WO2014123186A1 - 抗セマフォリン3a抗体、並びにこれを用いたアルツハイマー病及び免疫・炎症性疾患の治療 - Google Patents
抗セマフォリン3a抗体、並びにこれを用いたアルツハイマー病及び免疫・炎症性疾患の治療 Download PDFInfo
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Definitions
- the present invention relates to an anti-semaphorin 3A antibody or an antibody fragment thereof that is effective for the prevention and / or treatment of diseases involving semaphorin 3A protein. Furthermore, the present invention relates to a pharmaceutical composition using the anti-semaphorin 3A antibody or an antibody fragment thereof. Furthermore, the present invention relates to a method for measuring semaphorin 3A protein using an anti-semaphorin 3A antibody or an antibody fragment thereof.
- Alzheimer's disease is the most common form of dementia (loss of memory) in the elderly.
- Currently used treatments for Alzheimer's disease include cholinesterase inhibitors that improve cholinergic neurotransmission in the central nervous system, or excitotoxicity caused by ⁇ -methyl-D-aspartate (NMDA) glutamate receptor inhibitors. Suppression is the mechanism of action, but only slight improvement in symptoms has been observed.
- NMDA ⁇ -methyl-D-aspartate
- AD The major pathological injury of AD found in the brain is due to extracellular deposits of ⁇ -amyloid protein in the form of plaques and vascular disorders, and intracellular neurofibrillary tangles of aggregated hyperphosphorylated ⁇ protein is doing. Recent evidence indicates that elevated ⁇ -amyloid levels in the brain not only precede the pathology of ⁇ , but also correlate with cognitive decline. Recent studies have further suggested a possible role for ⁇ -amyloid in AD, revealing that aggregated ⁇ -amyloid is toxic to neurons in cell culture.
- ⁇ -amyloid protein is mainly composed of a peptide of 39 to 42 amino acids, and is produced from a larger precursor protein called amyloid precursor protein (APP) by the continuous action of protease, ⁇ and ⁇ -secretase.
- APP amyloid precursor protein
- APP amyloid precursor protein
- cases of early-onset AD result from gene mutations in APP that lead to overproduction of either the entire ⁇ -amyloid protein or its isoform.
- people with Down's syndrome have extra chromosomes that contain the gene encoding APP, have elevated ⁇ -amyloid levels, and inevitably develop AD with age.
- Semaphorin is an endogenous protein that has been identified as a factor that retreats the nerve growth cone and suppresses axonal elongation.
- Semaphorin genes are classified into eight gene subfamilies and classes based on their structures, and about 20 molecular species have been known so far (Non-patent Document 1).
- the most studied group is a group of subfamily genes called class 3 type, and the most studied is semaphorin 3A (Sema3A) (Non-patent Documents 2 and 3). It is known that Sema3A protein induces growth cone retraction of cultured neurons in a short time at a low concentration of 10 pM and inhibits nerve regeneration elongation.
- Sema3A protein is secreted from cancer cells / tissues and inhibits the signal pathway of mitogen-activated protein (MAP) kinase to suppress T cell activation, or Sema3A in cancer tissues of pancreatic cancer patients.
- MAP mitogen-activated protein
- DIC disseminated intravascular coagulation
- the main symptoms are bleeding symptoms and organ symptoms, but it is known that the prognosis becomes extremely poor when clinical symptoms appear.
- the underlying diseases that trigger DIC include sepsis, acute leukemia, solid cancer, early placenta detachment, amniotic fluid embolism, trauma, burns, collagen disease, shock, aortic aneurysm, fulminant hepatitis, cirrhosis, acute pancreatitis, lateral
- rhabdomyolysis, thrombosis, severe infection, etc. are known, the relationship with Sema3A has not been clarified.
- Sema3A protein activity inhibitors such as anti-Sema3A antibodies are effective in treating Alzheimer's and Parkinson's disease (see Patent Document 1), and effective in treating immune diseases and inflammatory diseases (see Patent Document 2).
- Patent Document 1 Sema3A protein activity inhibitors
- Patent Document 2 Anti-Sema3A antibodies
- Patent Document 3 Anti-Sema3A antibodies
- Sema3A protein is involved in various pathologies as described above, and a technique for measuring the Sema3A protein with high accuracy is indispensable for elucidating the pathology and developing therapeutic agents. ing.
- the present invention relates to Sema3A protein that can effectively prevent and / or treat disseminated intravascular coagulation syndrome and diseases involving Sema3A protein such as neurodegenerative diseases, autoimmune diseases, inflammatory diseases, cancer, infectious diseases and the like. It is an object to provide an antibody. Another object of the present invention is to provide a pharmaceutical composition that enables prevention and / or treatment of a disease associated with the Sema3A protein and alleviation of symptoms associated with the disease. Furthermore, another object of the present invention is to provide an antibody against the Sema3A protein that is effective for measuring the Sema3A protein, and a method for measuring the Sema3A protein using the antibody.
- an anti-Sema3A antibody having a complementarity determining region (CDR) having a specific amino acid sequence is a neurodegenerative disease such as Alzheimer's disease. It was found that the disease can be effectively prevented and / or treated, and the symptoms associated with the neurodegenerative disease are remarkably improved. Further, in a fatal sepsis model, the anti-Sema3A antibody showed a dramatic improvement in survival rate and an extension of survival period. In addition, the present inventors have found that the anti-Sema3A antibody reduces blood plasminogen activator inhibitor-1 (PAI-1), which is one of the exacerbation factors of disseminated intravascular coagulation syndrome.
- PAI-1 blood plasminogen activator inhibitor-1
- cancer cell migration / invasion and drug resistance are induced by Sema3A
- cancer cell migration / invasion and drug resistance can be suppressed by the anti-Sema3A antibody, so that malignancy of cancer can also be suppressed. I found.
