39 (SEQ ID NO: 93) and KOD plus DNA Polymerase (manufactured by Toyobo Co., Ltd.).
- PCR was carried out for 30 cycles of a reaction consisting of 94 ° C. for 30 seconds, 58 ° C. for 30 seconds, and 68 ° C. for 90 seconds.
- a reaction consisting of 94 ° C. for 30 seconds, 58 ° C. for 30 seconds, and 68 ° C. for 90 seconds.
- Primer 36 SEQ ID NO: 90
- Primer 39 SEQ ID NO: 93
- KOD plus DNA Polymerase Toyobo Co., Ltd.
- CH1-Hinge-CH2-CH3-AVM scFv was amplified.
- the PCR was carried out 30 cycles of a reaction consisting of 94 ° C. for 30 seconds, 58 ° C. for 30 seconds, and 68 ° C. for 2 minutes.
- the obtained gene fragment was inserted into the NheI-BamHI site of the above pCI-AVM-hLG4PE (R409K) _MOG01 scFv, to obtain pCI-AVM-hLG4PE (R409K) _AVM scFv.
- Example 4 Preparation of soluble MOG antigen and soluble HER2 antigen (1) Preparation of extracellular domain protein of rat MOG bound with FLAG-Fc As a soluble antigen of rat MOG, MOG with FLAG-Fc added to the C-terminus Extracellular domain proteins were produced by the method described below.
- the nucleotide sequence encoding rMOG is shown in SEQ ID NO: 67, and the amino acid sequence deduced from the nucleotide sequence is shown in SEQ ID NO: 68.
- INPEP4_rMOG-FLAG-Fc was introduced into floating 293 cells using Expi293TM Expression System (manufactured by Thermo Fisher Scientific) and cultured, and the protein was expressed by a transient expression system. Four days after vector introduction, the culture supernatant was recovered and filtered through a membrane filter (MILLIPORE) with a pore size of 0.22 ⁇ m.
- MILLIPORE membrane filter
- MOG-FLAG-Fc protein in the culture supernatant was affinity purified using Protein A resin (MabSelect SuRe, manufactured by GE Healthcare Biosciences). Phosphate buffer was used as a washing solution.
- Protein adsorbed to protein A was eluted with 20 mM sodium citrate, 50 mM NaCl buffer (pH 3.4), and collected in a tube containing 1 M Tris-HCl Buffer Solution (pH 8.0).
- extracellular domain protein of MOG Bound to GST As a soluble antigen of rat MOG, extracellular domain protein of MOG to which GST was added at the C terminus was prepared by the method described below.
- the plasmid vector N5_rMOG-GST to express was constructed.
- the nucleotide sequence of rMOG-GST is shown in SEQ ID NO: 71, and the amino acid sequence deduced from the nucleotide sequence is shown in SEQ ID NO: 72.
- an extracellular domain protein of HER2 to which GST was added at the C-terminus was prepared by the method described below.
- the extracellular domain of HER2 was synthesized by synthesizing the gene sequence of the extracellular domain of HER2 and inserting it into the BglII-KpnI site of the GST-inserted N5 vector (IDEC), the extracellular domain of HER2 with GST added to the C-terminal side
- the plasmid vector N5_hHER2-GST to be expressed was constructed.
- the nucleotide sequence of hHER2-GST is shown in SEQ ID NO: 71, and the amino acid sequence deduced from the nucleotide sequence is shown in SEQ ID NO: 72.
- N5_rMOG-GST and N5_hHER2-GST were introduced into floating 293 cells using Expi293TM Expression System (manufactured by Thermo Fisher Scientific) and cultured, and proteins were expressed by a transient expression system.
- Expi293TM Expression System manufactured by Thermo Fisher Scientific
- the culture supernatant was recovered and filtered through a membrane filter (MILLIPORE) with a pore size of 0.22 ⁇ m.
- the protein in the culture supernatant was affinity purified using Glutathione Sepharose 4B (manufactured by GE Healthcare Biosciences). Phosphate buffer was used as a washing solution. Protein adsorbed to Glutathione Sepharose 4B was eluted with 50 mM Tris-HCl, 10 mM reduced glutatione (pH 8.0).
- the solvent in the solution is replaced with PBS by ultrafiltration using NOVASPIN (manufactured by Sartrius stealin) and a NAP column (manufactured by GE Healthcare Bio-Sciences), and then a membrane filter (Millex-GV with a pore diameter of 0.22 ⁇ m).
- NOVASPIN manufactured by Sartrius stealin
- NAP column manufactured by GE Healthcare Bio-Sciences
- MILLIPORE MILLIPORE.
- concentration of purified rMOG-GST protein and hHER2-GST protein in the solution was measured by absorbance at 280 nm.
- Example 5 Preparation of Membrane-Type MOG Antigen Expression Vector
- rMOG rat MOG
- mMOG mouse MOG
- cMOG monkey MOG
- hMOG human MOG
- Plasmid vectors pEF6_rMOG, pEF6_mMOG, pEF6_cMOG and pEF6_hMOG for membrane expression of various MOGs were prepared by inserting into the BamHI-NotI site of / V5-His (manufactured by Thermo Fisher Scientific) vector.
- the nucleotide sequence encoding mMOG is shown in SEQ ID NO: 73, and the amino acid sequence deduced from the nucleotide sequence is shown in SEQ ID NO: 74.
- the nucleotide sequence encoding cMOG is shown in SEQ ID NO: 75, and the amino acid sequence deduced from the nucleotide sequence is shown in SEQ ID NO: 76.
- the nucleotide sequence encoding hMOG is shown in SEQ ID NO: 77, and the amino acid sequence deduced from the nucleotide sequence is shown in SEQ ID NO: 78.
- Example 6 antibody expression plasmid vector prepared in Preparation Example 2 and Example 3 of the various antibodies were cultured and introduced into planktonic 293 cells using Expi293 TM Expression System (Thermo Fisher Scientific, Inc.), one Antibodies were expressed in a transient expression system.
- the culture supernatant was recovered and filtered through a membrane filter (MILLIPORE) with a pore size of 0.22 ⁇ m.
- the proteins in the culture supernatant were affinity purified using Protein A resin (MabSelect SuRe, manufactured by GE Healthcare Biosciences). Phosphate buffer was used as a washing solution.
- the antibody adsorbed to protein A was eluted with 20 mM sodium citrate, 50 mM NaCl buffer (pH 3.4), and collected in a tube containing 1 M Tris-HCl Buffer Solution (pH 8.0).
- Anti-MOG Human IgG Antibodies Expressed with the Expression Vectors N5LG4PE_MOG01, N5LG4PE_MOG09, N5KG4PE_MOG14 and N5G4PEFc_iMOG-3Rim1-S32 of Anti-MOG Antibodies Described in Example 2
- AVM IgG4PE (R409K) _AVM dscFv antibody AVM IgG4PE (R409K) _MOG01 dscFv antibody
- Trastuzumab IgG4PE (R409K) _MOG01 scFv antibody Trastuzumab IgG4PE (R409K) _MOG01 scFv antibody.
- Example 7 Evaluation of Binding of Anti-MOG Antibody to MOG by Flow Cytometer
- the binding of the anti-MOG antibody MOG01 antibody, MOG09 antibody, MOG14 antibody and iMOG-3 Rim1 -S32 antibody obtained in Example 6 to MOG is described below Evaluation was performed by the fluorescence activated cell sorting (FACS) method according to
- the membrane type MOG antigen expression vector prepared in Example 5 is introduced into floating 293 cells using FreeStyleTM 293 Expression System (manufactured by Thermo Fisher Scientific) and cultured, and the membrane type is a transient expression system. The antigen was expressed. Using the above cells, the reactivity of the anti-MOG antibody was analyzed by the method described below.
- rMOG / HEK293F, mM OG / HEK293F, cMOG / HEK293 and hMOG / HEK293 cells were respectively suspended in a staining buffer (SB) containing 0.1% NaN 3 and 1% FBS in PBS at a concentration of 5 ⁇ 10 5 cells / mL , It distributed to the 96 hole round bottom plate (made by Becton Dickinson company).
- SB staining buffer
- Example 6 After centrifugation (2000 rpm, 4 ° C., 2 minutes), the supernatant is removed and 10 ⁇ g / mL of each antibody obtained in Example 6 is added to the pellet and suspended, and then left standing for 30 minutes under ice temperature. did. The supernatant is removed by further centrifugation (2000 rpm, 4 ° C., 2 minutes), and the pellet is washed with SB, and 1 ⁇ g / mL of RPE fluorescently labeled goat anti-human antibody (Southern Bioblot) is added, and the temperature is ice cold. Incubated for 30 minutes.
- the anti-MOG antibodies MOG01 antibody, MOG09 antibody, MOG14 antibody and iMOG-3Rim1-S32 antibody all show binding activity to rMOG / HEK293F cells and mMOG / HEK293F cells.
- anti-MOG human IgG antibodies MOG01 and MOG14 recognize and bind not only rat and mouse MOG but also all of cynomolgus monkey and human MOG.
- Example 8 Evaluation of the binding of anti-MOG antibody to MOG by surface plasmon resonance detection The affinity of the anti-MOG antibody MOG01 antibody, MOG09 antibody, MOG14 antibody and iMOG-3 Rim1 -S32 antibody obtained in Example 6 to rat MOG was measured using Biacore T-100 (GE Healthcare).
- the dissociation constant (KD value) of each anti-MOG antibody is 2.1 ⁇ 10 -11 (M) to 4.0 ⁇ 10 -8 (M), and both antibodies have good affinity It became clear to show.
- the dissociation rate constant kd is out of the instrument measurement range, and the KD value can not be determined uniquely.
- Example 9 Rat brain migration evaluation of anti-MOG antibody After tail vein (iv) administration to rat, tail vein blood was collected. After whole body perfusion under pentobarbital anesthesia on the same day as blood collection, brain tissue was collected and its weight was measured. Further, a buffer solution was added to the collected brain tissue to homogenize, and after centrifugation, the eluted antibody solution was collected in the supernatant. The volume was measured and the antibody concentration was measured by AlphaLISA (manufactured by PerkinElmer) to calculate the amount of antibody per unit brain weight.
- AlphaLISA manufactured by PerkinElmer
- the MOG01 antibody and MOG14 antibody have a dose of 1 mg / kg body weight
- the iMOG-3Rim1-S32 antibody has a dose of 5 mg / kg body weight
- FIG. 3 (A) and (B) The antibody concentration in serum and the amount of antibody per unit brain weight in brain tissue after 4 days of administration of the antibody are shown in FIG. 3 (A) and (B).
- the antibody concentration in serum was unchanged as compared to the negative control (AVM), but the antibody in the brain was The amount was shown to increase 5-10 fold.
- anti-MOG antibody MOG01 antibody, anti-transferrin receptor antibody OX26 antibody, and negative control anti-AVM antibody antibody concentration in serum and brain tissue 4 days and 10 days after administration of antibody in the amount of 5 mg / kg body weight The amount of antibody per unit brain weight is shown in FIGS. 4 (A) and (B).
- the antibody concentration in the serum after 4 days was the lowest among the evaluated antibodies, and after 10 days, the antibody's blood kinetics became worse, such as becoming lower than detection sensitivity.
- the anti-MOG antibody MOG01 antibody there was no significant change in serum antibody concentration 4 days and 10 days after administration, and the antibody concentration was similar to that of the negative control. From this, it can be said that the half life of the MOG01 antibody in blood is the negative control and the degree of identification.
- the negative control has the lowest antibody amount among the antibodies evaluated at 4 days after administration, but a slight amount after 10 days Furthermore, the amount of antibody decreased.
- the OX26 antibody rapidly decreases in the amount of antibody between 4 and 10 days after administration, and the amount of antibody after 10 days of administration becomes lower than that of the negative control, while for MOG01 antibody, between 4 and 10 days after administration
- the antibody amount increased, and the antibody amount after 4 days of administration was about 2.5 times that of the negative control, and the antibody amount after 10 days of administration was about 10 times that of the negative control.
- the anti-MOG antibody MOG01 antibody shows antibody concentration of negative control and identification degree in serum
- the antibody amount in brain is administered about 2.5 times that of negative control 4 days after administration 10 After the day, it was shown to be about 10 times higher than the negative control and OX26 antibody.
- Example 10 Evaluation of MOG Bispecific Antibody Binding to MOG or HER2 by Flow Cytometer Bispecific Antibody Trastuzumab IgG 4 PE (R409K) _ MOG 01 dscFv Antibody, MOG and AVM, which Binds to MOG and Her 2 Obtained in Example 6
- the binding of the scFv antibody to MOG or HER2 is performed according to the following procedure by fluorescence activated cell sorting (FACS) evaluated.
- FACS fluorescence activated cell sorting
- the membrane-type MOG antigen expression vector prepared in Example 5 is introduced into floating 293 cells using FreeStyleTM 293 Expression System (manufactured by Thermo Fisher Scientific) and cultured, and the membrane-type antigen is expressed in a transient expression system. It was expressed.
- HEK293F cells, rMOG / HEK293F cells, hMOG / HEK293F cells and human breast cancer cell line SK-BR-3 cells are stained at a concentration of 5 ⁇ 10 5 cells / mL with 0.1% NaN 3 , PBS containing 1% FBS The cells were suspended in Buffer (SB) and aliquoted into 96-well round bottom plates (manufactured by Becton Dickinson).
- SB Buffer
- Example 6 After centrifugation (2000 rpm, 4 ° C., 2 minutes), the supernatant is removed and 10 ⁇ g / mL of each antibody obtained in Example 6 is added to the pellet and suspended, and then left standing for 30 minutes under ice temperature. did. The supernatant is removed by further centrifugation (2000 rpm, 4 ° C., 2 minutes), and the pellet is washed with SB, and 1 ⁇ g / mL of RPE fluorescently labeled goat anti-human antibody (Southern Bioblot) is added, and the temperature is ice cold. Incubated for 30 minutes.
- Trastuzumab IgG4PE (R409K) _MOG01 dscFv antibody retains the binding to HER2 even in the form of a bispecific antibody.
- Example 11 Evaluation of MOG Bispecific Antibody Binding to MOG by Surface Plasmon Resonance Detection Affinity of MOG bispecific antibody to MOG was measured in the same manner as in Example 8 and the results are shown in Table 4. .
- the dissociation constant (KD value) of each bispecific antibody is AVM.
- bispecific antibodies of any MOG It turned out to show good affinity.
- Example 12 Evaluation of the binding of a bispecific antibody of MOG to HER2 by surface plasmon resonance detection A bispecific antibody Trastuzumab IgG4 PE (R409K) _MOG01 that binds to MOG and HER2 Biacore T-100 (affinity of dscFv antibody to HER2 It measured using GE Healthcare).
- the dissociation constant (KD value) of Trastuzumab IgG4PE (R409K) _MOG01 dscFv antibody to HER2 is 3.7 ⁇ 10 -9 (M), and it is revealed that the antibody exhibits a good affinity. Became.
- Example 13 Rat brain migration evaluation of bispecific antibodies of MOG Each bispecific antibody AVM IgG4PE (R409K) _MOG01 dcscFv antibody, Trastuzumab IgG4PE (R409K) _MOG01 dcscFv antibody, and AVM IgG4PE (R409K) _AVM dscFv antibody rat Evaluation of the brain transferability of was measured in the same manner as in Example 9. The antibody concentration in serum and the amount of antibody per unit brain weight in brain tissue 10 days after administration of the antibody in the amount of 5 mg / kg body weight are shown in FIGS. 7 (A) and (B).
- the AVM IgG4PE (R409K) _MOG01 dscFv antibody and Trastuzumab IgG4PE (R409K) _MOG01 dscFv antibody compared to AVM IgG4PE (R409K) _AVM dscFv antibody, which is a negative control of the bispecific antibody. There was no difference in antibody concentration in serum.
- AVM IgG4PE (R409K) _MOG01 dscFv antibody Trastuzumab IgG4PE (R409K) _MOG01 dscFv antibody as compared with AVM IgG4PE (R409K) _AVM dscFv antibody which is a negative control of the bispecific antibody.
- R409K Trastuzumab IgG4PE
- R409K Trastuzumab IgG4PE (R409K) _MOG01 dscFv antibody
- AVM IgG4PE (R409K) _AVM dscFv antibody which is a negative control of the bispecific antibody.
- bispecific antibodies that bind to MOG can increase the amount of antibodies in the brain by about 10 times compared to bispecific antibodies that do not bind to MOG, there is no change in blood half-life. It was done.
- mice Mouse brain migration evaluation of anti-MOG01 antibody (1) Measurement of antibody amount The tail vein blood was collected several days after tail vein (iv) administration of the antibody at 35 nmol / kg to mice. After whole body perfusion under pentobarbital anesthesia on the same day as blood collection, brain tissue was collected and its weight was measured. Further, a buffer solution was added to the collected brain tissue to homogenize, and after centrifugation, the eluted antibody solution was collected in the supernatant. The volume was measured and the antibody concentration was measured by AlphaLISA (manufactured by PerkinElmer) to calculate the amount of antibody per unit brain weight.