- Sema3A protein can be measured by ELISA using an anti-Sema3A antibody equipped with a CDR having a specific amino acid sequence.
- Item 2. The anti-semaphorin 3A antibody or antibody fragment according to Item 1, which is a chimeric antibody, a humanized antibody, or an antibody fragment thereof.
- Item 3. The anti-semaphorin 3A antibody or antibody fragment according to Item 1 or 2, which is used for promoting nerve regeneration.
- Item 4. Item 3.
- Item 5. Item 3. The anti-semaphorin 3A antibody or antibody fragment according to Item 1 or 2, which is used for prevention and / or treatment of sepsis.
- Item 6. Item 3. The anti-semaphorin 3A antibody or antibody fragment according to Item 1 or 2, which is used for prevention and / or treatment of cancer.
- Item 1 or used for prevention and / or treatment of at least one disease selected from the group consisting of central or peripheral nervous system diseases, autoimmune diseases, inflammatory diseases, infectious diseases, and allergic diseases 3.
- Item 9. Item 9.
- a pharmaceutical composition comprising the anti-semaphorin 3A antibody or antibody fragment according to any one of Items 1 to 8.
- Item 10. Item 10.
- Item 11. Item 10.
- Item 13. Item 10.
- Cancer is colon cancer, colorectal cancer, lung cancer, breast cancer, brain tumor, melanoma, renal cell carcinoma, leukemia, lymphoma, T cell lymphoma, stomach cancer, pancreatic cancer, cervical cancer, endometrial cancer, ovarian cancer, esophagus Item 14.
- Item 15. Item 10.
- Disseminated intravascular coagulation syndrome is sepsis, acute leukemia, solid cancer, early placenta detachment, amniotic fluid embolism, trauma, burn, collagen disease, shock, aortic aneurysm, fulminant hepatitis, cirrhosis, acute pancreatitis, striated muscle Item 16.
- the pharmaceutical composition according to Item 15 accompanied by at least one selected from the group consisting of thawing, thrombosis, and severe infection.
- Item 18. The pharmaceutical composition according to Item 17, wherein the central or peripheral nervous system disease is neuropathic pain, spinal cord injury, or neurodegenerative disease.
- the neurodegenerative disease is Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, progressive supranuclear palsy, nigrostriatal degeneration, Shy-Drager syndrome, Olive Bridge cerebellar atrophy, or spinal cord Item 19.
- Item 18 The pharmaceutical composition according to Item 17, wherein the autoimmune disease is rheumatoid arthritis, type I diabetes, inflammatory bowel disease, Crohn's disease, systemic lupus erythematosus, or multiple sclerosis.
- Item 22. Item 18.
- the infection is a bacterial infection, encephalitis, meningitis, endocarditis, hepatitis C, influenza, severe acute respiratory syndrome, pneumonia, sepsis, burns, or traumatic infection Pharmaceutical composition.
- Item 23 The pharmaceutical composition according to Item 17, wherein the allergic disease is allergic rhinitis, allergic conjunctivitis, atopic dermatitis, bronchial asthma, or food allergy.
- a nerve regeneration method comprising a step of administering the anti-semaphorin 3A antibody or antibody fragment according to item 1 or 2 to a patient for whom nerve regeneration is required.
- a method for treating Alzheimer's disease comprising a step of administering the anti-semaphorin 3A antibody or antibody fragment according to Item 1 or 2 to a patient suffering from Alzheimer's disease.
- Item 26 A method for treating Alzheimer's disease, comprising a step of administering the anti-semaphorin 3A antibody or antibody fragment according to Item 1 or 2 to a patient suffering from Alzheimer's disease.
- a method for treating sepsis comprising a step of administering the anti-semaphorin 3A antibody or antibody fragment according to Item 1 or 2 to a patient suffering from sepsis.
- Item 27 A method for treating cancer, comprising a step of administering the anti-semaphorin 3A antibody or antibody fragment according to Item 1 or 2 to a patient suffering from cancer.
- Item 28. Cancer is colon cancer, colorectal cancer, lung cancer, breast cancer, brain tumor, melanoma, renal cell carcinoma, leukemia, lymphoma, T cell lymphoma, stomach cancer, pancreatic cancer, cervical cancer, endometrial cancer, ovarian cancer, esophagus Item 28.
- Item 29. A method for treating disseminated intravascular coagulation syndrome, comprising a step of administering the anti-semaphorin 3A antibody or antibody fragment according to item 1 or 2 to a patient suffering from disseminated intravascular coagulation syndrome.
- Disseminated intravascular coagulation syndrome is sepsis, acute leukemia, solid cancer, early placenta detachment, amniotic fluid embolism, trauma, burn, collagen disease, shock, aortic aneurysm, fulminant hepatitis, cirrhosis, acute pancreatitis, striated muscle Item 30.
- a method for treating the disease comprising the step of administering an anti-semaphorin 3A antibody or antibody fragment.
- Item 3. Use of the anti-semaphorin 3A antibody or antibody fragment according to Item 1 or 2 for the manufacture of a medicament for nerve regeneration.
- Item 3. Use of the anti-semaphorin 3A antibody or antibody fragment according to Item 1 or 2 for the manufacture of an agent for preventing and / or treating Alzheimer's disease.
- Item 34 Use of the anti-semaphorin 3A antibody or antibody fragment according to Item 1 or 2 for the manufacture of an agent for preventing and / or treating sepsis.
- Item 34 Use of the anti-semaphorin 3A antibody or antibody fragment according to Item 1 or 2 for the manufacture of an agent for preventing and / or treating cancer.
- Item 35 Use of the anti-semaphorin 3A antibody or antibody fragment according to Item 1 or 2 for the manufacture of a preventive and / or therapeutic agent for disseminated intravascular coagulation syndrome.
- Item 36 Use of the anti-semaphorin 3A antibody or antibody fragment according to 2.
- Item 37 Use of the anti-semaphorin 3A antibody or antibody fragment according to 2.
- a method for measuring Sema3A protein comprising a step of measuring Sema3A protein in a sample by immunoassay using the anti-Sema3A antibody or antibody fragment according to Item 1 or 2.
- Item 3. A kit for measuring Sema3A protein, comprising the anti-Sema3A antibody or antibody fragment according to item 1 or 2.
- the anti-Sema3A antibody of the present invention neurodegenerative diseases such as Alzheimer's disease can be effectively prevented and / or treated, and the alleviation of symptoms associated with the neurodegenerative diseases can be remarkably improved.
- the anti-Sema3A antibody of the present invention inhibits Sema3A signaling by effectively inhibiting the function of the Sema3A protein, As a result, it is thought that accumulation of phosphorylated Collapsin Response Mediator Protein (CRMP) in neurofibrillary tangles is inhibited.
- CRMP Collapsin Response Mediator Protein
- the anti-Sema3A antibody of the present invention is effective for the prevention and / or treatment of central or peripheral nervous system diseases other than neurodegenerative diseases, autoimmune diseases, inflammatory diseases, infectious diseases, and allergic diseases. In particular, it can exhibit an excellent preventive and / or therapeutic effect on inflammatory diseases in which the immune system is destroyed by infection or strong inflammation-induced stimulation such as sepsis.
- the migration / invasion activity of cancer cells induced by Sema3A can be effectively suppressed, anti-cancer agent unresponsiveness induced by Sema3A is canceled, and drug sensitivity is reduced. Since it can be recovered, it is also effective in preventing and / or treating cancer.
- the anti-Sema3A antibody of the present invention has an action of suppressing an increase in blood PAI-1 level, and is also effective in the prevention and / or treatment of disseminated intravascular coagulation syndrome.
- the anti-Sema3A antibody of the present invention can also be used for measuring Sema3A protein.
- the Sema3A protein can be measured with high accuracy even in the presence of serum, and the Sema3A protein in a biological sample can be measured.
- FIG. 1 is a graph showing that 13 strains of anti-Sema3A antibody-producing clones were obtained in Example 1.
- FIG. 2 is a graph showing the results of evaluating the binding specificity of the anti-Sema3A tri-mouse chimeric antibody prepared in Example 1 for the Sema3A protein.
- FIG. 3 shows the results of CBB staining after electrophoresis of anti-Sema3A tri-mouse chimeric antibody, anti-Sema3A humanized antibody, and anti-Sema3A tri-human chimeric antibody.
- FIG. 1 is a graph showing that 13 strains of anti-Sema3A antibody-producing clones were obtained in Example 1.
- FIG. 2 is a graph showing the results of evaluating the binding specificity of the anti-Sema3A tri-mouse chimeric antibody prepared in Example 1 for the Sema3A protein.
- FIG. 3 shows the results of CBB staining after electrophore
- FIG. 4 shows the results of testing the reactivity of anti-Sema3A tri-mouse chimeric antibody, anti-Sema3A humanized antibody, and tri-human chimeric anti-Sema3A antibody against human Sema3A and mouse Sema3A antigen.
- FIG. 5 shows the results of measuring the affinity of the anti-Sema3A tri-mouse chimeric antibody, anti-Sema3A humanized antibody, and tri-human chimeric anti-Sema3A antibody for Sema3A protein by competitive ELISA.
- FIG. 6 shows the results of measuring Sema3A in a test sample not containing serum by sandwich ELISA using the anti-Sema3A antibody prepared in Examples 2-4.
- FIG. 7 shows the results of measuring Sema3A in a test sample containing serum by sandwich ELISA using the anti-Sema3A antibody prepared in Example 2.
- FIG. 8 shows the results of a regression assay using chicken-derived Sema3A protein and avian anti-Sema3A antibody.
- FIG. 9 shows the results of a regression assay using chicken-derived Sema3A protein and anti-Sema3A tri-mouse chimeric antibody.
- FIG. 10 shows the results of a regression assay using chicken-derived Sema3A protein and avian anti-Sema3A antibody.
- FIG. 11 shows the results of a regression assay using chicken-derived Sema3A protein and anti-Sema3A tri-mouse chimeric antibody.
- FIG. 12 shows the results of a binding assay for human-derived Sema3A protein with respect to the avian anti-Sema3A antibody.
- FIG. 13 shows the results of a binding assay for anti-Sema3A tri-mouse chimeric antibody against human-derived Sema3A protein.
- FIG. 14 shows the results of conducting a binding assay for human-derived Sema3F protein with respect to the avian anti-Sema3A antibody.
- FIG. 15 shows the results of Example 10.
- FIG. 15A is a graph showing the result of the access rate to the object B when trying to acquire the Novell Object Recognition Task.
- FIG. 15B is a graph showing a result of an access rate to the object C at the time of a test test of the Novell Object Recognition Task.
- FIG. 16 shows the results of Example 15.
- FIG. 3 is a diagram in which three tests administered with anti-Sema3A tri-mouse chimeric antibody were combined, and the survival curve was plotted by the Kaplan-Meier method.
- FIG. 17 shows the results of Example 16.
- FIG. 6 is a graph in which survival curves are plotted by the Kaplan-Meier method for a group administered with 500 ⁇ g of anti-Sema3A humanized antibody and a negative control group.
- FIG. 6 is a graph in which survival curves are plotted by the Kaplan-Meier method for a group administered with 500 ⁇ g of anti-Sema3A humanized antibody and a negative control group.
- FIG. 18 shows the results of Example 17.
- FIG. 2 is a graph plotting survival curves of a group administered with 500 ⁇ g of anti-Sema3A humanized antibody and a negative control group after 1 hour after inoculating mice with Lipopolysaccharide and inducing an inflammatory disease state by the Kaplan-Meier method.