- AlphaLISA manufactured by PerkinElmer
- anti-MOG01 human IgG antibody and negative control anti-AVM human IgG antibody antibody concentration in serum 3, 6, 10, 14, 21 and 28 days after administration of antibody and antibody per unit brain weight in brain tissue The amounts are shown in FIGS. 8 (A) and (B), respectively.
- the anti-MOG01 human IgG antibody did not have a difference in antibody concentration in serum as compared to the negative control.
- FIG. 8 (B) it was shown that the amount of antibody in the brain can be increased several tens of times over 28 days.
- the anti-MOG01 human IgG antibody and the negative control anti-AVM human IgG antibody were labeled with Alexa FluorR 488 Protein Labeling Kit (manufactured by Molecular Probes).
- the labeled antibody is referred to as AF488-MOG01 IgG4PE antibody or AF488-AVM IgG4PE antibody.
- mice Several days after tail vein (iv) administration, the antibody after labeling was administered to mice at 10 mg / kg, tomato lectin administration was performed, and buccal blood collection was performed. After blood collection, whole body perfusion was performed under pentobarbital anesthesia, and brain tissue was collected, and fluorescence intensity was measured with IVIS Spectrum (manufactured by Perkin Elmer).
- the brain imaging image after 6 days is shown in FIG. 9 (A), and the brain imaging image after 14 days is shown in FIG. 9 (B).
- the amount of fluorescence in the brain after correction with the fluorescence intensity of the administered antibody is shown in FIG. 9 (C).
- the anti-MOG01 antibody can increase the amount of antibody several dozen times over the entire brain as compared to the negative control.
- Example 15 Construction of various bispecific antibody expression vectors A bispecific antibody expression vector that binds to AVM and MOG having the structures described in FIGS. 10 (A) to (C) and FIGS. 11 (A) and 11 (B) It was produced by the following method.
- the name of the bispecific antibody and the name of the antibody expression vector are shown in Table 6, and the name of the antibody expression vector, the nucleotide sequence of the antibody, and the amino acid sequence deduced from the nucleotide sequence are shown in Table 7.
- the gene fragment of -Hinge-CH2-CH3 (R409K / S354C / T366W) region is amplified and inserted into the NheI-BamHI site of pCI-AVM-hLG4PE (R409K) _AVMscFv, pCI-AVM-hLG4PE (R409K / S354C / T366W) ) -FLAG tag vector was generated.
- the obtained gene fragment was inserted into pCI vector (Promega) to prepare pCI-MOG01-hLG4PE (R409K / Y349C / T366S / L368A / Y407V) -His tag vector.
- the gene fragment of the linker-MOG01 scFv-FLAG tag region was amplified by PCR using the above PCR product as a template.
- the gene fragments of CH1-Hinge-CH2-CH3 (R409K / S354C / T366W) -linker region and linker-MOG01scFv-FLAG tag region were inserted into the NheI-BamHI site of pCI-AVM-hLG4PE (R409K) AVM scFv, AVM-hLG4PE (R409K / S354C / T366W) -linker-MOG01 scFv-FLAG tag vector was constructed.
- the gene fragment of the CH1-Hinge-CH2-CH3-linker region and the VH and VL regions of MOG01 are inserted into the NheI-BamHI site of pCI-AVM-hLG4PE (R409K) _AVMscFv, pCI-AVM-hLG4PE (R409K ) _MOG01 scFv2 vector was produced.
- the gene fragment of the CH1-Hinge-CH2-CH3-linker region and the gene region of the VH and VL regions of AVM are inserted into the NheI-BamHI site of pCI-AVM-hLG4PE (R409K) _AVMscFv, and ) AVMscFv3 vector and pCI-AVM-hLG4PE (R409K) AVMscFv5 vector were constructed.
- Example 16 Preparation of Various Bispecific Antibodies AVM IgG4PE (R409K) _MOG01 Fab antibody, AVM IgG4PE (R409K) _MOG01dscFv2 antibody, AVM IgG4PE (R409K) _MOG01dscFv3 antibody, AVM IgG4PE (R409K) by the method described in Example 6.
- AVM IgG4PE (R409K) _MOG01dscFv5 antibody AVM IgG4PE (R409K) _MOG01dscFv6 antibody
- AVM IgG4PE (R409K) _MOG01dscFv7 antibody AVM lgGPE (R409K) _MOG01dscFv8 antibody
- OG01dscFv10 antibodies were AVM IgG4PE (R409K) _MOG01dscFv11 antibody, AVM IgG4PE (R409K) _AVM Fab antibodies, the AVM IgG4PE (R409K) _AVMdscFv3 antibody and AVM IgG4PE (R409K) _AVMdscFv5 antibody preparation.
- AVM-MOG01 IgG4PE (R409K) antibody, AVM IgG4PE (R409K) _MOG01 sscFv antibody and AVM IgG4PE (R409K) _AVMsscFv antibody were prepared by the method described below.
- the antibody expression plasmid vector was introduced into floating 293 cells using Expi 293 (trademark) Expression System (manufactured by Thermo Fisher Scientific) and cultured, and the antibody was expressed in a transient expression system.
- the culture supernatant was recovered and filtered through a membrane filter (MILLIPORE) with a pore size of 0.22 ⁇ m.
- MILLIPORE membrane filter
- the protein in the culture supernatant was affinity purified with His tag using Ni Sepharose resin (manufactured by GE Healthcare Bio-Sciences).
- Ni Sepharose resin manufactured by GE Healthcare Bio-Sciences.
- 20 mM Imidazole-phosphate buffer was used as a washing solution.
- the antibody adsorbed to Ni Sepharose resin was eluted with 500 mM Imidazole-phosphate buffer. Next, the solvent of the eluate was replaced with PBS by ultrafiltration using VIVASPIN (manufactured by Sartrius stealin) and a NAP column (manufactured by GE Healthcare Biosciences).
- the protein after His tag purification was affinity purified using FLAG antibody affinity gel (manufactured by Sigma-Aldrich). Phosphate buffer was used as a washing solution. The antibody adsorbed to the FLAG antibody affinity gel was eluted with 20 mM sodium citrate, 50 mM NaCl buffer (pH 3.4) and collected in a tube containing 1 M Tris-HCl Buffer Solution (pH 8.0).
- the solvent of the eluate was replaced with PBS by ultrafiltration using a VIV ASPIN (manufactured by Sartrius stealin) and a NAP column (manufactured by GE Healthcare Biosciences), and then a membrane filter (Millex-GV with a pore diameter of 0.22 ⁇ m). , And filter sterilization was performed by MILLIPORE. The absorbance at 280 nm of the antibody solution was measured to calculate the concentration of the purified antibody.
- Example 17 Evaluation of Binding Properties of Various Bispecific Antibodies to MOG by Flow Cytometer
- the binding of the various bispecific antibodies and negative control antibodies obtained in Example 6 and Example 16 to MOG was performed according to the following procedure: fluorescence activated cells It evaluated by the sorting (FACS) method.
- the pEF6_hMOG obtained in Example 5 was introduced into mouse connective tissue-derived fibroblast L929 [American Type Culture Collection (ATCC) No .: CCL-1] using HilyMax (manufactured by Dojin Kagaku Co., Ltd.). After selection of cells after gene transfer with antibiotic Blasticidin (manufactured by Invitrogen), cloning by limiting dilution is performed, and L929 cells expressing hMOG on the cell surface (hereinafter abbreviated as hMOG / L929) are used. The reactivity of various bispecific antibodies was analyzed by the method described below.
- hMOG / L 929 was suspended in Staining Buffer (SB) containing PBS containing 0.1% NaN 3 and 1% FBS, and aliquoted into a 96-well round bottom plate (manufactured by Becton Dickinson). After centrifugation (2000 rpm, 4 ° C., 2 minutes), the supernatant is removed, and the various MOG01 bispecific antibodies obtained in Example 6 and Example 16 are added to the pellet and suspended, and Let stand for a minute.
- SB Staining Buffer
- AVM IgG4PE (R409K) _MOG01Fab antibody [FIG. 10 (B), FIG. 12 (C)]
- AVM IgG4PE (R409K) _MOG01dscFv3 antibody AVM IgG4PE (R409K) _MOG01dscFv5 antibody
- AVM IgG4PE (R409K) _MOG01dscFv6 antibody A.
- Example 18 Evaluation of Binding Properties of Various Bispecific Antibodies to MOG by Surface Plasmon Resonance Detection
- the binding of various bispecific antibodies obtained in Example 6 and Example 16 to MOG was carried out in the same manner as in Example 8. evaluated. The obtained results are shown in Tables 10 and 11.
- the dissociation constant (KD value) of the bispecific antibody of each MOG is 1.2 ⁇ 10 ⁇ 8 (M) to 2.0 ⁇ 10 ⁇ 7 (M), whichever It turned out that the antibodies also show good affinity.
- Example 19 Mouse brain transferability evaluation of various bispecific antibodies The mouse brain transferability of various bispecific antibodies and negative control antibodies obtained in Example 6 and Example 16 was evaluated by the method of Example 14.
- AVM-MOG01 IgG4PE (R409K) antibody For AVM-MOG01 IgG4PE (R409K) antibody, AVM IgG4PE (R409K) _MOG01sscFv antibody and AVM IgG4PE (R409K) _MOG01 Fab antibody, the antibody is administered at 5 mg / kg, and the antibody concentration in serum and unit in brain tissue after 10 days The amount of antibody per brain weight is shown in FIG. 14 (A) to FIG. 16 (B).
- FIG. 14 (A), FIG. 15 (A) and FIG. 16 (A) there was no difference in the antibody concentration in serum as compared with the negative control, with any of the MOG01-modified antibodies.
- AVM-MOG01 IgG4PE (R409K) antibody is about 8 times as large as AVM IgG4PE (R409K) _MOG01 sscFv as compared with the negative control.
- the antibody amount in the brain was shown to increase about 12 times for the antibody and about 30 times for the AVM IgG4PE (R409K) _MOG01 Fab antibody.
- AVM IgG4PE (R409K) _MOG01dscFv antibody For AVM IgG4PE (R409K) _MOG01dscFv antibody, AVM IgG4PE (R409K) _MOG01dscFv3 antibody and AVM IgG4PE (R409K) _MOG01dscFv5 antibody, the antibody is administered at 5 mg / kg, and after 10 days and 28 days serum antibody concentration and in brain tissue The amount of antibody per unit brain weight is shown in FIGS. 17 (A) to (D).
- FIGS. 17 (A) and (C) there was no difference in the antibody concentration in serum as compared to the negative control for any of the bispecific antibodies.
- FIGS. 17B and 17D with the AVM IgG4PE (R409K) _MOG01 dscFv antibody, the AVM IgG4PE (R409K) _MOG01 dscFv3 antibody and the AVM IgG4 PE (R409K) _MOG01 dscFv5 antibody, the amount of antibody in the brain was determined over 28 days. It has been shown that it can be enhanced several dozen times. Further, as shown in FIG. 17 (D), the bispecific antibody in which the amount of antibody in the brain is high after 28 days has high binding ability to MOG (Table 11), and the MOG binding activity is correlated with the amount of antibody in brain. It became clear.
- Example 20 Preparation of novel MOG antibody showing stronger MOG binding ability than anti-MOG01 antibody
- Soluble human MOG antigen to which FLAG-Fc is bound and extracellular domain protein of soluble mouse MOG antigen Preparation
- the plasmid vectors INPEP4_hMOG-FLAG-Fc and INPEP4_mMOG-FLAG-Fc expressing the extracellular domain protein of MOG to which FLAG-Fc is added at the C-terminus as soluble antigens of human MOG and mouse MOG are described in Example 4. It was made by the method.
- the nucleotide sequence of hMOG-FLAG-Fc is shown in SEQ ID NO: 100, and the amino acid sequence deduced from the nucleotide sequence is shown in SEQ ID NO: 101.
- the nucleotide sequence of mMOG-FLAG-Fc is deduced from SEQ ID NO: 102, from the nucleotide sequence
- the amino acid sequence is shown in SEQ ID NO: 103.
- the extracellular domain protein of MOG bound with FLAG-Fc was transiently expressed and purified by the method described in Example 4.
- hMOG-GST and mMOG-GST were immunized three times.
- the individual injected intraperitoneally was dissected and spleen was collected, and after removing erythrocytes with erythrocyte removal reagent (manufactured by SIGMA), CELLBANKER 1 (manufactured by Nippon Zenyaku Kogyo Co., Ltd.) Frozen at.
- the inguinal lymph nodes were collected by dissection, red blood cells were removed with a red blood cell removing reagent, and then frozen in CELLBANKER 1.
- RNA was extracted from the obtained spleen cells and cells of the inguinal lymph node using RNeasy Plus Mini kit (manufactured by QIAGEN), and cDNA was synthesized by using a SMARTer RACE cDNA amplification kit (manufactured by Clontech). Using the synthesized cDNA, a human antibody-producing mouse-derived phage library was produced by the method described in Example 1.
- the antibody is a antibody capable of generating an antibody such as a antibody capable of generating an antibody such as a antibody capable of It describes as an antibody, MOG 310 antibody, MOG 312 antibody, MOG 326 antibody, MOG 329 antibody, MOG 446 antibody, MOG 456 antibody and MOG 473 antibody.
- nucleotide sequences encoding VH or VL of various anti-MOG antibodies and the amino acid sequences deduced from the nucleotide sequences are shown in Table 12.
- nucleotide sequences encoding VH or VL of similar clones are shown in Table 13, and the comparison of the amino acid sequences of similar clones is shown in FIG. 18 to FIG. 22 (B).
- Example 21 Preparation of Anti-MOG scFv-Fc Antibody Using the phagemid vector pCANTAB_MOG01 as a template, gene fragments in the scFv region were amplified by PCR. The gene fragment of the Hinge-CH2-CH3 region was amplified by PCR using the synthetic gene of heavy chain constant region as a template. The obtained gene fragment was inserted into the N5KG4PE vector (described in WO 2002/088186) to prepare the N5-MOG01 scFv-hG4PE vector.
- the gene fragment of the scFv region was amplified by PCR using the phagemid vector pCANTAB_MOG301 as a template.
- the gene fragment of the Hinge-CH2-CH3 region was amplified by PCR using the synthetic gene of heavy chain constant region as a template.
- the resulting gene fragment was inserted into pCI vector (Promega) to prepare pCI-MOG301 scFv-hG4PE (R409K) vector.
- antibody expression vectors into which gene fragments of scFv regions of various anti-MOG antibodies shown in Table 12 were inserted were prepared, and pCI-MOG303 scFv-hG4PE (R409K) and pCI-MOG307 scFv-hG4PE (R409K), respectively.
- the prepared expression vector of anti-MOG antibody was prepared by the method described in Example 6.
- Expression vector for anti-MOG antibody pCI-MOG301 scFv-hG4PE (R409K), pCI-MOG303 scFv-hG4PE (R409K), pCI-MOG307 scFv-hG4PE (R409K), pCI-MOG310 scFv-hG4PE (R409K), pCI-MOG312 scFv -HG4PE (R409K), pCI-MOG326 scFv-hG4PE (R409K), pCI-MOG329 scFv-hG4PE (R409K), pCI-MOG446 scFv-hG4PE (R409K), pCI-MOG456 scFv-hG4PE (R409K) and pCI-MOG473 sc
- Example 22 Evaluation of Anti-MOG Antibody Binding to MOG by Flow Cytometer The binding of the anti-MOG antibody obtained in Example 21 to MOG was evaluated in the same manner as in Example 7. The results are shown in Figures 23-25.
- MOG01 scFv-hG4PE, MOG301 scFv-hG4PE (R409K), MOG303 scFv-hG4PE (R409K), MOG307 scFv-hG4PE (R409K), MOG310 scFv-hG4PE (R409K), MOG312 scFv-hG4PE (R409K), MOG326 scFv-hG4PE (R409K), MOG329 scFv-hG4PE (R409K), MOG446 scFv-hG4PE (R409K), MOG456 scFv-hG4PE (R409K) and MOG473 scFv-hG4PE (R409K) are all hMOG / Expi293F It showed binding activity to cells and mM OG / Expi293F
- Example 23 Evaluation of the binding of anti-MOG antibody to MOG by surface plasmon resonance detection MOG01 scFv-hG4PE, MOG301 scFv-hG4PE (R409K), MOG303 scFv-hG4PE (R409K), MOG307 scFv- obtained in Example 21
- the binding of hG4PE (R409K), MOG329 scFv-hG4PE (R409K), MOG446 scFv-hG4PE (R409K), MOG456 scFv-hG4PE (R409K) and MOG 473 scFv-hG4PE (R409K) to human MOG and mouse MOG is described with Example 8 It evaluated by the same method.
- hMOG-GST and mMOG-GST were used.
- the results of the evaluation of the binding to human MOG are shown in Table 14, and the results of the evaluation of
- the dissociation constant (KD value) for human MOG of each anti-MOG antibody is 1.0 ⁇ 10 -10 (M) to 3.6 ⁇ 10 -9 (M), for mouse MOG
- the dissociation constant (KD value) was 1.9 ⁇ 10 -10 (M) to 6.9 ⁇ 10 -9 (M), and it became clear that all antibodies exhibited good affinity.
- the binding rate constant ka falls outside the instrumental measurement range, and the KD value can not be determined uniquely.
- Example 24 Preparation of Enzyme-Fused Antibody An enzyme-fused antibody in which acid sphingomyelinase (ASM) was fused to the C-terminal of anti-MOG01 IgG antibody and anti-AVM IgG antibody was prepared by the method described below.
- PCI-MOG01-hLG4PE (R409K) _ASM for the antibody expression vector in which ASM is fused to the C-terminus of anti-MOG01 IgG antibody
- pCI-AVM-hLG4PE R409K
- the gene fragment of the linker-ASM region was amplified by PCR using the synthetic gene of ASM shown in SEQ ID NO: 150 as a template.
- gene fragments of the CH1-Hinge-CH2-CH3 (R409K) region were synthesized by PCR using the synthetic gene as a template.
- the gene fragment of the MOG01 light chain region and the gene fragment of the MOG01 VH region were amplified by PCR using N5LG4PE_MOG01 as a template.
- the obtained gene fragment was inserted into pCI vector (Promega) to prepare pCI-MOG01-hLG4PE (R409K) _ASM vector.
- the gene fragment of the CH2-CH3 region was amplified by PCR using the synthetic gene as a template.
- Gene fragments of CH2-CH3 region and linker-ASM region were inserted into PmlI-BamHI site of pCI-AVM-hLG4PE (R409K) vector to prepare pCI-AVM-hLG4PE (R409K) _ASM.
- PCI-MOG01-hLG4PE (R409K) _ASM and pCI-AVM-hLG4PE (R409K) _ASM were expressed and purified by the method described in Example 6.
- the antibody obtained by expressing pCI-MOG01-hLG4PE (R409K) _ASM is expressed as MOG01 IgG4PE (R409K) -ASM
- the antibody obtained by expressing pCI-AVM-hLG4PE (R409K) _ASM is expressed as AVM IgG4PE (R409K) -ASM I named it.
- Example 25 Activity Evaluation of Enzyme-Fused Antibody
- the binding property of MOG01 IgG4 PE (R409K) -ASM to MOG-expressing cells was confirmed by the same method as in Example 23, and the results are shown in FIG. Further, the binding property to MOG soluble antigen was confirmed by the same method as in Example 8.
- the dissociation constant (KD value) of MOG01 IgG4PE (R409K) -ASM is 2.9 ⁇ 10 ⁇ 9 (M), It showed good affinity.
- MOG01 IgG4PE (R409K) -ASM and AVM IgG4PE (R409K) -ASM are immobilized (100 ng / 50 ⁇ L) on MAXISORP (manufactured by NUNC), and MOG01 IgG4 PE (manufactured by Thermo) is used with SuperBlock Blockig Buffer (manufactured by Thermo) R409K) -ASM and AVM IgG4PE (R409K) -ASM blocked sites not bound.
- a plate on which anti-MOG01 IgG antibody and anti-AVM IgG antibody were immobilized 50 ng / 50 ⁇ L was also prepared. After adding anti-ASM antibody diluted with PBS-T to a concentration of 0.2, 1, 5 ⁇ g / mL to each well and reacting at room temperature for 1 hour, each well was washed with PBS-T.
- the generated ASM fusion antibody was shown to be recognized and bound by the anti-ASM antibody.
- the prepared ASM fusion antibody is It was confirmed to have an enzyme activity. From the above results, it was confirmed that both the antigen binding activity and the enzyme activity are retained in the enzyme fusion antibody in which the enzyme is fused to the MOG antibody.
- Example 26 Evaluation of Mouse Brain Transferability of Enzyme-Fused Antibody
- the mouse brain transferability of the ASM-fused antibody obtained in Example 24 was evaluated in the same manner as in Example 14.
- the ASM fusion antibody was administered at 5 mg / kg, and the serum antibody concentration and the amount of antibody per unit brain weight in brain tissue after 10 days are shown in FIG.
- MOG01 IgG4PE (R409K) -ASM did not have a difference in the antibody concentration in serum as compared to AVM IgG4PE (R409K) -ASM.
- MOG01 IgG4PE (R409K) -ASM was shown to increase the amount of antibody in the brain by about 58 times compared to AVM IgG4PE (R409K) -ASM.
- SEQ ID NO: 3 Description of artificial sequence: amino acid sequence of VH of MOG01 excluding signal sequence
- SEQ ID NO: 4 Description of artificial sequence: amino acid sequence of HCDR1 of MOG01 SEQ ID NO: 5-description of artificial sequence: amino acid sequence of HCDR2 of MOG01 SEQ ID NO: 6-Description of artificial sequence: amino acid sequence of HCDR3 of MOG01 SEQ ID NO: 9-Description of artificial sequence: amino acid sequence of VL of MOG01 excluding signal sequence SEQ ID NO: 10-Description of artificial sequence: amino acid sequence of LCDR1 of MOG01 SEQ ID NO: 11-Description of artificial sequence: amino acid sequence of LCDR2 of MOG01 SEQ ID NO: 12-Description of artificial sequence: amino acid sequence of LCDR3 of MOG01 SEQ ID NO: 15-Description of artificial sequence: amino acid sequence of VH of MOG09 excluding signal sequence SEQ ID NO: 16-Description of artificial sequence: amino acid sequence of HCDR1 of MOG09 SEQ ID NO: 17-
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Abstract
Description
(1)ミエリンオリゴデンドロサイト糖タンパク質(以下MOGと記載)に結合する抗体または該抗体断片。
(2)抗体が脳滞留性を有する(1)に記載の抗体または該抗体断片。
(3)抗体が下記(a)~(r)からなる群より選ばれる1である、(1)または(2)に記載の抗体または該抗体断片。
(a)重鎖可変領域(以下VHと記載する)の相補性決定領域(以下、CDR)1~3のアミノ酸配列が、それぞれ配列番号4、5および6に記載されるアミノ酸配列を含み、かつ軽鎖可変領域(VL)のCDR1~3のアミノ酸配列が、それぞれ配列番号10、11および12に記載されるアミノ酸配列を含む抗体、
(b)VHのCDR1~3のアミノ酸配列が、それぞれ配列番号16、17および18に記載されるアミノ酸配列を含み、かつVLのCDR1~3のアミノ酸配列が、それぞれ配列番号22、23および24に記載されるアミノ酸配列を含む抗体、
(c)VHのCDR1~3のアミノ酸配列が、それぞれ配列番号28、29および30に記載されるアミノ酸配列を含み、かつVLのCDR1~3のアミノ酸配列が、それぞれ配列番号34、35および36に記載されるアミノ酸配列を含む抗体、
(d)重鎖抗体の重鎖可変領域(以下VHHと記載する)のCDR1~3のアミノ酸配列が、それぞれ配列番号40、41および42に記載されるアミノ酸配列を含む抗体断片、
(e)VHのCDR1~3のアミノ酸配列が、それぞれ配列番号153、154および155に記載されるアミノ酸配列を含み、かつVLのCDR1~3のアミノ酸配列が、それぞれ配列番号158、159および160に記載されるアミノ酸配列を含む抗体、
(f)VHのCDR1~3のアミノ酸配列が、それぞれ配列番号163、164および165に記載されるアミノ酸配列を含み、かつVLのCDR1~3のアミノ酸配列が、それぞれ配列番号168、169および170に記載されるアミノ酸配列を含む抗体、
(g)VHのCDR1~3のアミノ酸配列が、それぞれ配列番号173、174および175に記載されるアミノ酸配列を含み、かつVLのCDR1~3のアミノ酸配列が、それぞれ配列番号178、179および180に記載されるアミノ酸配列を含む抗体、
(h)VHのCDR1~3のアミノ酸配列が、それぞれ配列番号183、184および185に記載されるアミノ酸配列を含み、かつVLのCDR1~3のアミノ酸配列が、それぞれ配列番号188、189および190に記載されるアミノ酸配列を含む抗体、
(i)VHのCDR1~3のアミノ酸配列が、それぞれ配列番号193、194および195に記載されるアミノ酸配列を含み、かつVLのCDR1~3のアミノ酸配列が、それぞれ配列番号198、199および200に記載されるアミノ酸配列を含む抗体、
(j)VHのCDR1~3のアミノ酸配列が、それぞれ配列番号203、204および205に記載されるアミノ酸配列を含み、かつVLのCDR1~3のアミノ酸配列が、それぞれ配列番号208、209および210に記載されるアミノ酸配列を含む抗体、
(k)VHのCDR1~3のアミノ酸配列が、それぞれ配列番号213、214および215に記載されるアミノ酸配列を含み、かつVLのCDR1~3のアミノ酸配列が、それぞれ配列番号218、219および220に記載されるアミノ酸配列を含む抗体、
(l)VHのCDR1~3のアミノ酸配列が、それぞれ配列番号223、224および225に記載されるアミノ酸配列を含み、かつVLのCDR1~3のアミノ酸配列が、それぞれ配列番号228、229および230に記載されるアミノ酸配列を含む抗体、
(m)VHのCDR1~3のアミノ酸配列が、それぞれ配列番号233、234および235に記載されるアミノ酸配列を含み、かつVLのCDR1~3のアミノ酸配列が、それぞれ配列番号238、239および240に記載されるアミノ酸配列を含む抗体、
(n)VHのCDR1~3のアミノ酸配列が、それぞれ配列番号243、244および245に記載されるアミノ酸配列を含み、かつVLのCDR1~3のアミノ酸配列が、それぞれ配列番号248、249および250に記載されるアミノ酸配列を含む抗体、
(o)前記(a)~(n)に記載の少なくとも1つの抗体と、MOGへの結合について競合する抗体、
(p)前記(a)~(n)に記載のいずれか1つの抗体が結合するエピトープを含むエピトープに結合する抗体、および
(q)前記(a)~(n)に記載のいずれか1つの抗体が結合するエピトープと同じエピトープに結合する抗体。
(r)前記(a)~(n)に記載のいずれか1つの抗体のアミノ酸配列と85%以上の相同性を有するアミノ酸配列を含む抗体。
(4)抗体が下記(a)~(n)、(o1)~(o22)および(p)からなる群より選ばれる1である、(1)~(3)のいずれか1つに記載の抗体または該抗体断片。
(a)VHのアミノ酸配列が配列番号3に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号9に記載されるアミノ酸配列を含む抗体、
(b)VHのアミノ酸配列が配列番号15に記載されるアミノ酸配列含み、かつVLのアミノ酸配列が配列番号21に記載されるアミノ酸配列を含む抗体、
(c)VHのアミノ酸配列が配列番号27に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号33に記載されるアミノ酸配列を含む抗体、
(d)VHHのアミノ酸配列が配列番号39に記載されるアミノ酸配列を含む抗体断片、
(e)VHのアミノ酸配列が配列番号152に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号157に記載されるアミノ酸配列を含む抗体、
(f)VHのアミノ酸配列が配列番号162に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号167に記載されるアミノ酸配列を含む抗体、
(g)VHのアミノ酸配列が配列番号172に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号177に記載されるアミノ酸配列を含む抗体、
(h)VHのアミノ酸配列が配列番号182に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号187に記載されるアミノ酸配列を含む抗体、
(i)VHのアミノ酸配列が配列番号192に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号197に記載されるアミノ酸配列を含む抗体、
(j)VHのアミノ酸配列が配列番号202に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号207に記載されるアミノ酸配列を含む抗体、
(k)VHのアミノ酸配列が配列番号212に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号217に記載されるアミノ酸配列を含む抗体、
(l)VHのアミノ酸配列が配列番号222に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号227に記載されるアミノ酸配列を含む抗体、
(m)VHのアミノ酸配列が配列番号232に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号237に記載されるアミノ酸配列を含む抗体、
(n)VHのアミノ酸配列が配列番号242に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号247に記載されるアミノ酸配列を含む抗体、
(o1)VHのアミノ酸配列が配列番号252に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号254に記載されるアミノ酸配列を含む抗体、
(o2)VHのアミノ酸配列が配列番号256に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号258に記載されるアミノ酸配列を含む抗体、
(o3)VHのアミノ酸配列が配列番号260に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号262に記載されるアミノ酸配列を含む抗体、
(o4)VHのアミノ酸配列が配列番号264に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号266に記載されるアミノ酸配列を含む抗体、
(o5)VHのアミノ酸配列が配列番号268に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号270に記載されるアミノ酸配列を含む抗体、
(o6)VHのアミノ酸配列が配列番号272に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号274に記載されるアミノ酸配列を含む抗体、
(o7)VHのアミノ酸配列が配列番号276に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号278に記載されるアミノ酸配列を含む抗体、
(o8)VHのアミノ酸配列が配列番号280に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号282に記載されるアミノ酸配列を含む抗体、
(o9)VHのアミノ酸配列が配列番号284に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号286に記載されるアミノ酸配列を含む抗体、