- FIG. 19 shows the results of Example 18. It is a figure which shows the result of having measured the plasminogen activator inhibitor-1 blood concentration of a mouse
- FIG. 20 shows the results of Example 19.
- FIG. 21 is a diagram showing the results of Example 20. It is a figure which shows the result of having performed cell invasion assay using the human pancreatic cancer cell line (MIAPaCa-2).
- FIG. 22 shows the results of Example 20. It is a figure which shows the result of having performed cell invasion assay using the human glioblastoma cell line (U87MG).
- FIG. 23 shows the results of Example 20. It is a figure which shows the result of having performed cell invasion assay using the mouse-derived lung cancer cell line (3LL).
- FIG. 24 shows the results of Example 20.
- FIG. It is the image which performed the cell invasion assay using the mouse-derived lung cancer cell line (3LL), and observed the stained infiltrating cell under a microscope.
- FIG. 25 shows the results of Example 21.
- FIG. It is the result of having analyzed the influence which Sema3A protein and / or an anti-Sema3A humanized antibody have about the sensitivity of the gemcitabine hydrochloride in a human pancreatic cancer cell (MIAPaCa-2).
- Anti-Sema3A antibody and antibody fragment thereof comprises a heavy chain variable region in which CDR1 to CDR3 have a specific amino acid sequence, and a light chain variable region in which CDR1 to CDR3 has a specific amino acid sequence.
- the anti-Sema3A antibody of the present invention specifically binds to the Sema3A protein and can effectively inhibit the function of the protein.
- the Sema3A protein is encoded by a gene belonging to the class 3 subfamily of semaphorins, and is an endogenous protein identified as a factor that retreats the nerve growth cone and suppresses axon elongation.
- the amino acid sequence of the Sema3A protein is known (GenBank accession number; NP_006071.1 from human, NP_033178.2 from mouse).
- a preferred embodiment of the anti-Sema3A antibody of the present invention includes those comprising the heavy chain variable region and the light chain variable region shown in the following (A) to (E).
- C a heavy chain variable region comprising CDR1 comprising the amino acid sequence shown in SEQ ID NO: 68, CDR2 containing the amino acid sequence shown in SEQ ID NO: 69, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 70; and the amino acid shown in SEQ ID NO: 72
- the anti-Sema3A antibody of the present invention comprises the amino acid sequences of CDR1 to CDR3 (SEQ ID NOs: 1 to 6, 60 to 62, 64 to 66, 68 to 70, 72 to 74, 76 to 78, 80 in the heavy chain and the light chain. -82, 84-86 and 88-90) may have one or several amino acids substituted, deleted, added and / or inserted.
- the number of amino acids to be substituted, deleted, added and / or inserted is not particularly limited, but preferably 1 to 3, more preferably 1 to 2, particularly preferably 1 per CDR.
- one of the amino acid sequences of CDR1 to CDR3 in the heavy chain and the light chain may be substituted, deleted, added and / or inserted into one amino acid sequence. Substitutions, deletions, additions and / or insertions may be made to the above amino acid sequences.
- substitution of amino acids in the amino acid sequence of the CDR substitution with similar amino acids (ie, conservative amino acid substitution) is preferable since it is predicted that no change is caused in the binding activity of the antibody. Specifically, the following classification has been established based on the properties of amino acid side chains.
- Basic amino acids lysine, arginine, histidine
- Acidic amino acids glutamic acid, aspartic acid
- Neutral amino acids glycine, alanine, serine, threonine, methionine, cysteine, phenylalanine, tryptophan, tyrosine, leucine, isoleucine, valine, glutamine, asparagine, proline more
- the neutral amino acid has a polar side chain (asparagine, glutamine, serine, threonine, tyrosine, cysteine), and has a nonpolar side chain (glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine) , Tryptophan), those having amide-containing side chains (asparagine, glutamine), those having sulfur-containing side chains (methionine, cysteine), those having aromatic side chains (pheny It is also possible to classify them into
- Examples of the method for substituting one or several amino acid residues with other amino acids of interest include, for example, site-directed mutagenesis (Hashimoto-Gotoh T. et al., Gene, Vol. 152, p. 271-275). (1995); Zoller MJ. Et al., Methods Enzymol. Vol.100, p.468-500 (1983), Kramer W. et al., Nucleic Acids Res.Vol.12, p.9441-9456 (1984). Kramer W. et al., Methods. Enzymol. Vol.154, p.350-367 (1987); Kunkel TA., Proc Natl Acad Sci USA., Vol.82, p.488.
- the framework region in the variable region and the amino acid sequence of the constant region are not particularly limited as long as they do not substantially affect the binding activity to the Sema3A protein.
- the anti-Sema3A antibody of the present invention may be an avian antibody, preferably a chimeric antibody and a humanized antibody.
- Chimeric antibodies are antibodies in which different regions derived from each other are linked.
- the anti-Sema3A antibody of the present invention when used as a chimeric antibody, when used as a pharmaceutical composition or a therapeutic agent for various diseases, a tri-human chimeric antibody comprising a variable region derived from an avian antibody and a constant region derived from a human antibody or A tri-mouse chimeric antibody composed of a variable region derived from an avian antibody and a constant region derived from a mouse antibody is desirable, and a tri-human chimeric antibody is more preferable.
- a tri-rabbit composed of a variable region derived from an avian antibody and a constant region derived from a rabbit antibody
- a tri-goat chimeric antibody composed of a chimeric antibody or a variable region derived from an avian antibody and a constant region derived from a goat antibody can also be used.
- Examples of the amino acid sequence of the heavy chain variable region used in the tri-human chimeric antibody, tri-mouse chimeric antibody, tri-rabbit chimeric antibody or tri-rabbit chimeric antibody are SEQ ID NOs: 7, 8, 59, 67, 75, and 83.