(o10)VHのアミノ酸配列が配列番号288に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号290に記載されるアミノ酸配列を含む抗体、
(o11)VHのアミノ酸配列が配列番号292に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号294に記載されるアミノ酸配列を含む抗体、
(o12)VHのアミノ酸配列が配列番号296に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号298に記載されるアミノ酸配列を含む抗体、
(o13)VHのアミノ酸配列が配列番号300に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号302に記載されるアミノ酸配列を含む抗体、
(o14)VHのアミノ酸配列が配列番号304に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号306に記載されるアミノ酸配列を含む抗体、
(o15)VHのアミノ酸配列が配列番号308に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号310に記載されるアミノ酸配列を含む抗体、
(o16)VHのアミノ酸配列が配列番号312に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号314に記載されるアミノ酸配列を含む抗体、
(o17)VHのアミノ酸配列が配列番号316に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号318に記載されるアミノ酸配列を含む抗体、
(o18)VHのアミノ酸配列が配列番号320に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号322に記載されるアミノ酸配列を含む抗体、
(o19)VHのアミノ酸配列が配列番号324に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号326に記載されるアミノ酸配列を含む抗体、
(o20)VHのアミノ酸配列が配列番号328に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号330に記載されるアミノ酸配列を含む抗体、
(o21)VHのアミノ酸配列が配列番号332に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号334に記載されるアミノ酸配列を含む抗体、および
(o22)VHのアミノ酸配列が配列番号336に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号338に記載されるアミノ酸配列を含む抗体。
(p)前記(a)~(n)及び(о1)~(о22)に記載のいずれか1つの抗体のアミノ酸配列と85%以上の相同性を有するアミノ酸配列を含む抗体。
(5)抗体または該抗体断片がバイスペシフィック抗体である、(1)~(4)のいずれか1つに記載の抗体または該抗体断片。
(6)バイスペシフィック抗体がMOGと脳に存在する抗原に結合する、(5)に記載のバイスペシフィック抗体。
(7)バイスペシフィック抗体がMOGに結合する抗原結合部位と、脳に存在する抗原に結合する抗原結合部位とを含む、(5)または(6)に記載のバイスペシフィック抗体。
(8)抗体断片がFab、Fab’、F(ab’)2、一本鎖抗体(scFv)、二量体化V領域(diabody)、ジスルフィド安定化V領域(dsFv)、VHHおよびCDRを含むペプチドからなる群より選ばれる1である、(1)~(7)のいずれか1つに記載の抗体断片。
(9)抗体が遺伝子組換え抗体である、(1)~(8)のいずれか1つに記載の抗体および該抗体断片。
(10)抗体がマウス抗体、ラット抗体、ラビット抗体、アルパカ抗体、ラクダ抗体、ラマ抗体、キメラ抗体、ヒト化抗体およびヒト抗体からなる群より選ばれる1である、(1)~(9)のいずれか1つに記載の抗体および該抗体断片。
(11)(1)~(10)のいずれか1つに記載のMOGに結合する抗体または該抗体断片に、下記(a)~(c)からなる群より選ばれる少なくとも1つを結合させた融合抗体または融合抗体断片。
(a)親水性高分子、
(b)両親媒性高分子、および
(c)機能性分子。
(12)(1)~(11)のいずれか1つに記載の抗体を産生するハイブリドーマ。
(13)(1)~(11)のいずれか1つに記載の抗体をコードする塩基配列を含む核酸。
(14)(13)に記載の核酸を含むベクターを含む形質転換細胞。
(15)(12)に記載のハイブリドーマまたは(14)に記載の形質転換細胞を培養し、培養液から(1)~(11)のいずれか1つに記載の抗体または該抗体断片を採取することを含む、(1)~(11)のいずれか1つに記載の抗体または該抗体断片の製造方法。
(16)(1)~(11)のいずれか1つに記載の抗体または該抗体断片を含む、組成物。
(17)脳に存在する抗原の検出または測定用の組成物である、(16)に記載の組成物。
(18)脳疾患の診断または治療をするための組成物である、(16)に記載の組成物。
(19)(1)~(11)のいずれか1つに記載の抗体若しくは該抗体断片、または(16)に記載の組成物を用いて、脳に存在する抗原を検出または測定する方法。
(20)(1)~(11)のいずれか1つに記載の抗体若しくは該抗体断片、または(16)に記載の組成物を用いて、脳疾患を診断または治療する方法。
(21)(1)~(11)のいずれか1つに記載の抗体または該抗体断片若しくは融合抗体または融合抗体断片、または(16)に記載の組成物を用いて、抗体または該抗体断片若しくは融合抗体または融合抗体断片の脳滞留性を向上させる方法。
(22)(1)~(11)のいずれか1つに記載の抗体または該抗体断片若しくは融合抗体または融合抗体断片、または(16)に記載の組成物を用いて、脳内の抗体量または該抗体断片量若しくは融合抗体量または融合抗体断片量を増加させる方法。
(a)VHのCDR1~3のアミノ酸配列が、それぞれ配列番号4、5および6に記載されるアミノ酸配列であって、かつVLのCDR1~3のアミノ酸配列が、それぞれ配列番号10、11および12に記載されるアミノ酸配列を含む抗体、
(b)VHのCDR1~3のアミノ酸配列が、それぞれ配列番号16、17および18に記載されるアミノ酸配列であって、かつVLのCDR1~3のアミノ酸配列が、それぞれ配列番号22、23および24に記載されるアミノ酸配列を含む抗体、
(c)VHのCDR1~3のアミノ酸配列が、それぞれ配列番号28、29および30に記載されるアミノ酸配列であって、かつVLのCDR1~3のアミノ酸配列が、それぞれ配列番号34、35および36に記載されるアミノ酸配列を含む抗体、
(d)VHHのCDR1~3のアミノ酸配列が、それぞれ配列番号40、41および42に記載されるアミノ酸配列を含む抗体断片、
(e)VHのCDR1~3のアミノ酸配列が、それぞれ配列番号153、154および155に記載されるアミノ酸配列を含み、かつVLのCDR1~3のアミノ酸配列が、それぞれ配列番号158、159および160に記載されるアミノ酸配列を含む抗体、
(f)VHのCDR1~3のアミノ酸配列が、それぞれ配列番号163、164および165に記載されるアミノ酸配列を含み、かつVLのCDR1~3のアミノ酸配列が、それぞれ配列番号168、169および170に記載されるアミノ酸配列を含む抗体、
(g)VHのCDR1~3のアミノ酸配列が、それぞれ配列番号173、174および175に記載されるアミノ酸配列を含み、かつVLのCDR1~3のアミノ酸配列が、それぞれ配列番号178、179および180に記載されるアミノ酸配列を含む抗体、
(h)VHのCDR1~3のアミノ酸配列が、それぞれ配列番号183、184および185に記載されるアミノ酸配列を含み、かつVLのCDR1~3のアミノ酸配列が、それぞれ配列番号188、189および190に記載されるアミノ酸配列を含む抗体、
(i)VHのCDR1~3のアミノ酸配列が、それぞれ配列番号193、194および195に記載されるアミノ酸配列を含み、かつVLのCDR1~3のアミノ酸配列が、それぞれ配列番号198、199および200に記載されるアミノ酸配列を含む抗体、
(j)VHのCDR1~3のアミノ酸配列が、それぞれ配列番号203、204および205に記載されるアミノ酸配列を含み、かつVLのCDR1~3のアミノ酸配列が、それぞれ配列番号208、209および210に記載されるアミノ酸配列を含む抗体、
(k)VHのCDR1~3のアミノ酸配列が、それぞれ配列番号213、214および215に記載されるアミノ酸配列を含み、かつVLのCDR1~3のアミノ酸配列が、それぞれ配列番号218、219および220に記載されるアミノ酸配列を含む抗体、
(l)VHのCDR1~3のアミノ酸配列が、それぞれ配列番号223、224および225に記載されるアミノ酸配列を含み、かつVLのCDR1~3のアミノ酸配列が、それぞれ配列番号228、229および230に記載されるアミノ酸配列を含む抗体、
(m)VHのCDR1~3のアミノ酸配列が、それぞれ配列番号233、234および235に記載されるアミノ酸配列を含み、かつVLのCDR1~3のアミノ酸配列が、それぞれ配列番号238、239および240に記載されるアミノ酸配列を含む抗体、
(n)VHのCDR1~3のアミノ酸配列が、それぞれ配列番号243、244および245に記載されるアミノ酸配列を含み、かつVLのCDR1~3のアミノ酸配列が、それぞれ配列番号248、249および250に記載されるアミノ酸配列を含む抗体、
(o)前記(a)~(n)に記載の少なくとも1つの抗体と、MOGへの結合について競合する抗体、
(p)前記(a)~(n)に記載のいずれか1つの抗体が結合するエピトープを含むエピトープに結合する抗体、および
(q)前記(a)~(n)に記載のいずれか1つの抗体が結合するエピトープと同じエピトープに結合する抗体。
(a)VHのアミノ酸配列が配列番号3に記載されるアミノ酸配列であって、かつVLのアミノ酸配列が配列番号9に記載されるアミノ酸配列を含む抗体、
(b)VHのアミノ酸配列が配列番号15に記載されるアミノ酸配列であって、かつVLのアミノ酸配列が配列番号21に記載されるアミノ酸配列を含む抗体、
(c)VHのアミノ酸配列が配列番号27に記載されるアミノ酸配列であって、かつVLのアミノ酸配列が配列番号33に記載されるアミノ酸配列を含む抗体、
(d)VHHのアミノ酸配列が配列番号39に記載されるアミノ酸配列を含む抗体、
(e)VHのアミノ酸配列が配列番号152に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号157に記載されるアミノ酸配列を含む抗体、
(f)VHのアミノ酸配列が配列番号162に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号167に記載されるアミノ酸配列を含む抗体、
(g)VHのアミノ酸配列が配列番号172に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号177に記載されるアミノ酸配列を含む抗体、
(h)VHのアミノ酸配列が配列番号182に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号187に記載されるアミノ酸配列を含む抗体、
(i)VHのアミノ酸配列が配列番号192に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号197に記載されるアミノ酸配列を含む抗体、
(j)VHのアミノ酸配列が配列番号202に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号207に記載されるアミノ酸配列を含む抗体、
(k)VHのアミノ酸配列が配列番号212に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号217に記載されるアミノ酸配列を含む抗体、
(l)VHのアミノ酸配列が配列番号222に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号227に記載されるアミノ酸配列を含む抗体、
(m)VHのアミノ酸配列が配列番号232に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号237に記載されるアミノ酸配列を含む抗体、
(n)VHのアミノ酸配列が配列番号242に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号247に記載されるアミノ酸配列を含む抗体、
(o1)VHのアミノ酸配列が配列番号252に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号254に記載されるアミノ酸配列を含む抗体、
(o2)VHのアミノ酸配列が配列番号256に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号258に記載されるアミノ酸配列を含む抗体、
(o3)VHのアミノ酸配列が配列番号260に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号262に記載されるアミノ酸配列を含む抗体、
(o4)VHのアミノ酸配列が配列番号264に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号266に記載されるアミノ酸配列を含む抗体、
(o5)VHのアミノ酸配列が配列番号268に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号270に記載されるアミノ酸配列を含む抗体、
(o6)VHのアミノ酸配列が配列番号272に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号274に記載されるアミノ酸配列を含む抗体、
(o7)VHのアミノ酸配列が配列番号276に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号278に記載されるアミノ酸配列を含む抗体、
(o8)VHのアミノ酸配列が配列番号280に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号282に記載されるアミノ酸配列を含む抗体、
(o9)VHのアミノ酸配列が配列番号284に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号286に記載されるアミノ酸配列を含む抗体、
(o10)VHのアミノ酸配列が配列番号288に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号290に記載されるアミノ酸配列を含む抗体、
(o11)VHのアミノ酸配列が配列番号292に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号294に記載されるアミノ酸配列を含む抗体、
(o12)VHのアミノ酸配列が配列番号296に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号298に記載されるアミノ酸配列を含む抗体、
(o13)VHのアミノ酸配列が配列番号300に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号302に記載されるアミノ酸配列を含む抗体、
(o14)VHのアミノ酸配列が配列番号304に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号306に記載されるアミノ酸配列を含む抗体、
(o15)VHのアミノ酸配列が配列番号308に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号310に記載されるアミノ酸配列を含む抗体、
(o16)VHのアミノ酸配列が配列番号312に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号314に記載されるアミノ酸配列を含む抗体、
(o17)VHのアミノ酸配列が配列番号316に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号318に記載されるアミノ酸配列を含む抗体、
(o18)VHのアミノ酸配列が配列番号320に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号322に記載されるアミノ酸配列を含む抗体、
(o19)VHのアミノ酸配列が配列番号324に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号326に記載されるアミノ酸配列を含む抗体、
(o20)VHのアミノ酸配列が配列番号328に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号330に記載されるアミノ酸配列を含む抗体、
(o21)VHのアミノ酸配列が配列番号332に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号334に記載されるアミノ酸配列を含む抗体、および
(o22)VHのアミノ酸配列が配列番号336に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号338に記載されるアミノ酸配列を含む抗体。
本発明の抗体断片は、上述したいずれの抗体断片又は該部分断片を含みかつMOG結合活性を有する抗体断片であればいずれのものも含む。
(1)抗体の二つの重鎖のうち、一方の重鎖(重鎖A)のCH3にS354C/T366W、もう一方の重鎖(重鎖B)のCH3にY349C/T366S/L368A/Y407Vのアミノ酸改変を加えたバイスペシフィック抗体。
(2)抗体のC末端に抗体断片を融合させたバイスペシフィック抗体。
(3)抗体のN末端に抗体断片を融合させたバイスペシフィック抗体。
(1)抗原の調製
抗原となるMOGまたはMOG発現細胞は、MOG全長またはその部分長をコードするcDNAを含む発現ベクターを、大腸菌、酵母、昆虫細胞または動物細胞などに導入することで得ることができる。また、MOGは、MOGを多量に発現している各種動物細胞株、動物細胞および動物組織などからMOGを精製することによっても得ることができる。
3~20週令のマウス、ラット、ラビットまたはハムスターなどの動物に、(1)で得られる抗原を免疫して、その動物の脾、リンパ節、末梢血中の抗体産生細胞を採取する。また被免疫動物としてラマ、アルパカ、ラクダなどの動物を用いることもできる。
骨髄腫細胞としては、マウスから得られた株化細胞を用い、例えば、8-アザグアニン耐性マウス(BALB/c由来)骨髄腫細胞株P3-X63Ag8-U1(P3-U1)[Current Topics in Microbiology and Immunology, 18, 1 (1978)]、P3-NS1/1-Ag41(NS-1)[European J. Immunology, 6, 511 (1976)]、SP2/0-Ag14(SP-2)[Nature, 276, 269 (1978)]、P3-X63-Ag8653(653)[J. Immunology, 123, 1548 (1979)]またはP3-X63-Ag8(X63)[Nature, 256, 495 (1975)]などが用いられる。
(2)で得られる融合用抗体産生細胞と(3)で得られる骨髄腫細胞をMinimu Essential Medium(MEM)培地またはPBS(リン酸二ナトリウム1.83g、リン酸一カリウム0.21g、食塩7.65g、蒸留水1リットル、pH7.2)でよく洗浄し、細胞数が、融合用抗体産生細胞:骨髄腫細胞=5~10:1になるよう混合し、遠心分離した後、上清を除く。
プリスタン処理[2,6,10,14-テトラメチルペンタデカン(Pristane)0.5mLを腹腔内投与し、2週間飼育する]した8~10週令のマウスまたはヌードマウスに、(4)で得られるモノクローナル抗体産生ハイブリドーマを腹腔内に注射する。10~21日でハイブリドーマは腹水癌化する。
抗体の選択は以下に示すように、フローサイトメトリーを用いて、MOG発現細胞への抗体の結合性を測定することなどにより行う。MOG発現細胞は、細胞表面上にMOGが発現していればいずれの細胞でもよく、例えば、動物細胞、動物細胞株および(1)で得られるMOG強制発現細胞株などが挙げられる。
(7-1)抗体ファージライブラリの作製方法
本発明において、抗体ファージライブラリは免疫ライブラリ、ナイーブライブラリおよび合成ライブラリを用いることができる。各ライブラリの作製方法を以下に記載する。
(7-1)で作製した抗体ファージライブラリからの抗体ファージクローンの選択は、以下に示すELISA法を用いて行うことができる。
遺伝子組換え抗体の作製例として、以下にヒト型キメラ抗体およびヒト化抗体の作製方法を示す。遺伝子組換えのマウス抗体、ラット抗体、ラビット抗体、ハムスター抗体、ラクダ抗体、ラマ抗体、アルパカ抗体、およびヒト抗体、各種キメラ抗体、ならびに重鎖抗体なども同様の方法で作製することができる。
遺伝子組換え抗体発現用ベクターは、ヒト抗体のCHおよびCLをコードするDNAが組み込まれた動物細胞用発現ベクターであり、動物細胞用発現ベクターにヒト抗体のCHおよびCLをコードするDNAをそれぞれクローニングすることにより構築することができる。
非ヒト抗体のVHおよびVLをコードするcDNAの取得およびアミノ酸配列の解析は以下のようにして行うことができる。
非ヒト抗体を産生するハイブリドーマ細胞よりmRNAを抽出し、cDNAを合成する。合成したcDNAをファージまたはプラスミドなどのベクターにクローニングしてcDNAライブラリを作製する。
選択したファージクローンのプラスミドベクターから、ベクター部分またはV領域部分をコードするDNAをプローブとして用い、VHまたはVLの全塩基配列をそれぞれ決定し、塩基配列よりVHまたはVLの全アミノ酸配列をそれぞれ推定することができる。
(1)で得られる遺伝子組換え抗体発現用ベクターのヒト抗体のCHまたはCLをコードするそれぞれの遺伝子の上流に、それぞれ非ヒト抗体のVHまたはVLをコードするcDNAをそれぞれクローニングすることで、ヒト型キメラ抗体発現ベクターを構築することができる。
ヒト化抗体のVHまたはVLをコードするcDNAは、以下のようにして構築することができる。
ヒト化抗体は、非ヒト抗体のVHおよびVLのCDRのみをヒト抗体のVHおよびVLのFRに移植しただけでは、その抗原結合活性は元の非ヒト抗体に比べて低下する[BIO/TECHNOLOGY, 9, 266 (1991)]。
(1)で得られる遺伝子組換え抗体発現用ベクターのヒト抗体のCHまたはCLをコードするそれぞれの遺伝子の上流に、構築した遺伝子組換え抗体のVHまたはVLをコードするcDNAをそれぞれクローニングし、ヒト化抗体発現ベクターを構築することができる。
(3)および(6)で得られる遺伝子組換え抗体発現ベクター、またはそれらを改変した発現ベクターを用いて遺伝子組換え抗体の一過性発現を行い、作製した多種類のヒト型キメラ抗体、ヒト化抗体の抗原結合活性を効率的に評価することができる。
(3)および(6)で得られた遺伝子組換え抗体発現ベクターを適当な宿主細胞に導入することにより遺伝子組換え抗体を安定に発現する形質転換株を得ることができる。
宿主細胞への発現ベクターの導入には、エレクトロポレーション法[日本国特開平2-257891号公報、Cytotechnology, 3, 133 (1990)]などを用いる。
本発明の抗体断片は、公知の方法に従い作製することができる。本発明の抗体断片は、上記(1)~(8)にて記載した方法に従い作製した抗体を、酵素などで切断することにより作製してもよいし、所望の抗体断片をコードする塩基配列を調製し、遺伝子工学的な手法で作製してもよい。
本発明において、一価抗体は、国際公開第2014/054804号、国際公開第2011/090754号、国際公開第2007/048037号、および国際公開第2012/116927号などに記載する方法などで作製することができる。
本発明のバイスペシフィック抗体またはマルチスペシフィック抗体は、上述した抗体の製造方法に準じて作製することができる。例えば、国際公開第2009/131239号、国際公開第2014/054804号、国際公開第01/077342号、米国特許出願公開第2007/0071675号明細書、国際公開第2007/024715、Wu et al.,[Nature Biotechnology,2007,25(11),p.1290-1297]、Labrijn et al.,[PNAS 2013, vol.110, no.13, p5145-5150]、Jong et al., [http://dx.doi.org/10.1371/journal.pbio.1002344]、Kontermann et al., [mAbs 2012, vol.4, issue2, p182-197]、Spiess et al., [Molecular Immunology 67 (2015) 95-106]、Ridgway et al., [Protein engineering, 1996 vol.9 no.7 pp617-621、国際公開第2009/080251、国際公開第2010/151792および国際公開第2014/033074などに記載される方法を用いて作製することができる。
本発明において、抗体または該抗体断片の活性評価は、以下のように行うことができる。
本発明の抗体または該抗体断片のMOGに対する結合活性は、前述の1-(6)に記載のフローサイトメトリー、ELISA、および表面プラズモン共鳴検出などを用いて測定する。また、蛍光抗体法[Cancer Immunol. Immunother., 36, 373 (1993)]を用いて測定することもできる。
本発明の抗体または該抗体断片の脳滞留性は以下に記載する方法で測定することができる。
ヒトMOG発現細胞またはMOGと脳に存在する抗原が発現している細胞に対する本発明の抗体または該抗体断片のCDC、またはADCCは公知の測定方法[Cancer Immunol. Immunother., 36, 373(1993); Current protocols in Immunology, Chapter7. Immunologic studies in humans, Editor, John E, Coligan et al., John Wiley & Sons,Inc.,(1993)]により測定することができる。
本発明の抗体または該抗体断片のエフェクター活性を制御する方法としては、抗体またはFcを含む該抗体断片のFc領域の297番目のアスパラギン(Asn)に結合するN結合複合型糖鎖の還元末端に存在するN-アセチルグルコサミン(GlcNAc)にα1,6結合するフコース(コアフコースともいう)の量を制御する方法(国際公開第2005/035586号、国際公開第2002/31140号、国際公開第00/61739号)、または抗体若しくは該抗体断片のFc領域のアミノ酸残基を改変することで制御する方法などが知られている。本発明の抗体または該抗体断片にはいずれの方法を用いても、エフェクター活性を制御することができる。
本発明の抗体または該抗体断片は、脳内にMOGが発現している動物の脳疾患の治療に用いることができる。
本発明の抗体または該抗体断片を用いて、MOG、またはMOGと脳に存在する抗原とを検出または測定することができる。また、MOG、またはMOGと脳に存在する抗原とを検出または測定することにより、脳内にMOGが発現している動物の脳疾患を診断することができる。
(1)ヒト抗体ファージライブラリでの抗体の取得
ヒトPBMC由来のcDNAから、PCRにてVH遺伝子断片、VL遺伝子断片を増幅させた。VH遺伝子断片とVL遺伝子断片をファージミドベクターpCANTAB 5E(Amersham Pharmacia社製)にそれぞれ挿入し、大腸菌TG1(Lucigen社製)を形質転換してプラスミドを得た。
免疫原としてrMOG-FLAG_Fcと初回はコンプリートアジュバント、2および3回目はインコンプリートアジュバントとのエマルジョンを作製し、アルパカに免疫した。
(1)抗MOG抗体の発現ベクターの構築
ヒトIgG型の抗MOG抗体を作製するために、実施例1で取得したヒト抗体ファージライブラリ由来抗MOG scFv抗体の各可変領域のアミノ酸配列をコードするDNA配列をヒトIgG抗体定常領域のアミノ酸配列をコードするアミノ酸配列に組みこんだ各種抗MOG抗体の発現ベクターを以下に記載する方法で作製した。
ファージミドベクターpCANTAB_MOG01を鋳型として、プライマー1(配列番号43)およびプライマー2(配列番号44)、ならびにKOD plus DNA Polymerase(東洋紡社製)を用いて、PCRによりVL領域の遺伝子断片を増幅した。PCRは、94℃で30秒間、58℃で30秒間、68℃で45秒間からなる反応を30サイクル実施した。実施例2に記載するPCRは、特に記載がない限り、上記の条件で行った。
上記(1-1)と同様の方法でN5LG4PE_MOG09を作製した。ファージミドベクターpCANTAB_MOG09を鋳型として、VL領域の遺伝子断片の増幅には、プライマー7(配列番号49)およびプライマー8(配列番号50)、VL領域の遺伝子断片にシグナル配列を付加するときには、プライマー3(配列番号45)およびプライマー8(配列番号50)、VH領域の遺伝子断片を増幅には、プライマー9(配列番号51)およびプライマー10(配列番号52)、VH領域の遺伝子断片にシグナル配列を付加するときには、プライマー6(配列番号48)およびプライマー10(配列番号52)を用いた。
上記(1-1)と同様の方法でN5KG4PE_MOG14を作製した。ファージミドベクターpCANTAB_MOG14を鋳型として、VL領域の遺伝子断片の増幅には、プライマー11(配列番号53)およびプライマー12(配列番号54)、VL領域の遺伝子断片にシグナル配列を付加するときには、プライマー3(配列番号45)およびプライマー12(配列番号54)を使用した。得られたシグナル配列が付加されたVL領域の遺伝子断片をN5KG4PEベクターのBglII-BsiWIサイトに挿入し、N5KG4PE_MOG14VLを得た。
ヒトIgG4PEのFc領域をコードする遺伝子にシグナル配列を付加した配列を合成し、プライマー25(配列番号79)およびプライマー26(配列番号80)、ならびにKOD plus DNA Polymerase(東洋紡社製)を用いて、PCRによりヒトFc領域の遺伝子断片を増幅した。
ネガティブコントロール抗体として、キメラ抗Avermectin(AVM)抗体を上記(1-1)と同様の方法で作製した。AVM抗体のVH、VLのアミノ酸配列をコードする塩基配列をN5LG4PEに挿入した発現ベクターを、N5LG4PE_AVMと命名した。
抗ラットトランスフェリン受容体抗体のポジティブコントロール抗体として[Protein Engineering, 12, 787-796, 1999]に記載された抗ラットトランスフェリン受容体抗体OX26抗体を作製した。OX26抗体のVH、VLのアミノ酸配列をコードする塩基配列をN5KG4PE(R409K)(国際公開第2002/088186号に記載)に挿入した発現ベクターを、上記(1-1)と同様の方法で作製し、N5KG4PE(R409K)_OX26と命名した。
(1)Her2とMOGに結合するバイスペシフィック抗体の発現ベクターの作製
HER2とMOGに結合するバイスペシフィック抗体の発現ベクターpCI-Trastuzumab-hKG4PE(R409K)_MOG01scFvを以下の方法で作製した。当該バイスペシフィック抗体は、抗HER2抗体のIgGの2本のH鎖のC末端に抗MOG抗体のscFvが融合したものである。
また、AVMとMOGに結合するバイスペシフィック抗体の発現ベクターをpCI-AVM-hLG4PE(R409K)_MOG01scFvを以下に記載する方法で作製した。当該バイスペシフィック抗体は、抗AVM抗体のIgGのC末端に抗MOG抗体のscFvが融合したものである。
ネガティブコントロール抗体として、抗AVM抗体のIgGのC末端に抗AVM抗体のscFvが融合した抗体の発現ベクターをpCI-AVM-hLG4PE(R409K)_AVM scFvと命名した。
可溶型MOG抗原、可溶型HER2抗原の作製
(1)FLAG-Fcが結合したラットMOGの細胞外ドメインタンパク質の作製
ラットMOGの可溶性抗原として、C末端にFLAG-Fcが付加されたMOGの細胞外ドメインタンパク質を以下に記載する方法で作製した。rMOGをコードする塩基配列を配列番号67に、当該塩基配列から推定されるアミノ酸配列を配列番号68に示す。
ラットMOGの可溶性抗原として、C末端にGSTが付加されたMOGの細胞外ドメインタンパク質を以下に記載する方法で作製した。
ラットMOG(rMOG)、マウスMOG(mMOG)、サルMOG(cMOG)およびヒトMOG(hMOG)の全長遺伝子配列を合成し、それぞれの遺伝子配列をpEF6/V5-His(Thermo Fisher Scientific社製)ベクターのBamHI-NotIサイトに挿入することにより、各種MOGの膜発現用プラスミドベクターpEF6_rMOG、pEF6_mMOG、pEF6_cMOGおよびpEF6_hMOGを作製した。
実施例2および実施例3で作製した抗体発現プラスミドベクターをExpi293TM Expression System(Thermo Fisher Scientific社製)を用いて浮遊性293細胞に導入して培養し、一過性発現系で抗体を発現させた。
実施例6で得た抗MOG抗体MOG01抗体、MOG09抗体、MOG14抗体およびiMOG-3Rim1-S32抗体のMOGへの結合を以下の手順に従いfluoresence activated cell sorting (FACS)法により評価した。
実施例6で得た抗MOG抗体MOG01抗体、MOG09抗体、MOG14抗体およびiMOG-3Rim1-S32抗体のラットMOGへのアフィニティーをBiacore T-100(GE Healthcare)を用いて測定した。
ラットに抗体を尾静脈(i.v.)投与後、尾静脈採血を行った。採血と同じ日に、ペントバルビタール麻酔下にて全身灌流後、脳組織を回収し、その重さを測定した。また、回収した脳組織にバッファー溶液を加えホモジナイズし、遠心分離後、上清に溶出された抗体溶液を回収した。その容量を測定するとともに抗体濃度をAlphaLISA(PerkinElmer社製)により測定し、単位脳重量あたりの抗体量を算出した。
実施例6で得たMOGとHer2に結合するバイスペシフィック抗体Trastuzumab IgG4PE(R409K)_MOG01 dscFv抗体、MOGとAVMに結合するバイスペシフィック抗体AVM IgG4PE(R409K)_MOG01 dscFv抗体、およびAVMに結合する抗体AVM IgG4PE(R409K)_AVM dscFv抗体のMOGまたはHER2への結合を以下の手順に従いfluoresence activated cell sorting(FACS)法により評価した。
MOGのバイスペシフィック抗体のMOGへのアフィニティーを実施例8と同様の方法で測定し、結果を表4に示す。
IgG4PE(R409K)_MOG01 dscFv抗体で2.0×10-7(M)、Trastuzumab IgG4PE(R409K)_MOG01 dscFv抗体で1.0×10-7(M)であり、いずれのMOGのバイスペシフィック抗体についても良好なアフィニティーを示すことが明らかになった。
MOGとHER2に結合するバイスペシフィック抗体Trastuzumab IgG4PE(R409K)_MOG01 dscFv抗体のHER2へのアフィニティーをBiacore T-100(GE Healthcare)を用いて測定した。
各バイスペシフィック抗体AVM IgG4PE(R409K)_MOG01 dscFv抗体、Trastuzumab IgG4PE(R409K)_MOG01 dscFv 抗体、およびAVM IgG4PE(R409K)_AVM dscFv抗体のラットでの脳移行性評価を実施例9と同様の方法で測定した。5mg/kg体重の量で抗体を投与して10日後の血清中の抗体濃度および脳組織中の単位脳重量あたりの抗体量を図7(A)および(B)に示す。
(1)抗体量測定
マウスに抗体を35nmol/kgで尾静脈(i.v.)投与して数日後、尾静脈採血を行った。採血と同じ日に、ペントバルビタール麻酔下にて全身灌流後、脳組織を回収し、その重さを測定した。また、回収した脳組織にバッファー溶液を加えホモジナイズし、遠心分離後、上清に溶出された抗体溶液を回収した。その容量を測定するとともに抗体濃度をAlphaLISA(PerkinElmer社製)により測定し、単位脳重量あたりの抗体量を算出した。
抗MOG01ヒトIgG抗体ならびにネガティブコントロールの抗AVMヒトIgG抗体について、Alexa FluorR 488 Protein Labeling Kit(Molecular Probes社製)にて標識を行った。標識後の抗体を、AF488-MOG01 IgG4PE抗体、AF488-AVM IgG4PE抗体とする。
図10(A)~(C)並びに図11(A)および(B)に記載する構造を有するAVMとMOGに結合するバイスペシフィック抗体発現ベクターを以下の方法で作製した。当該バイスペシフィック抗体の名称および抗体発現ベクターの名称を表6に、抗体発現ベクターの名称と抗体の塩基配列、当該塩基配列から推定されるアミノ酸配列を表7に示す。
(1-1)pCI-AVM-hLG4PE(R409K/S354C/T366W)-FLAG tagベクターの構築
合成遺伝子を鋳型として、PCRによりCH1-Hinge-CH2-CH3(R409K/S354C/T366W)領域の遺伝子断片を増幅し、pCI-AVM-hLG4PE(R409K)_AVMscFvのNheI-BamHIサイトに挿入し、pCI-AVM-hLG4PE(R409K/S354C/T366W)-FLAG tagベクターを作製した。
合成遺伝子を鋳型として、PCRによりCH1-Hinge-CH2-CH3(R409K/Y349C/T366S/L368A/Y407V)-His tag領域の遺伝子断片を増幅した。また、N5LG4PE_MOG01を鋳型として、MOG01軽鎖領域の遺伝子断片とMOG01 VH領域の遺伝子断片をPCRにより増幅した。得られた遺伝子断片をpCIベクター(Promega社製)に挿入し、pCI-MOG01-hLG4PE(R409K/Y349C/T366S/L368A/Y407V)-His tagベクターを作製した。
(2-1)pCI-AVM-hLG4PE(R409K)-linker-MOG01VL-CLベクターの構築
合成遺伝子を鋳型として、PCRによりCH1-Hinge-CH2-CH3-linker-MOG01VL-CL領域の遺伝子断片を増幅し、pCI-AVM-hLG4PE(R409K)_AVMscFvのNheI-BamHIサイトに挿入し、pCI-AVM-hLG4PE(R409K)-linker-MOG01VL-CLベクターを作製した。
合成遺伝子を鋳型として、PCRによりMOG01VH-CH領域の遺伝子断片を増幅し、pCIベクター(Promega社製)に挿入し、pCI-MOG01VH-CHベクターを作製した。
(3-1)pCI-AVM-hLG4PE(R409K/S354C/T366W)-linker-MOG01scFv-FLAG tagベクターの構築