- Examples of the amino acid sequence of the light chain variable region used in the tri-human chimeric antibody, tri-mouse chimeric antibody, tri-rabbit chimeric antibody or tri-rabbit chimeric antibody include SEQ ID NOs: 9 and 10, 63, 71, 79, The amino acid sequence shown in 87 is mentioned.
- variable regions SEQ ID NOs: 7 to 10, 59, 63, 67, 71, 75, 79, 83, and 87
- One or several amino acids may be substituted, deleted, added and / or inserted.
- the number of amino acids to be substituted, deleted, added and / or inserted is not particularly limited.
- the heavy chain variable region SEQ ID NO: 7, 8, 59, 67, 75, 83
- the light chain variable region In SEQ ID NO: 9, 10, 63, 71, 79, 87), 1 to 21, preferably 1 to 14, and more preferably 1 to 3 may be mentioned.
- substitution, deletion In the heavy chain variable region (SEQ ID NO: 7, 8, 59, 67, 75, 83) and / or the light chain variable region (SEQ ID NO: 9, 10, 63, 71, 79, 87), substitution, deletion,
- the added and / or inserted amino acid is preferably a region other than the amino acid sequences of CDR1 to CDR3.
- the amino acid substitution in the amino acid sequence of the variable region of the heavy chain and light chain is preferably the conservative amino acid substitution described above.
- the humanized antibody is obtained by grafting a non-human-derived CDR sequence onto the framework region of a human antibody.
- the CDR of the non-human-derived antibody, the framework region derived from a human antibody, and the constant region derived from a human antibody An antibody composed of Since the antigenicity in the human body is reduced, the humanized antibody is suitable when the anti-Sema3A antibody of the present invention is used for pharmaceutical use.
- An example of the amino acid sequence of the heavy chain variable region used for the humanized antibody is the amino acid sequence shown in SEQ ID NO: 11.
- Examples of the amino acid sequence of the light chain variable region used in the humanized antibody include the amino acid sequences shown in SEQ ID NOs: 12 and 13.
- variable regions In the amino acid sequences of these variable regions (SEQ ID NOs: 11 to 13), one or several amino acids are substituted, deleted, added and / or inserted if the binding activity to the Sema3A protein is equivalent to that before modification. Also good.
- the number of amino acids to be substituted, deleted, added and / or inserted is not particularly limited.
- 1 to 21 may be mentioned, preferably 1 to 14 and more preferably 1 to 3.
- the amino acids to be substituted, deleted, added and / or inserted are other than the amino acid sequences of CDR1 to CDR3 above. It is desirable that The amino acid substitution in the amino acid sequence of the variable region of the heavy chain and light chain is preferably the conservative amino acid substitution described above.
- a chimeric antibody can be produced, for example, by substituting the constant region of an avian antibody having the heavy chain variable region and the light chain variable region having the amino acid sequences of the CDR1 to CDR3 with a constant region of a human antibody (for example, , Morrison et al., Proc. Natl. Acad. Sci., Vol.81, p.6851-6855 (1984); Neuberger et al., Nature, Vol.312, p.604-608 (1984); al., Nature, Vol. 314, p.452-454 (1985)).
- a human antibody for example, , Morrison et al., Proc. Natl. Acad. Sci., Vol.81, p.6851-6855 (1984); Neuberger et al., Nature, Vol.312, p.604-608 (1984); al., Nature, Vol. 314, p.452-454 (1985)).
- the nucleotide sequence of DNA encoding SEQ ID NO: 7, 8, 59, 67, 75, and 83, which is the amino acid sequence of the heavy chain variable region used in the tri-human chimeric antibody, is represented by SEQ ID NOs: 14, 15, 103, 111, 119. And 127, respectively.
- the nucleotide sequence of DNA encoding SEQ ID NO: 9, 10, 63, 71, 79, and 87, which is the amino acid sequence of the light chain variable region used in the tri-human chimeric antibody is represented by SEQ ID NO: 16, 17, 104, 112. , 120, and 128, respectively.
- Known constant regions of human antibodies can be used.
- a tri-human chimeric antibody can be produced by the following method.
- DNA encoding a tri heavy chain variable region containing CDR having a predetermined amino acid sequence is prepared by chemical synthesis, biochemical cleavage, recombination, or the like.
- the obtained heavy chain variable region DNA is ligated with a human heavy chain constant region DNA and incorporated into an expression vector to prepare a heavy chain expression vector.
- a light chain expression vector is prepared in a similar manner.
- host cells such as HEK293 cell line, CHO cell, and SP2 / 0 cell are cotransformed. After culturing the transformant, the target chimeric antibody is separated from the culture solution of the transformant.
- amino acids in the framework region in the variable region of the antibody may be substituted so that the CDR of the tri-human chimeric antibody forms an appropriate antigen-binding site (Sato, K. et al., Cancer Research, Vol. 53). , P. 851-856 (1993)).
- a humanized antibody can be prepared, for example, by grafting CDR1 to CDR3 containing each of the amino acid sequences onto the framework region of a human antibody (for example, Jones et al., Nature, Vol. 321, p.522). -525 (1986); Riechmann et al., Nature, Vol.332, p.323-327 (1988); Verhoeyen et al., Science, Vol.239, p.1534-1536 (1988)).
- the nucleotide sequences of DNAs encoding heavy chain CDR1 (SEQ ID NOs: 1, 60, 68, 76, and 84) are shown in SEQ ID NOs: 18, 97, 105, 113, and 121, respectively.
- the nucleotide sequences of DNAs encoding heavy chain CDR2 are shown in SEQ ID NOs: 19, 98, 106, 114, and 122, respectively.
- the nucleotide sequences of DNAs encoding heavy chain CDR3 are shown in SEQ ID NOs: 20, 99, 107, 115, and 123, respectively.