合成遺伝子を鋳型として、PCRによりCH1-Hinge-CH2-CH3(R409K/S354C/T366W)-linker領域の遺伝子断片を増幅した。また、MOG01scFvを鋳型として、PCRによりlinker-MOG01scFv領域の遺伝子断片を増幅した。さらに上記のPCR産物を鋳型として、PCRによりlinker-MOG01scFv-FLAG tag領域の遺伝子断片を増幅した。CH1-Hinge-CH2-CH3(R409K/S354C/T366W)-linker領域とlinker-MOG01scFv-FLAG tag領域の遺伝子断片をpCI-AVM-hLG4PE(R409K)_AVM scFvのNheI-BamHIサイトに挿入し、pCI-AVM-hLG4PE(R409K/S354C/T366W)-linker-MOG01scFv-FLAG tagベクターを作製した。
合成遺伝子を鋳型として、PCRによりCH1-Hinge-CH2-CH3(R409K/Y349C/T366S/L368A/Y407V)-His tag領域の遺伝子断片を増幅し、pCI-AVM-hLG4PE(R409K)_AVMscFvのNheI-BamHIサイトに挿入し、pCI-AVM-hLG4PE(R409K/Y349C/T366S/L368A/Y407V)-His tagベクターを作製した。
(4-1)pCI-AVM-hLG4PE(R409K)_MOG01scFvベクターの構築
合成遺伝子を鋳型として、PCRによりCH1-Hinge-CH2-CH3-linker領域の遺伝子断片を増幅した。また、MOG01scFvを鋳型として、PCRによりMOG01のVH領域、VL領域の遺伝子断片を増幅した。CH1-Hinge-CH2-CH3-linker領域の遺伝子断片とMOG01のVH領域、VL領域の遺伝子断片をpCI-AVM-hLG4PE(R409K)_AVMscFvのNheI-BamHIサイトに挿入し、pCI-AVM-hLG4PE(R409K)_MOG01scFv2ベクターを作製した。
ネガティブコントロールとなる抗体を以下の方法で作製した。当該抗体の名称および抗体発現ベクターの名称を表8に、抗体発現ベクターの名称と抗体の塩基配列、当該塩基配列から推定されるアミノ酸配列を表9に示す。
合成遺伝子を鋳型として、PCRによりAVMのVH領域、VL領域ならびに抗体定常領域の遺伝子断片を増幅し、pCIベクター(Promega社製)に挿入し、pCI-AVM-hLG4PE(R409K)ベクターを作製した。
合成遺伝子を鋳型として、PCRによりCH1-Hinge-CH2-CH3-linker-AVMVL-CL領域の遺伝子断片を増幅し、pCI-AVM-hLG4PE(R409K)_AVM scFvのNheI-BamHIサイトに挿入し、pCI-AVM-hLG4PE(R409K)-linker-AVMVL-CLベクターを作製した。
合成遺伝子を鋳型として、PCRによりAVMVH-CH領域の遺伝子断片を増幅し、pCIベクター(Promega社製)に挿入し、pCI-AVMVH-CHベクターを作製した。
合成遺伝子を鋳型として、PCRによりCH1-Hinge-CH2-CH3(R409K/S354C/T366W)-linker領域の遺伝子断片を増幅した。また、N5LG4PE_AVMを鋳型として、PCRによりlinker-AVMscFv-FLAG tag領域の遺伝子断片を増幅した。CH1-Hinge-CH2-CH3(R409K/S354C/T366W)-linker領域とlinker-AVMscFv-FLAG tag領域の遺伝子断片をpCI-AVM-hLG4PE(R409K)_AVM scFvのNheI-BamHIサイトに挿入し、pCI-AVM-hLG4PE(R409K/S354C/T366W)-linker-AVMscFv-FLAG tagベクターを作製した。
合成遺伝子を鋳型として、PCRによりCH1-Hinge-CH2-CH3-linker領域の遺伝子断片を増幅した。また、N5LG4PE_AVMを鋳型として、PCRによりAVMのVH領域、VL領域の遺伝子断片を増幅した。CH1-Hinge-CH2-CH3-linker領域の遺伝子断片とAVMのVH領域、VL領域の遺伝子断片をpCI-AVM-hLG4PE(R409K)_AVMscFvのNheI-BamHIサイトに挿入し、pCI-AVM-hLG4PE(R409K)_AVMscFv3ベクターとpCI-AVM-hLG4PE(R409K)_AVMscFv5ベクターを作製した。
実施例6に記載した方法で、AVM IgG4PE(R409K)_MOG01 Fab抗体、AVM IgG4PE(R409K)_MOG01dscFv2抗体、AVM IgG4PE(R409K)_MOG01dscFv3抗体、AVM IgG4PE(R409K)_MOG01dscFv4抗体、AVM IgG4PE(R409K)_MOG01dscFv5抗体、AVM IgG4PE(R409K)_MOG01dscFv6抗体、AVM IgG4PE(R409K)_MOG01dscFv7抗体、AVM IgG4PE(R409K)_MOG01dscFv8抗体、AVM IgG4PE(R409K)_MOG01dscFv9抗体、AVM IgG4PE(R409K)_MOG01dscFv10抗体、AVM IgG4PE(R409K)_MOG01dscFv11抗体、AVM IgG4PE(R409K)_AVM Fab抗体、AVM IgG4PE(R409K)_AVMdscFv3抗体およびAVM IgG4PE(R409K)_AVMdscFv5抗体を調製した。
実施例6および実施例16で得た各種バイスペシフィック抗体およびネガティブコントロール抗体のMOGに対する結合を以下の手順に従いfluoresence activated cell sorting(FACS)法により評価した。
実施例6および実施例16で得られた各種バイスペシフィック抗体のMOGへの結合を実施例8と同様の方法で評価した。得られた結果を表10および表11に示す。
実施例6および実施例16で得た各種バイスペシフィック抗体およびネガティブコントロール抗体のマウス脳移行性を実施例14の方法で評価した。
(1)FLAG-Fcが結合した可溶型ヒトMOG抗原および可溶型マウスMOG抗原の細胞外ドメインタンパク質の作製
ヒトMOGとマウスMOGの可溶性抗原として、C末端にFLAG-Fcが付加されたMOGの細胞外ドメインタンパク質を発現するプラスミドベクターINPEP4_hMOG-FLAG-FcとINPEP4_mMOG-FLAG-Fcを実施例4に記載する方法で作製した。hMOG-FLAG-Fcの塩基配列を配列番号100に、当該塩基配列から推定されるアミノ酸配列を配列番号101に示し、mMOG-FLAG-Fcの塩基配列を配列番号102に、当該塩基配列から推定されるアミノ酸配列を配列番号103に示した。FLAG-Fcが結合したMOGの細胞外ドメインタンパク質は、実施例4に記載する方法で一過性発現させ、精製して取得した。
ヒトMOGとマウスMOGの可溶性抗原として、C末端にGSTが付加されたMOGの細胞外ドメインタンパク質を発現するプラスミドベクターN5_hMOG-GSTとN5_mMOG-GSTを実施例4に記載する方法で作製した。hMOG-GSTの塩基配列を配列番号104に、当該塩基配列から推定されるアミノ酸配列を配列番号105に示し、mMOG-GSTの塩基配列を配列番号106に、当該塩基配列から推定されるアミノ酸配列を配列番号107に示した。GSTが結合したMOGの細胞外ドメインタンパク質は、実施例4に記載する方法で一過性発現させ、精製して取得した。
ヒト抗体産生マウス(Ishida& Lonberg, IBC’s 11th Antibody Engineering, Abstract 2000; Ishida, I. et al., Cloning & Stem Cells 4, 85-96 (2002)および石田 功 (2002) 実験医学 20, 6, 846-851)に、hMOG-GSTとmMOG-GSTを百日咳ワクチンとアラムゲルと混合して、腹腔内または皮内に投与した。
ファージミドベクターpCANTAB_MOG01を鋳型として、PCRによりscFv領域の遺伝子断片を増幅した。重鎖定常領域の合成遺伝子を鋳型として、PCRによりHinge-CH2-CH3領域の遺伝子断片を増幅した。得られた遺伝子断片をN5KG4PEベクター(国際公開第2002/088186号に記載)に挿入し、N5-MOG01 scFv-hG4PEベクターを作製した。
実施例21で得た抗MOG抗体のMOGへの結合を実施例7と同様の方法で評価した。結果を図23~25に示す。
実施例21で得たMOG01 scFv-hG4PE、MOG301 scFv-hG4PE(R409K)、MOG303 scFv-hG4PE(R409K)、MOG307 scFv-hG4PE(R409K)、MOG329 scFv-hG4PE(R409K)、MOG446 scFv-hG4PE(R409K)、MOG456 scFv-hG4PE(R409K)およびMOG473 scFv-hG4PE(R409K)のヒトMOGおよびマウスMOGへの結合を実施例8と同様の方法で評価した。アナライトには、hMOG-GSTとmMOG-GSTを用いた。ヒトMOGへの結合性評価の結果を表14に、マウスMOGへの結合性評価の結果を表15に示す。
抗MOG01IgG抗体および抗AVMIgG抗体のC末端に酸性スフィンゴミエリナーゼ(Acid Sphingomyelinase;ASM)が融合した酵素融合抗体を以下に記載する方法で作製した。抗MOG01IgG抗体のC末端にASMが融合した抗体の発現ベクターをpCI-MOG01-hLG4PE(R409K)_ASM、抗AVMIgG抗体のC末端にASMが融合した抗体の発現ベクターをpCI-AVM-hLG4PE(R409K)_ASMと命名した。
MOG01 IgG4PE(R409K)-ASMのMOG発現細胞への結合性を実施例23と同様の方法により確認した結果を図26に示す。また、MOG可溶性抗原への結合性を実施例8と同様の方法により確認した結果、MOG01 IgG4PE(R409K)-ASMの解離定数(KD値)は2.9×10-9(M)であり、良好なアフィニティーを示した。
以上の結果から、MOG抗体に酵素を融合させた酵素融合抗体は抗原結合活性、酵素活性いずれも保持されていることが確認された。
実施例24で得たASM融合抗体のマウス脳移行性を実施例14と同様の方法で評価した。ASM融合抗体を5mg/kgで投与し、10日後での血清中抗体濃度および脳組織中の単位脳重量あたりの抗体量を図28に示す。
配列番号4-人工配列の説明:MOG01のHCDR1のアミノ酸配列
配列番号5-人工配列の説明:MOG01のHCDR2のアミノ酸配列
配列番号6-人工配列の説明:MOG01のHCDR3のアミノ酸配列
配列番号9-人工配列の説明:シグナル配列を除いたMOG01のVLのアミノ酸配列
配列番号10-人工配列の説明:MOG01のLCDR1のアミノ酸配列
配列番号11-人工配列の説明:MOG01のLCDR2のアミノ酸配列
配列番号12-人工配列の説明:MOG01のLCDR3のアミノ酸配列
配列番号15-人工配列の説明:シグナル配列を除いたMOG09のVHのアミノ酸配列
配列番号16-人工配列の説明:MOG09のHCDR1のアミノ酸配列
配列番号17-人工配列の説明:MOG09のHCDR2のアミノ酸配列
配列番号18-人工配列の説明:MOG09のHCDR3のアミノ酸配列
配列番号21-人工配列の説明:シグナル配列を除いたMOG09のVLのアミノ酸配列
配列番号22-人工配列の説明:MOG09のLCDR1のアミノ酸配列
配列番号23-人工配列の説明:MOG09のLCDR2のアミノ酸配列
配列番号24-人工配列の説明:MOG09のLCDR3のアミノ酸配列
配列番号27-人工配列の説明:シグナル配列を除いたMOG14のVHのアミノ酸配列
配列番号28-人工配列の説明:MOG14のHCDR1のアミノ酸配列
配列番号29-人工配列の説明:MOG14のHCDR2のアミノ酸配列
配列番号30-人工配列の説明:MOG14のHCDR3のアミノ酸配列
配列番号33-人工配列の説明:シグナル配列を除いたMOG14のVLのアミノ酸配列
配列番号34-人工配列の説明:MOG14のLCDR1のアミノ酸配列
配列番号35-人工配列の説明:MOG14のLCDR2のアミノ酸配列
配列番号36-人工配列の説明:MOG14のLCDR3のアミノ酸配列
配列番号37-人工配列の説明:シグナル配列を含むiMOG_3Rim1_S32のVHHの塩基配列
配列番号38-人工配列の説明:合成コンストラクトのアミノ酸配列
配列番号39-人工配列の説明:シグナル配列を除いたiMOG_3Rim1_S32のVHHのアミノ酸配列
配列番号40-人工配列の説明:iMOG_3Rim1_S32のCDR1のアミノ酸配列
配列番号41-人工配列の説明:iMOG_3Rim1_S32のCDR2のアミノ酸配列配
列番号42-人工配列の説明:iMOG_3Rim1_S32のCDR3のアミノ酸配列
配列番号43-人工配列の説明:プライマー1の塩基配列
配列番号44-人工配列の説明:プライマー2の塩基配列
配列番号45-人工配列の説明:プライマー3の塩基配列
配列番号46-人工配列の説明:プライマー4の塩基配列
配列番号47-人工配列の説明:プライマー5の塩基配列
配列番号48-人工配列の説明:プライマー6の塩基配列
配列番号49-人工配列の説明:プライマー7の塩基配列
配列番号50-人工配列の説明:プライマー8の塩基配列
配列番号51-人工配列の説明:プライマー9の塩基配列
配列番号52-人工配列の説明:プライマー10の塩基配列
配列番号53-人工配列の説明:プライマー11の塩基配列
配列番号54-人工配列の説明:プライマー12の塩基配列
配列番号55-人工配列の説明:プライマー13の塩基配列
配列番号56-人工配列の説明:プライマー14の塩基配列
配列番号57-人工配列の説明:プライマー15の塩基配列
配列番号58-人工配列の説明:プライマー16の塩基配列
配列番号59-人工配列の説明:プライマー17の塩基配列
配列番号60-人工配列の説明:プライマー18の塩基配列
配列番号61-人工配列の説明:プライマー19の塩基配列
配列番号62-人工配列の説明:プライマー20の塩基配列
配列番号63-人工配列の説明:プライマー21の塩基配列
配列番号64-人工配列の説明:プライマー22の塩基配列
配列番号65-人工配列の説明:プライマー23の塩基配列
配列番号66-人工配列の説明:プライマー24の塩基配列
配列番号69-人工配列の説明:rMOG-FLAG-Fcの塩基配列
配列番号70-人工配列の説明:合成コンストラクトのアミノ酸配列
配列番号71-人工配列の説明:rMOG-GSTの塩基配列
配列番号72-人工配列の説明:合成コンストラクトのアミノ酸配列
配列番号79-人工配列の説明:プライマー25の塩基配列
配列番号80-人工配列の説明:プライマー26の塩基配列
配列番号81-人工配列の説明:プライマー27の塩基配列
配列番号82-人工配列の説明:プライマー28の塩基配列
配列番号83-人工配列の説明:プライマー29の塩基配列
配列番号84-人工配列の説明:プライマー30の塩基配列
配列番号85-人工配列の説明:プライマー31の塩基配列
配列番号86-人工配列の説明:プライマー32の塩基配列
配列番号87-人工配列の説明:プライマー33の塩基配列
配列番号88-人工配列の説明:プライマー34の塩基配列
配列番号89-人工配列の説明:プライマー35の塩基配列
配列番号90-人工配列の説明:プライマー36の塩基配列
配列番号91-人工配列の説明:プライマー37の塩基配列
配列番号92-人工配列の説明:プライマー38の塩基配列
配列番号93-人工配列の説明:プライマー39の塩基配列
配列番号94-人工配列の説明:プライマー40の塩基配列
配列番号95-人工配列の説明:プライマー41の塩基配列
配列番号96-人工配列の説明:プライマー42の塩基配列
配列番号97-人工配列の説明:プライマー43の塩基配列
配列番号98-人工配列の説明:hHER2-GSTの塩基配列
配列番号99-人工配列の説明:合成コンストラクトのアミノ酸配列
配列番号100-人工配列の説明:hMOG-FLAG-Fcの塩基配列(シグナル配列含む)
配列番号101-人工配列の説明:hMOG-FLAG-Fcのアミノ酸配列(シグナル配列含む)
配列番号102-人工配列の説明:mMOG-FLAG-Fcの塩基配列(シグナル配列含む)
配列番号103-人工配列の説明:mMOG-FLAG-Fcのアミノ酸配列(シグナル配列含む)
配列番号104-人工配列の説明:hMOG-GSTの塩基配列(シグナル配列含む)
配列番号105-人工配列の説明:hMOG-GSTのアミノ酸配列(シグナル配列含む)
配列番号106-人工配列の説明:mMOG-GSTの塩基配列(シグナル配列含む)
配列番号107-人工配列の説明:mMOG-GSTのアミノ酸配列(シグナル配列含む)
配列番号108-人工配列の説明:pCI-AVM-hLG4PE(R409K/S354C/T366W)-FLAG tagの抗体配列の塩基配列(シグナル配列除く)
配列番号109-人工配列の説明:pCI-AVM-hLG4PE(R409K/S354C/T366W)-FLAG tagの抗体配列のアミノ酸配列(シグナル配列除く)
配列番号110-人工配列の説明:pCI-MOG01-hLG4PE(R409K/Y349C/T366S/L368A/Y407V)-His tagの抗体配列の塩基配列(シグナル配列除く)
配列番号111-人工配列の説明:pCI-MOG01-hLG4PE(R409K/Y349C/T366S/L368A/Y407V)-His tagの抗体配列のアミノ酸配列(シグナル配列除く)
配列番号112-人工配列の説明:pCI-AVM-hLG4PE(R409K)-linker-MOG01VL-CLの抗体配列の塩基配列(シグナル配列除く)
配列番号113-人工配列の説明:pCI-AVM-hLG4PE(R409K)-linker-MOG01VL-CLの抗体配列のアミノ酸配列(シグナル配列除く)
配列番号114-人工配列の説明:pCI-MOG01VH-CHの抗体配列の塩基配列(シグナル配列除く)
配列番号115-人工配列の説明:pCI-MOG01VH-CHの抗体配列のアミノ酸配列(シグナル配列除く)
配列番号116-人工配列の説明:pCI-AVM-hLG4PE(R409K/S354C/T366W)-linker-MOG01scFv-FLAG tagの抗体配列の塩基配列(シグナル配列除く)
配列番号117-人工配列の説明:pCI-AVM-hLG4PE(R409K/S354C/T366W)-linker-MOG01scFv-FLAG tagの抗体配列のアミノ酸配列(シグナル配列除く)
配列番号118-人工配列の説明:pCI-AVM-hLG4PE(R409K/Y349C/T366S/L368A/Y407V)-His tagの抗体配列の塩基配列(シグナル配列除く)
配列番号119-人工配列の説明:pCI-AVM-hLG4PE(R409K/Y349C/T366S/L368A/Y407V)-His tagの抗体配列のアミノ酸配列(シグナル配列除く)
配列番号120-人工配列の説明:pCI-AVM-hLG4PE(R409K)_MOG01scFv2の抗体配列の塩基配列(シグナル配列除く)
配列番号121-人工配列の説明:pCI-AVM-hLG4PE(R409K)_MOG01scFv2の抗体配列のアミノ酸配列(シグナル配列除く)
配列番号122-人工配列の説明:pCI-AVM-hLG4PE(R409K)_MOG01scFv3の抗体配列の塩基配列(シグナル配列除く)
配列番号123-人工配列の説明:pCI-AVM-hLG4PE(R409K)_MOG01scFv3の抗体配列のアミノ酸配列(シグナル配列除く)
配列番号124-人工配列の説明:pCI-AVM-hLG4PE(R409K)_MOG01scFv4の抗体配列の塩基配列(シグナル配列除く)
配列番号125-人工配列の説明:pCI-AVM-hLG4PE(R409K)_MOG01scFv4の抗体配列のアミノ酸配列(シグナル配列除く)
配列番号126-人工配列の説明:pCI-AVM-hLG4PE(R409K)_MOG01scFv5の抗体配列の塩基配列(シグナル配列除く)
配列番号127-人工配列の説明:pCI-AVM-hLG4PE(R409K)_MOG01scFv5の抗体配列のアミノ酸配列(シグナル配列除く)
配列番号128-人工配列の説明:pCI-AVM-hLG4PE(R409K)_MOG01scFv6の抗体配列の塩基配列(シグナル配列除く)
配列番号129-人工配列の説明:pCI-AVM-hLG4PE(R409K)_MOG01scFv6の抗体配列のアミノ酸配列(シグナル配列除く)
配列番号130-人工配列の説明:pCI-AVM-hLG4PE(R409K)_MOG01scFv7の抗体配列の塩基配列(シグナル配列除く)
配列番号131-人工配列の説明:pCI-AVM-hLG4PE(R409K)_MOG01scFv7の抗体配列のアミノ酸配列(シグナル配列除く)
配列番号132-人工配列の説明:pCI-AVM-hLG4PE(R409K)_MOG01scFv8の抗体配列の塩基配列(シグナル配列除く)
配列番号133-人工配列の説明:pCI-AVM-hLG4PE(R409K)_MOG01scFv8の抗体配列のアミノ酸配列(シグナル配列除く)
配列番号134-人工配列の説明:pCI-AVM-hLG4PE(R409K)_MOG01scFv9の抗体配列の塩基配列(シグナル配列除く)
配列番号135-人工配列の説明:pCI-AVM-hLG4PE(R409K)_MOG01scFv9の抗体配列のアミノ酸配列(シグナル配列除く)
配列番号136-人工配列の説明:pCI-AVM-hLG4PE(R409K)_MOG01scFv10の抗体配列の塩基配列(シグナル配列除く)
配列番号137-人工配列の説明:pCI-AVM-hLG4PE(R409K)_MOG01scFv10の抗体配列のアミノ酸配列(シグナル配列除く)
配列番号138-人工配列の説明:pCI-AVM-hLG4PE(R409K)_MOG01scFv11の抗体配列の塩基配列(シグナル配列除く)
配列番号139-人工配列の説明:pCI-AVM-hLG4PE(R409K)_MOG01scFv11の抗体配列のアミノ酸配列(シグナル配列除く)
配列番号140-人工配列の説明:pCI-AVM-hLG4PE(R409K)-linker-AVMVL-CLの抗体配列の塩基配列(シグナル配列除く)
配列番号141-人工配列の説明:pCI-AVM-hLG4PE(R409K)-linker-AVMVL-CLの抗体配列のアミノ酸配列(シグナル配列除く)
配列番号142-人工配列の説明:pCI-AVMVH-CHの抗体配列の塩基配列(シグナル配列除く)
配列番号143-人工配列の説明:pCI-AVMVH-CHの抗体配列のアミノ酸配列(シグナル配列除く)
配列番号144-人工配列の説明:pCI-AVM-hLG4PE(R409K/S354C/T366W)-linker-AVMscFv-FLAG tagの抗体配列の塩基配列(シグナル配列除く)
配列番号145-人工配列の説明:pCI-AVM-hLG4PE(R409K/S354C/T366W)-linker-AVMscFv-FLAG tagの抗体配列のアミノ酸配列(シグナル配列除く)
配列番号146-人工配列の説明:pCI-AVM-hLG4PE(R409K)_AVMscFv3の抗体配列の塩基配列(シグナル配列除く)
配列番号147-人工配列の説明:pCI-AVM-hLG4PE(R409K)_AVMscFv3の抗体配列のアミノ酸配列(シグナル配列除く)
配列番号148-人工配列の説明:pCI-AVM-hLG4PE(R409K)_AVMscFv5の抗体配列の塩基配列(シグナル配列除く)
配列番号149-人工配列の説明:pCI-AVM-hLG4PE(R409K)_AVMscFv5の抗体配列のアミノ酸配列(シグナル配列除く)
配列番号150-人工配列の説明:Acid Sphingomyelinase(ASM)の塩基配列
配列番号151-人工配列の説明:MOG301のVH(シグナル配列を除く)をコードする塩基配列
配列番号152-人工配列の説明:MOG301のVH(シグナル配列を除く)のアミノ酸配列
配列番号153-人工配列の説明:MOG301のHCDR1のアミノ酸配列
配列番号154-人工配列の説明:MOG301のHCDR2のアミノ酸配列
配列番号155-人工配列の説明:MOG301のHCDR3のアミノ酸配列
配列番号156-人工配列の説明:MOG301のVL(シグナル配列を除く)をコードする塩基配列
配列番号157-人工配列の説明:MOG301のVL(シグナル配列を除く)のアミノ酸配列
配列番号158-人工配列の説明:MOG301のLCDR1のアミノ酸配列
配列番号159-人工配列の説明:MOG301のLCDR2のアミノ酸配列
配列番号160-人工配列の説明:MOG301のLCDR3のアミノ酸配列
配列番号161-人工配列の説明:MOG303のVH(シグナル配列を除く)をコードする塩基配列
配列番号162-人工配列の説明:MOG303のVH(シグナル配列を除く)のアミノ酸配列
配列番号163-人工配列の説明:MOG303のHCDR1のアミノ酸配列
配列番号164-人工配列の説明:MOG303のHCDR2のアミノ酸配列
配列番号165-人工配列の説明:MOG303のHCDR3のアミノ酸配列
配列番号166-人工配列の説明:MOG303のVL(シグナル配列を除く)をコードする塩基配列
配列番号167-人工配列の説明:MOG303のVL(シグナル配列を除く)のアミノ酸配列
配列番号168-人工配列の説明:MOG303のLCDR1のアミノ酸配列
配列番号169-人工配列の説明:MOG303のLCDR2のアミノ酸配列
配列番号170-人工配列の説明:MOG303のLCDR3のアミノ酸配列
配列番号171-人工配列の説明:MOG307のVH(シグナル配列を除く)をコードする塩基配列
配列番号172-人工配列の説明:MOG307のVH(シグナル配列を除く)のアミノ酸配列
配列番号173-人工配列の説明:MOG307のHCDR1のアミノ酸配列
配列番号174-人工配列の説明:MOG307のHCDR2のアミノ酸配列
配列番号175-人工配列の説明:MOG307のHCDR3のアミノ酸配列
配列番号176-人工配列の説明:MOG307のVL(シグナル配列を除く)をコードする塩基配列
配列番号177-人工配列の説明:MOG307のVL(シグナル配列を除く)のアミノ酸配列
配列番号178-人工配列の説明:MOG307のLCDR1のアミノ酸配列
配列番号179-人工配列の説明:MOG307のLCDR2のアミノ酸配列
配列番号180-人工配列の説明:MOG307のLCDR3のアミノ酸配列
配列番号181-人工配列の説明:MOG310のVH(シグナル配列を除く)をコードする塩基配列
配列番号182-人工配列の説明:MOG310のVH(シグナル配列を除く)のアミノ酸配列
配列番号183-人工配列の説明:MOG310のHCDR1のアミノ酸配列
配列番号184-人工配列の説明:MOG310のHCDR2のアミノ酸配列
配列番号185-人工配列の説明:MOG310のHCDR3のアミノ酸配列
配列番号186-人工配列の説明:MOG310のVL(シグナル配列を除く)をコードする塩基配列
配列番号187-人工配列の説明:MOG310のVL(シグナル配列を除く)のアミノ酸配列
配列番号188-人工配列の説明:MOG310のLCDR1のアミノ酸配列
配列番号189-人工配列の説明:MOG310のLCDR2のアミノ酸配列
配列番号190-人工配列の説明:MOG310のLCDR3のアミノ酸配列
配列番号191-人工配列の説明:MOG312のVH(シグナル配列を除く)をコードする塩基配列
配列番号192-人工配列の説明:MOG312のVH(シグナル配列を除く)のアミノ酸配列
配列番号193-人工配列の説明:MOG312のHCDR1のアミノ酸配列
配列番号194-人工配列の説明:MOG312のHCDR2のアミノ酸配列
配列番号195-人工配列の説明:MOG312のHCDR3のアミノ酸配列
配列番号196-人工配列の説明:MOG312のVL(シグナル配列を除く)をコードする塩基配列
配列番号197-人工配列の説明:MOG312のVL(シグナル配列を除く)のアミノ酸配列
配列番号198-人工配列の説明:MOG312のLCDR1のアミノ酸配列
配列番号199-人工配列の説明:MOG312のLCDR2のアミノ酸配列
配列番号200-人工配列の説明:MOG312のLCDR3のアミノ酸配列
配列番号201-人工配列の説明:MOG326のVH(シグナル配列を除く)をコードする塩基配列
配列番号202-人工配列の説明:MOG326のVH(シグナル配列を除く)のアミノ酸配列
配列番号203-人工配列の説明:MOG326のHCDR1のアミノ酸配列
配列番号204-人工配列の説明:MOG326のHCDR2のアミノ酸配列
配列番号205-人工配列の説明:MOG326のHCDR3のアミノ酸配列
配列番号206-人工配列の説明:MOG326のVL(シグナル配列を除く)をコードする塩基配列
配列番号207-人工配列の説明:MOG326のVL(シグナル配列を除く)のアミノ酸配列
配列番号208-人工配列の説明:MOG326のLCDR1のアミノ酸配列
配列番号209-人工配列の説明:MOG326のLCDR2のアミノ酸配列
配列番号210-人工配列の説明:MOG326のLCDR3のアミノ酸配列
配列番号211-人工配列の説明:MOG329のVH(シグナル配列を除く)をコードする塩基配列
配列番号212-人工配列の説明:MOG329のVH(シグナル配列を除く)のアミノ酸配列
配列番号213-人工配列の説明:MOG329のHCDR1のアミノ酸配列
配列番号214-人工配列の説明:MOG329のHCDR2のアミノ酸配列
配列番号215-人工配列の説明:MOG329のHCDR3のアミノ酸配列
配列番号216-人工配列の説明:MOG329のVL(シグナル配列を除く)をコードする塩基配列
配列番号217-人工配列の説明:MOG329のVL(シグナル配列を除く)のアミノ酸配列
配列番号218-人工配列の説明:MOG329のLCDR1のアミノ酸配列
配列番号219-人工配列の説明:MOG329のLCDR2のアミノ酸配列
配列番号220-人工配列の説明:MOG329のLCDR3のアミノ酸配列
配列番号221-人工配列の説明:MOG446のVH(シグナル配列を除く)をコードする塩基配列
配列番号222-人工配列の説明:MOG446のVH(シグナル配列を除く)のアミノ酸配列
配列番号223-人工配列の説明:MOG446のHCDR1のアミノ酸配列
配列番号224-人工配列の説明:MOG446のHCDR2のアミノ酸配列
配列番号225-人工配列の説明:MOG446のHCDR3のアミノ酸配列
配列番号226-人工配列の説明:MOG446のVL(シグナル配列を除く)をコードする塩基配列
配列番号227-人工配列の説明:MOG446のVL(シグナル配列を除く)のアミノ酸配列
配列番号228-人工配列の説明:MOG446のLCDR1のアミノ酸配列
配列番号229-人工配列の説明:MOG446のLCDR2のアミノ酸配列
配列番号230-人工配列の説明:MOG446のLCDR3のアミノ酸配列
配列番号231-人工配列の説明:MOG456のVH(シグナル配列を除く)をコードする塩基配列
配列番号232-人工配列の説明:MOG456のVH(シグナル配列を除く)のアミノ酸配列
配列番号233-人工配列の説明:MOG456のHCDR1のアミノ酸配列
配列番号234-人工配列の説明:MOG456のHCDR2のアミノ酸配列
配列番号235-人工配列の説明:MOG456のHCDR3のアミノ酸配列
配列番号236-人工配列の説明:MOG456のVL(シグナル配列を除く)をコードする塩基配列
配列番号237-人工配列の説明:MOG456のVL(シグナル配列を除く)のアミノ酸配列
配列番号238-人工配列の説明:MOG456のLCDR1のアミノ酸配列
配列番号239-人工配列の説明:MOG456のLCDR2のアミノ酸配列
配列番号240-人工配列の説明:MOG456のLCDR3のアミノ酸配列
配列番号241-人工配列の説明:MOG473のVH(シグナル配列を除く)をコードする塩基配列
配列番号242-人工配列の説明:MOG473のVH(シグナル配列を除く)のアミノ酸配列
配列番号243-人工配列の説明:MOG473のHCDR1のアミノ酸配列
配列番号244-人工配列の説明:MOG473のHCDR2のアミノ酸配列
配列番号245-人工配列の説明:MOG473のHCDR3のアミノ酸配列
配列番号246-人工配列の説明:MOG473のVL(シグナル配列を除く)をコードする塩基配列
配列番号247-人工配列の説明:MOG473のVL(シグナル配列を除く)のアミノ酸配列
配列番号248-人工配列の説明:MOG473のLCDR1のアミノ酸配列
配列番号249-人工配列の説明:MOG473のLCDR2のアミノ酸配列
配列番号250-人工配列の説明:MOG473のLCDR3のアミノ酸配列
配列番号251-人工配列の説明:MOG426のVH(シグナル配列を除く)をコードする塩基配列
配列番号252-人工配列の説明:MOG426のVH(シグナル配列を除く)のアミノ酸配列
配列番号253-人工配列の説明:MOG426のVL(シグナル配列を除く)をコードする塩基配列
配列番号254-人工配列の説明:MOG426のVL(シグナル配列を除く)のアミノ酸配列
配列番号255-人工配列の説明:MOG428のVH(シグナル配列を除く)をコードする塩基配列
配列番号256-人工配列の説明:MOG428のVH(シグナル配列を除く)のアミノ酸配列
配列番号257-人工配列の説明:MOG428のVL(シグナル配列を除く)をコードする塩基配列
配列番号258-人工配列の説明:MOG428のVL(シグナル配列を除く)のアミノ酸配列
配列番号259-人工配列の説明:MOG313のVH(シグナル配列を除く)をコードする塩基配列
配列番号260-人工配列の説明:MOG313のVH(シグナル配列を除く)のアミノ酸配列
配列番号261-人工配列の説明:MOG313のVL(シグナル配列を除く)をコードする塩基配列
配列番号262-人工配列の説明:MOG313のVL(シグナル配列を除く)のアミノ酸配列
配列番号263-人工配列の説明:MOG314のVH(シグナル配列を除く)をコードする塩基配列
配列番号264-人工配列の説明:MOG314のVH(シグナル配列を除く)のアミノ酸配列
配列番号265-人工配列の説明:MOG314のVL(シグナル配列を除く)をコードする塩基配列
配列番号266-人工配列の説明:MOG314のVL(シグナル配列を除く)のアミノ酸配列
配列番号267-人工配列の説明:MOG315のVH(シグナル配列を除く)をコードする塩基配列
配列番号268-人工配列の説明:MOG315のVH(シグナル配列を除く)のアミノ酸配列
配列番号269-人工配列の説明:MOG315のVL(シグナル配列を除く)をコードする塩基配列
配列番号270-人工配列の説明:MOG315のVL(シグナル配列を除く)のアミノ酸配列
配列番号271-人工配列の説明:MOG331のVH(シグナル配列を除く)をコードする塩基配列
配列番号272-人工配列の説明:MOG331のVH(シグナル配列を除く)のアミノ酸配列
配列番号273-人工配列の説明:MOG331のVL(シグナル配列を除く)をコードする塩基配列
配列番号274-人工配列の説明:MOG331のVL(シグナル配列を除く)のアミノ酸配列
配列番号275-人工配列の説明:MOG357のVH(シグナル配列を除く)をコードする塩基配列
配列番号276-人工配列の説明:MOG357のVH(シグナル配列を除く)のアミノ酸配列
配列番号277-人工配列の説明:MOG357のVL(シグナル配列を除く)をコードする塩基配列
配列番号278-人工配列の説明:MOG357のVL(シグナル配列を除く)のアミノ酸配列
配列番号279-人工配列の説明:MOG476のVH(シグナル配列を除く)をコードする塩基配列
配列番号280-人工配列の説明:MOG476のVH(シグナル配列を除く)のアミノ酸配列
配列番号281-人工配列の説明:MOG476のVL(シグナル配列を除く)をコードする塩基配列
配列番号282-人工配列の説明:MOG476のVL(シグナル配列を除く)のアミノ酸配列
配列番号283-人工配列の説明:MOG323のVH(シグナル配列を除く)をコードする塩基配列
配列番号284-人工配列の説明:MOG323のVH(シグナル配列を除く)のアミノ酸配列
配列番号285-人工配列の説明:MOG323のVL(シグナル配列を除く)をコードする塩基配列
配列番号286-人工配列の説明:MOG323のVL(シグナル配列を除く)のアミノ酸配列
配列番号287-人工配列の説明:MOG341のVH(シグナル配列を除く)をコードする塩基配列
配列番号288-人工配列の説明:MOG341のVH(シグナル配列を除く)のアミノ酸配列
配列番号289-人工配列の説明:MOG341のVL(シグナル配列を除く)をコードする塩基配列
配列番号290-人工配列の説明:MOG341のVL(シグナル配列を除く)のアミノ酸配列
配列番号291-人工配列の説明:MOG354のVH(シグナル配列を除く)をコードする塩基配列
配列番号292-人工配列の説明:MOG354のVH(シグナル配列を除く)のアミノ酸配列
配列番号293-人工配列の説明:MOG354のVL(シグナル配列を除く)をコードする塩基配列
配列番号294-人工配列の説明:MOG354のVL(シグナル配列を除く)のアミノ酸配列
配列番号295-人工配列の説明:MOG355のVH(シグナル配列を除く)をコードする塩基配列
配列番号296-人工配列の説明:MOG355のVH(シグナル配列を除く)のアミノ酸配列
配列番号297-人工配列の説明:MOG355のVL(シグナル配列を除く)をコードする塩基配列
配列番号298-人工配列の説明:MOG355のVL(シグナル配列を除く)のアミノ酸配列
配列番号299-人工配列の説明:MOG308のVH(シグナル配列を除く)をコードする塩基配列
配列番号300-人工配列の説明:MOG308のVH(シグナル配列を除く)のアミノ酸配列
配列番号301-人工配列の説明:MOG308のVL(シグナル配列を除く)をコードする塩基配列
配列番号302-人工配列の説明:MOG308のVL(シグナル配列を除く)のアミノ酸配列
配列番号303-人工配列の説明:MOG316のVH(シグナル配列を除く)をコードする塩基配列
配列番号304-人工配列の説明:MOG316のVH(シグナル配列を除く)のアミノ酸配列
配列番号305-人工配列の説明:MOG316のVL(シグナル配列を除く)をコードする塩基配列
配列番号306-人工配列の説明:MOG316のVL(シグナル配列を除く)のアミノ酸配列
配列番号307-人工配列の説明:MOG319のVH(シグナル配列を除く)をコードする塩基配列
配列番号308-人工配列の説明:MOG319のVH(シグナル配列を除く)のアミノ酸配列
配列番号309-人工配列の説明:MOG319のVL(シグナル配列を除く)をコードする塩基配列
配列番号310-人工配列の説明:MOG319のVL(シグナル配列を除く)のアミノ酸配列
配列番号311-人工配列の説明:MOG320のVH(シグナル配列を除く)をコードする塩基配列
配列番号312-人工配列の説明:MOG320のVH(シグナル配列を除く)のアミノ酸配列
配列番号313-人工配列の説明:MOG320のVL(シグナル配列を除く)をコードする塩基配列
配列番号314-人工配列の説明:MOG320のVL(シグナル配列を除く)のアミノ酸配列
配列番号315-人工配列の説明:MOG338のVH(シグナル配列を除く)をコードする塩基配列
配列番号316-人工配列の説明:MOG338のVH(シグナル配列を除く)のアミノ酸配列
配列番号317-人工配列の説明:MOG338のVL(シグナル配列を除く)をコードする塩基配列
配列番号318-人工配列の説明:MOG338のVL(シグナル配列を除く)のアミノ酸配列
配列番号319-人工配列の説明:MOG352のVH(シグナル配列を除く)をコードする塩基配列
配列番号320-人工配列の説明:MOG352のVH(シグナル配列を除く)のアミノ酸配列
配列番号321-人工配列の説明:MOG352のVL(シグナル配列を除く)をコードする塩基配列
配列番号322-人工配列の説明:MOG352のVL(シグナル配列を除く)のアミノ酸配列