- the nucleotide sequences of DNAs encoding the light chain CDR1 are shown in SEQ ID NOs: 21, 100, 108, 116, and 124, respectively.
- the nucleotide sequences of DNAs encoding the light chain CDR2 are shown in SEQ ID NOs: 22, 101, 109, 117, and 125, respectively.
- the nucleotide sequences of DNAs encoding the light chain CDR3 are shown in SEQ ID NOs: 23, 102, 110, 118, and 126, respectively.
- a humanized antibody can be produced by the following method.
- a DNA encoding a heavy chain variable region in which CDRs 1 to 3 having a predetermined amino acid sequence and four human antibody-derived framework regions are linked in a predetermined order is prepared by chemical synthesis, biochemical cleavage, recombination, or the like.
- mutations such as substitution, deletion and / or addition of amino acids in the framework region may be added so that each CDR of the humanized antibody can form an appropriate antigen-binding site (Sato, K. et al. Cancer Research, Vol. 53, p. 851-856 (1993)).
- the obtained heavy chain variable region DNA is ligated with a human heavy chain constant region DNA and incorporated into an expression vector to prepare a heavy chain expression vector.
- a light chain heavy chain expression vector is prepared. Using the obtained heavy chain expression vector and light chain expression vector, host cells such as Free Style 293 cell line (Life Technologies), CHO cells, SP2 / 0 cells and the like are cotransformed. After culturing the transformant, the desired humanized antibody is separated from the culture medium of the transformant.
- SEQ ID NO: 24 shows the base sequence of DNA encoding the amino acid sequence of SEQ ID NO: 11, which is a heavy chain variable region used for humanized antibodies.
- the nucleotide sequences of DNAs encoding the amino acid sequences of SEQ ID NOs: 12 and 13, which are amino acid sequences of the light chain variable region used in the humanized antibody are shown in SEQ ID NOs: 25 and 26, respectively.
- Known constant regions of human antibodies can be used.
- the isotype of the anti-Sema3A antibodies of the present invention is not particularly limited, for example, IgG (IgG 1, IgG 2 , IgG 3, IgG 4), IgA (IgA 1, IgA 2), IgM, IgD, and IgE Is mentioned. Among these, IgG is preferable.
- an antibody fragment thereof can also be used as long as it contains the antigen-binding region of the anti-Sema3A antibody.
- antibody fragments include Fab, Fab ′, F (ab ′) 2 , scFv, dsFv and the like. These antibody fragments can be prepared according to a conventionally known method.
- the anti-Sema3A antibody or antibody fragment thereof of the present invention may be a conjugated antibody or conjugated antibody fragment bound to various compounds such as polyethylene glycol, radioactive substance, and toxin.
- the anti-Sema3A antibody or antibody fragment thereof of the present invention may be modified in the sugar chain to which it is bound, or may be fused with other proteins as necessary.
- compositions comprising the anti-Sema3A antibody or antibody fragment.
- the pharmaceutical composition of the present invention can effectively inhibit the function of the Sema3A protein by the anti-Sema3A antibody or antibody fragment, and can exhibit various pharmacological effects.
- the anti-Sema3A antibody or antibody fragment contained as an active ingredient may be any of the above-described embodiments (A) to (E), but the function of the Sema3A protein can be more effectively achieved.
- the anti-Sema3A antibody or antibody fragment of the embodiment (A) is preferable from the viewpoint of inhibiting and exerting an excellent drug effect.
- the pharmaceutical composition of the present invention only needs to contain an effective amount of the anti-Sema3A antibody or antibody fragment, and may further contain a pharmaceutically acceptable carrier or additive.
- a pharmaceutically acceptable carrier or additive examples include surfactants, excipients, coloring agents, flavoring agents, preservatives, stabilizers, buffering agents, pH buffering agents, disintegrating agents, solubilizing agents, solubilizing agents, Examples include isotonic agents, binders, disintegrants, lubricants, diluents, and flavoring agents.
- the carrier or additive generally used in the pharmaceutical composition can be used suitably.
- the administration form of the pharmaceutical composition of the present invention may be either oral or parenteral, specifically, oral administration; intravenous administration, intramuscular administration, intraperitoneal administration, subcutaneous administration. And parenteral administration such as nasal administration, pulmonary administration, transdermal administration, transmucosal administration, and intraocular administration.
- the formulation form of the pharmaceutical composition of the present invention can be appropriately set according to the dosage form employed.
- it when used for oral administration, it may be prepared into a pharmaceutical form such as powder, granule, capsule, syrup, suspension, etc.
- a pharmaceutical form such as powder, granule, capsule, syrup, suspension, etc.
- parenteral administration liquid, What is necessary is just to prepare in formulation forms, such as suspension, an emulsion, a spray agent, a suppository, and eye drops.
- the pharmaceutical composition of the present invention can effectively inhibit the function of the Sema3A protein by the action of the anti-Sema3A antibody or antibody fragment, it is useful for the prevention and / or treatment of diseases involving the Sema3A protein.
- diseases involving the Sema3A protein include central or peripheral nervous system diseases, autoimmune diseases, inflammatory diseases, infectious diseases, allergic diseases, cancer and the like.
- central or peripheral nervous system diseases include neuropathic pain, spinal cord injury, neurodegenerative diseases (eg, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, advanced nuclear Paralysis, substantia nigra degeneration, Shy-Drager syndrome, olive bridge cerebellar atrophy, spinocerebellar degeneration, etc.).
- neurodegenerative diseases eg, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, advanced nuclear Paralysis, substantia nigra degeneration, Shy-Drager syndrome, olive bridge cerebellar atrophy, spinocerebellar degeneration, etc.
- autoimmune disease include rheumatoid arthritis, type I diabetes, inflammatory bowel disease, Crohn's disease, systemic lupus erythematosus, and multiple sclerosis.
- inflammatory diseases include sepsis, chronic obstructive pulmonary disease, asthma, arthritis, hepatitis, spondyloarthritis, and Sjogren's syndrome.
- infections include bacterial infections, encephalitis / meningitis, endocarditis, hepatitis C, influenza / severe acute respiratory syndrome (SARS), pneumonia, sepsis, burn, traumatic infection Symptoms and the like.
- SARS severe acute respiratory syndrome
- allergic diseases include allergic rhinitis, allergic conjunctivitis, atopic dermatitis, bronchial asthma, food allergy, and the like.
- cancer examples include colorectal cancer, colorectal cancer, lung cancer, breast cancer, brain tumor, melanoma, renal cell cancer, leukemia, lymphoma, T cell lymphoma, gastric cancer, pancreatic cancer, cervical cancer, endometrium
- cancer examples include cancer, ovarian cancer, esophageal cancer, liver cancer, squamous cell carcinoma of the head and neck, skin cancer, urinary tract cancer, prostate cancer, choriocarcinoma, pharyngeal cancer, and laryngeal cancer.
- the anti-Sema3A antibody or antibody fragment can effectively suppress the nerve regeneration inhibitory ability of the Sema3A protein and can promote nerve regeneration and elongation. It is useful as a pharmaceutical composition for the purpose of regeneration elongation (ie, pharmaceutical composition for nerve regeneration elongation).
- the pharmaceutical composition of the present invention is particularly useful as a pharmaceutical composition for preventing and / or treating neurodegenerative diseases because the nerve itself can be regenerated and elongated by the anti-Sema3A antibody or antibody fragment. is there.
- the anti-Sema3A antibody or antibody fragment may be used to treat sepsis or other diseases involving cytokine storms such as graft-versus-host disease (GVHD), acute respiratory distress syndrome (ARDS), avian influenza, smallpox, systemic inflammation syndrome (SIRS), drug-induced cytokine storms, etc., which exhibit excellent preventive and / or therapeutic effects on inflammatory diseases in which the immune mechanism fails due to infection or strong inflammation-induced stimulation, so prevention and / or treatment of these diseases It is also highly useful as a pharmaceutical composition.
- GVHD graft-versus-host disease
- ARDS acute respiratory distress syndrome
- SIRS systemic inflammation syndrome
- drug-induced cytokine storms etc.
- the anti-Sema3A antibody or antibody fragment exerts an action of effectively suppressing the migration / invasion activity of cancer cells induced by Sema3A, and is excellent in prevention and / or prevention of pathological conditions related to cancer exacerbation and progression. Since it shows a therapeutic effect, it is highly useful as a pharmaceutical composition for preventing and / or treating cancer diseases. Moreover, when using the pharmaceutical composition of this invention for the prevention and / or treatment use of cancer, you may use together with another anticancer agent. In particular, the anti-Sema3A antibody or antibody fragment can cancel the anti-cancer agent unresponsiveness of cancer cells induced by Sema3A and restore the drug sensitivity. In combination with cancer drugs, excellent cancer prevention and / or treatment effects can be demonstrated.
- the anti-Sema3A antibody or antibody fragment and the other anticancer agent may be formulated in the same pharmaceutical composition, or The anti-Sema3A antibody or antibody fragment and another anticancer agent may be contained in separate pharmaceutical compositions to be formulated.
- the pharmaceutical composition of the present invention can suppress an increase in blood PAI-1 level by the action of the anti-Sema3A antibody or antibody fragment, it is also effective for the prevention and / or treatment of disseminated intravascular coagulation syndrome.
- Basic diseases associated with disseminated intravascular coagulation syndrome include sepsis, acute leukemia, solid cancer, early placenta detachment, amniotic fluid embolism, trauma, burns, collagen disease, shock, aortic aneurysm, fulminant hepatitis, cirrhosis, acute Examples include pancreatitis, thrombosis, and severe infection.
- the dosage and frequency of administration of the pharmaceutical composition of the present invention vary depending on the dosage form, the age and weight of the patient, the type of disease, the degree of symptoms, etc., and cannot be defined uniformly, but are usually the number for nerve regeneration. Since a period of several months or more is required from the day, it is preferable to administer as many times as necessary to suppress the activity of semaphorin during that period. For example, if an amount corresponding to 0.1 mg to 1000 mg, preferably 1 mg to 500 mg, in terms of weight of the anti-Sema3A antibody or antibody fragment is administered once per administration, the dose may be administered about once every 1 to 30 days. Good.
- a sustained-release preparation can be used, or it can be administered in small amounts over a long period of time using an osmotic pump or the like.
- the present invention provides a method for measuring Sema3A protein using the anti-Sema3A antibody or antibody fragment.
- Sema3A in a sample is immunoassay using an antigen-antibody reaction between the anti-Sema3A antibody or antibody fragment and Sema3A in the sample.
- the anti-Sema3A antibody or antibody fragment reacted with Sema3A in the sample may be any of the above-described aspects (A) to (E), but the Sema3A protein is measured with higher accuracy.
- the anti-Sema3A antibody or antibody fragment of the embodiment (A) is preferable.
- the sample is not particularly limited as long as Sema3A measurement is required.
- Sema3A measurement is required.
- biological specimens such as various tissue extracts.
- the measurement method of the present invention may employ any immunoassay such as a sandwich method, a competition method, and an agglutination method.
- a capture antibody that captures an antigen and a primary antibody that binds to the antigen bound to the capture antibody are used.
- the sandwich method is employed in the measurement method of the present invention, the (A Among the anti-Sema3A antibodies or antibody fragments of the embodiments)) to (E), one embodiment may be used as a capture antibody, and the other embodiment may be used as a primary antibody.