配列番号323-人工配列の説明:MOG359のVH(シグナル配列を除く)をコードする塩基配列
配列番号324-人工配列の説明:MOG359のVH(シグナル配列を除く)のアミノ酸配列
配列番号325-人工配列の説明:MOG359のVL(シグナル配列を除く)をコードする塩基配列
配列番号326-人工配列の説明:MOG359のVL(シグナル配列を除く)のアミノ酸配列
配列番号327-人工配列の説明:MOG478のVH(シグナル配列を除く)をコードする塩基配列
配列番号328-人工配列の説明:MOG478のVH(シグナル配列を除く)のアミノ酸配列
配列番号329-人工配列の説明:MOG478のVL(シグナル配列を除く)をコードする塩基配列
配列番号330-人工配列の説明:MOG478のVL(シグナル配列を除く)のアミノ酸配列
配列番号331-人工配列の説明:MOG470のVH(シグナル配列を除く)をコードする塩基配列
配列番号332-人工配列の説明:MOG470のVH(シグナル配列を除く)のアミノ酸配列
配列番号333-人工配列の説明:MOG470のVL(シグナル配列を除く)をコードする塩基配列
配列番号334-人工配列の説明:MOG470のVL(シグナル配列を除く)のアミノ酸配列
配列番号335-人工配列の説明:MOG418のVH(シグナル配列を除く)をコードする塩基配列
配列番号336-人工配列の説明:MOG418のVH(シグナル配列を除く)のアミノ酸配列
配列番号337-人工配列の説明:MOG418のVL(シグナル配列を除く)をコードする塩基配列
配列番号338-人工配列の説明:MOG418のVL(シグナル配列を除く)のアミノ酸配列
Claims (22)
- ミエリンオリゴデンドロサイト糖タンパク質(以下MOGと記載)に結合する抗体または該抗体断片。
- 抗体が脳滞留性を有する請求項1に記載の抗体または該抗体断片。
- 抗体が下記(a)~(r)からなる群より選ばれる1である、請求項1または2に記載の抗体または該抗体断片。
(a)重鎖可変領域(以下VHと記載する)の相補性決定領域(以下、CDR)1~3のアミノ酸配列が、それぞれ配列番号4、5および6に記載されるアミノ酸配列を含み、かつ軽鎖可変領域(VL)のCDR1~3のアミノ酸配列が、それぞれ配列番号10、11および12に記載されるアミノ酸配列を含む抗体、
(b)VHのCDR1~3のアミノ酸配列が、それぞれ配列番号16、17および18に記載されるアミノ酸配列を含み、かつVLのCDR1~3のアミノ酸配列が、それぞれ配列番号22、23および24に記載されるアミノ酸配列を含む抗体、
(c)VHのCDR1~3のアミノ酸配列が、それぞれ配列番号28、29および30に記載されるアミノ酸配列を含み、かつVLのCDR1~3のアミノ酸配列が、それぞれ配列番号34、35および36に記載されるアミノ酸配列を含む抗体、
(d)重鎖抗体の重鎖可変領域(以下VHHと記載する)のCDR1~3のアミノ酸配列が、それぞれ配列番号40、41および42に記載されるアミノ酸配列を含む抗体断片、
(e)VHのCDR1~3のアミノ酸配列が、それぞれ配列番号153、154および155に記載されるアミノ酸配列を含み、かつVLのCDR1~3のアミノ酸配列が、それぞれ配列番号158、159および160に記載されるアミノ酸配列を含む抗体、
(f)VHのCDR1~3のアミノ酸配列が、それぞれ配列番号163、164および165に記載されるアミノ酸配列を含み、かつVLのCDR1~3のアミノ酸配列が、それぞれ配列番号168、169および170に記載されるアミノ酸配列を含む抗体、
(g)VHのCDR1~3のアミノ酸配列が、それぞれ配列番号173、174および175に記載されるアミノ酸配列を含み、かつVLのCDR1~3のアミノ酸配列が、それぞれ配列番号178、179および180に記載されるアミノ酸配列を含む抗体、
(h)VHのCDR1~3のアミノ酸配列が、それぞれ配列番号183、184および185に記載されるアミノ酸配列を含み、かつVLのCDR1~3のアミノ酸配列が、それぞれ配列番号188、189および190に記載されるアミノ酸配列を含む抗体、
(i)VHのCDR1~3のアミノ酸配列が、それぞれ配列番号193、194および195に記載されるアミノ酸配列を含み、かつVLのCDR1~3のアミノ酸配列が、それぞれ配列番号198、199および200に記載されるアミノ酸配列を含む抗体、
(j)VHのCDR1~3のアミノ酸配列が、それぞれ配列番号203、204および205に記載されるアミノ酸配列を含み、かつVLのCDR1~3のアミノ酸配列が、それぞれ配列番号208、209および210に記載されるアミノ酸配列を含む抗体、
(k)VHのCDR1~3のアミノ酸配列が、それぞれ配列番号213、214および215に記載されるアミノ酸配列を含み、かつVLのCDR1~3のアミノ酸配列が、それぞれ配列番号218、219および220に記載されるアミノ酸配列を含む抗体、
(l)VHのCDR1~3のアミノ酸配列が、それぞれ配列番号223、224および225に記載されるアミノ酸配列を含み、かつVLのCDR1~3のアミノ酸配列が、それぞれ配列番号228、229および230に記載されるアミノ酸配列を含む抗体、
(m)VHのCDR1~3のアミノ酸配列が、それぞれ配列番号233、234および235に記載されるアミノ酸配列を含み、かつVLのCDR1~3のアミノ酸配列が、それぞれ配列番号238、239および240に記載されるアミノ酸配列を含む抗体、
(n)VHのCDR1~3のアミノ酸配列が、それぞれ配列番号243、244および245に記載されるアミノ酸配列を含み、かつVLのCDR1~3のアミノ酸配列が、それぞれ配列番号248、249および250に記載されるアミノ酸配列を含む抗体、
(o)前記(a)~(n)に記載の少なくとも1つの抗体と、MOGへの結合について競合する抗体、
(p)前記(a)~(n)に記載のいずれか1つの抗体が結合するエピトープを含むエピトープに結合する抗体、および
(q)前記(a)~(n)に記載のいずれか1つの抗体が結合するエピトープと同じエピトープに結合する抗体。
(r)前記(a)~(n)に記載のいずれか1つの抗体のアミノ酸配列と85%以上の相同性を有するアミノ酸配列を含む抗体。 - 抗体が下記(a)~(n)、(o1)~(o22)および(p)からなる群より選ばれる1である、請求項1~3のいずれか1項に記載の抗体または該抗体断片。
(a)VHのアミノ酸配列が配列番号3に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号9に記載されるアミノ酸配列を含む抗体、
(b)VHのアミノ酸配列が配列番号15に記載されるアミノ酸配列含み、かつVLのアミノ酸配列が配列番号21に記載されるアミノ酸配列を含む抗体、
(c)VHのアミノ酸配列が配列番号27に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号33に記載されるアミノ酸配列を含む抗体、
(d)VHHのアミノ酸配列が配列番号39に記載されるアミノ酸配列を含む抗体断片、
(e)VHのアミノ酸配列が配列番号152に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号157に記載されるアミノ酸配列を含む抗体、
(f)VHのアミノ酸配列が配列番号162に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号167に記載されるアミノ酸配列を含む抗体、
(g)VHのアミノ酸配列が配列番号172に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号177に記載されるアミノ酸配列を含む抗体、
(h)VHのアミノ酸配列が配列番号182に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号187に記載されるアミノ酸配列を含む抗体、
(i)VHのアミノ酸配列が配列番号192に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号197に記載されるアミノ酸配列を含む抗体、
(j)VHのアミノ酸配列が配列番号202に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号207に記載されるアミノ酸配列を含む抗体、
(k)VHのアミノ酸配列が配列番号212に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号217に記載されるアミノ酸配列を含む抗体、
(l)VHのアミノ酸配列が配列番号222に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号227に記載されるアミノ酸配列を含む抗体、
(m)VHのアミノ酸配列が配列番号232に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号237に記載されるアミノ酸配列を含む抗体、
(n)VHのアミノ酸配列が配列番号242に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号247に記載されるアミノ酸配列を含む抗体、
(o1)VHのアミノ酸配列が配列番号252に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号254に記載されるアミノ酸配列を含む抗体、
(o2)VHのアミノ酸配列が配列番号256に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号258に記載されるアミノ酸配列を含む抗体、
(o3)VHのアミノ酸配列が配列番号260に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号262に記載されるアミノ酸配列を含む抗体、
(o4)VHのアミノ酸配列が配列番号264に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号266に記載されるアミノ酸配列を含む抗体、
(o5)VHのアミノ酸配列が配列番号268に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号270に記載されるアミノ酸配列を含む抗体、
(o6)VHのアミノ酸配列が配列番号272に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号274に記載されるアミノ酸配列を含む抗体、
(o7)VHのアミノ酸配列が配列番号276に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号278に記載されるアミノ酸配列を含む抗体、
(o8)VHのアミノ酸配列が配列番号280に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号282に記載されるアミノ酸配列を含む抗体、
(o9)VHのアミノ酸配列が配列番号284に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号286に記載されるアミノ酸配列を含む抗体、
(o10)VHのアミノ酸配列が配列番号288に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号290に記載されるアミノ酸配列を含む抗体、
(o11)VHのアミノ酸配列が配列番号292に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号294に記載されるアミノ酸配列を含む抗体、
(o12)VHのアミノ酸配列が配列番号296に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号298に記載されるアミノ酸配列を含む抗体、
(o13)VHのアミノ酸配列が配列番号300に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号302に記載されるアミノ酸配列を含む抗体、
(o14)VHのアミノ酸配列が配列番号304に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号306に記載されるアミノ酸配列を含む抗体、
(o15)VHのアミノ酸配列が配列番号308に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号310に記載されるアミノ酸配列を含む抗体、
(o16)VHのアミノ酸配列が配列番号312に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号314に記載されるアミノ酸配列を含む抗体、
(o17)VHのアミノ酸配列が配列番号316に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号318に記載されるアミノ酸配列を含む抗体、
(o18)VHのアミノ酸配列が配列番号320に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号322に記載されるアミノ酸配列を含む抗体、
(o19)VHのアミノ酸配列が配列番号324に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号326に記載されるアミノ酸配列を含む抗体、
(o20)VHのアミノ酸配列が配列番号328に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号330に記載されるアミノ酸配列を含む抗体、
(o21)VHのアミノ酸配列が配列番号332に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号334に記載されるアミノ酸配列を含む抗体、および
(o22)VHのアミノ酸配列が配列番号336に記載されるアミノ酸配列を含み、かつVLのアミノ酸配列が配列番号338に記載されるアミノ酸配列を含む抗体。
(p)前記(a)~(n)及び(о1)~(о22)に記載のいずれか1つの抗体のアミノ酸配列と85%以上の相同性を有するアミノ酸配列を含む抗体。 - 抗体または該抗体断片がバイスペシフィック抗体である、請求項1~4のいずれか1項に記載の抗体または該抗体断片。
- バイスペシフィック抗体がMOGと脳に存在する抗原に結合する、請求項5に記載のバイスペシフィック抗体。
- バイスペシフィック抗体がMOGに結合する抗原結合部位と、脳に存在する抗原に結合する抗原結合部位とを含む、請求項5または6に記載のバイスペシフィック抗体。
- 抗体断片がFab、Fab’、F(ab’)2、一本鎖抗体(scFv)、二量体化V領域(diabody)、ジスルフィド安定化V領域(dsFv)、VHHおよびCDRを含むペプチドからなる群より選ばれる1である、請求項1~7のいずれか1項に記載の抗体断片。
- 抗体が遺伝子組換え抗体である、請求項1~8のいずれか1項に記載の抗体および該抗体断片。
- 抗体がマウス抗体、ラット抗体、ラビット抗体、アルパカ抗体、ラクダ抗体、ラマ抗体、キメラ抗体、ヒト化抗体およびヒト抗体からなる群より選ばれる1である、請求項1~9のいずれか1項に記載の抗体および該抗体断片。
- 請求項1~10のいずれか1項に記載のMOGに結合する抗体または該抗体断片に、下記(a)~(c)からなる群より選ばれる少なくとも1つを結合させた融合抗体または融合抗体断片。
(a)親水性高分子、
(b)両親媒性高分子、および
(c)機能性分子。 - 請求項1~11のいずれか1項に記載の抗体を産生するハイブリドーマ。
- 請求項1~11のいずれか1項に記載の抗体をコードする塩基配列を含む核酸。
- 請求項13に記載の核酸を含むベクターを含む形質転換細胞。
- 請求項12に記載のハイブリドーマまたは請求項14に記載の形質転換細胞を培養し、培養液から請求項1~11のいずれか1項に記載の抗体または該抗体断片を採取することを含む、請求項1~11のいずれか1項に記載の抗体または該抗体断片の製造方法。
- 請求項1~11のいずれか1項に記載の抗体または該抗体断片を含む、組成物。
- 脳に存在する抗原の検出または測定用の組成物である、請求項16に記載の組成物。
- 脳疾患の診断または治療するための組成物である、請求項16に記載の組成物。
- 請求項1~11のいずれか1項に記載の抗体若しくは該抗体断片、または請求項16に記載の組成物を用いて、脳に存在する抗原を検出または測定する方法。
- 請求項1~11のいずれか1項に記載の抗体若しくは該抗体断片、または請求項16に記載の組成物を用いて、脳疾患を診断または治療する方法。
- 請求項1~11のいずれか1項に記載の抗体または該抗体断片若しくは融合抗体または融合抗体断片、または請求項16に記載の組成物を用いて、抗体または該抗体断片若しくは融合抗体または融合抗体断片の脳滞留性を向上させる方法。
- 請求項1~11のいずれか1項に記載の抗体または該抗体断片若しくは融合抗体または融合抗体断片、または請求項16に記載の組成物を用いて、脳内の抗体量または該抗体断片量若しくは融合抗体量または融合抗体断片量を増加させる方法。
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-
2017
- 2017-12-25 CA CA3048601A patent/CA3048601A1/en active Pending
- 2017-12-25 CN CN201780081320.6A patent/CN110536901A/zh active Pending
- 2017-12-25 JP JP2018559466A patent/JP7291339B2/ja active Active
- 2017-12-25 EP EP17885914.6A patent/EP3560955A4/en active Pending
- 2017-12-25 AU AU2017388346A patent/AU2017388346A1/en active Pending
- 2017-12-25 KR KR1020197018130A patent/KR20190097067A/ko not_active Application Discontinuation
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- 2017-12-25 WO PCT/JP2017/046445 patent/WO2018123979A1/ja active Application Filing
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2021
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US20220025044A1 (en) | 2022-01-27 |
US11117963B2 (en) | 2021-09-14 |
WO2018123979A1 (ja) | 2018-07-05 |
JPWO2018123979A1 (ja) | 2020-02-06 |
TW201829458A (zh) | 2018-08-16 |
AU2017388346A1 (en) | 2019-07-11 |
EP3560955A4 (en) | 2020-12-23 |
US20190352397A1 (en) | 2019-11-21 |
US20240124577A1 (en) | 2024-04-18 |
CN110536901A (zh) | 2019-12-03 |
JP7291339B2 (ja) | 2023-06-15 |
EP3560955A1 (en) | 2019-10-30 |
CA3048601A1 (en) | 2018-07-05 |
KR20190097067A (ko) | 2019-08-20 |
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