- the anti-Sema3A antibody or antibody fragment of the aspect (A) is used as a capture antibody, and the anti-Sema3A antibody or antibody fragment of the aspect (B) or (C) is used as a primary antibody. It is particularly preferable because it enables measurement of Sema3A protein with higher accuracy, particularly measurement of Sema3A protein in the presence of serum.
- immunoassay includes enzyme immunoassay (ELISA), fluorescent immunoassay, radioisotope immunoassay, etc. depending on the type of label, and any of these is used in the measurement method of the present invention. May be. From the viewpoint of simplicity of measurement and rapidity, an enzyme immunoassay is preferable.
- ELISA enzyme immunoassay
- fluorescent immunoassay fluorescent immunoassay
- radioisotope immunoassay radioisotope immunoassay
- Immunoassay using antigen-antibody reaction is known per se, and the measurement method of the present invention can be performed by a known technique according to the measurement principle of immunoassay and the type of label.
- the present invention also provides a measurement kit used in the measurement method.
- the measurement kit of the present invention contains the anti-Sema3A antibody or antibody fragment.
- the measurement kit of the present invention may further contain other reagents and instruments depending on the measurement principle of immunoassay and the type of label.
- a measurement plate, a chromogenic substrate solution, a reaction stop solution, a washing solution, a standard solution, and the like may be included in addition to the anti-Sema3A antibody or antibody fragment.
- the sandwich method the anti-Sema3A antibody or antibody fragment used as a capture antibody may be provided in a state immobilized on a solid phase.
- Example 1 Preparation of anti-Sema3A antibody (avian antibody, tri-mouse chimeric antibody) 1) Cell culture Cell culture of DT40 cells derived from chicken B cells was performed by the following method. The incubator was a CO 2 thermostatic chamber and cultured at 39.5 ° C. in the presence of 5% CO 2 . The medium used was IMDM medium (Life Technologies), and 10 vol% FBS, 1 vol% chicken serum, penicillin 100 units / mL, streptomycin 100 ⁇ g / mL, 2-mercaptoethanol 55 ⁇ M were used.
- IMDM medium Life Technologies
- Trichostatin A (Wako Pure Chemical Industries, Ltd.) is stocked with DMSO dissolved at 5 mg / mL, and is diluted with a suitable medium so that the final concentration is 1.25 ng / mL or 2.5 ng / mL. Using.
- ADLib Autonomous Diversifying Library
- the cells bound to the antigen-fixed magnetic beads were suspended in 500 ⁇ L medium, added to 20 mL medium, dispensed 200 ⁇ L each into a 96-well plate, and cultured at 39.5 ° C.
- the medium after this operation was IMDM medium (Life Technologies) supplemented with 10 vol% FBS, penicillin 100 units / mL, streptomycin 100 ⁇ g / mL, 2-mercaptoethanol 55 ⁇ M.
- the plate was washed 5 times with 120 ⁇ L of ELISA wash buffer (PBS containing 0.05 wt% Tween 20), and 25 ⁇ L of each of the colony-derived culture supernatants selected by the above 2-2) was added and incubated at room temperature for 1 hour. After washing 5 times with 120 ⁇ L of ELISA washing solution, 25 ⁇ L of secondary antibody diluted 2000 times with blocking buffer was added and incubated at room temperature for 45 minutes. As the secondary antibody, anti-chicken IgM-HRP (BETHYL) was used. After washing 5 times with 120 ⁇ L of ELISA washing solution, 25 ⁇ L of TMB + (Dako) was added and incubated for 30 minutes. Thereafter, the reaction was stopped with 25 ⁇ L of 1N sulfuric acid, and the absorbance at 450 nm was measured.
- ELISA wash buffer PBS containing 0.05 wt% Tween 20
- 25 ⁇ L of secondary antibody diluted 2000 times with blocking buffer was added and incubated
- Each strain is prepared to 1 ⁇ 10 3 cells / mL, 150 ⁇ L is added to 60 mL of medium, and 96 ⁇ l plate (Thermo Fisher Scientific Co.) is sprinkled at 200 ⁇ L / well and left for 7 days. Culture was performed. Twenty clones in which cell colonies were confirmed from each strain were screened for mouse Sema3A antibody-producing clones by the method described in 2-3). For each strain, 2 clones were selected from clones showing positive mouse Sema3A antibody production, and high-density culture was performed.
- each clone was expanded and grown until the number of cells reached 4 ⁇ 10 7 .
- the cells were cultured in CELLine CL-1000 (BD) using AIM-V medium (Life Technologies) containing 20 vol% chicken serum components.
- the chicken serum component is antibody-removed chicken serum.
- immunoglobulin was removed from chicken serum (Life Technologies) with 50% saturated ammonium sulfate as a precipitate, the supernatant was dialyzed against PBS buffer, and the volume increase caused by dialysis was concentrated by Centri Prep (Amicon). It was corrected with. After culturing for about 96 hours, the culture was continued until the survival rate reached 50% or less while measuring the cell survival rate. When the survival rate reached 50% or less, the culture supernatant was collected.
- the culture supernatant whose titer was analyzed by ELISA was prepared as follows in order to remove serum-derived IgM and the like.
- Chicken serum components were added to AIM-V serum-free medium (Life Technologies) at a concentration of 3%.
- Cells were added to a concentration of about 1 ⁇ 10 6 cells / mL, and cultured for 2 days to obtain a culture supernatant.
- a sense primer (primer 1: GAAGATCTAAGCTTGCCATGGCCCTGGCTCCCTCTCCTCCT (SEQ ID NO: 27)
- a sequence of a tri-antibody ⁇ light chain variable region and a sequence of a mouse antibody light chain constant region
- the light chain variable region gene was amplified by PCR using the included antisense primer (Primer 2: TGGCGAAGACTTCCGCTGGCCTAGGAC (SEQ ID NO: 28)).
- a sense primer (primer 3: GAAGATCTAAGCTTACCATGAGCCCACTCG (SEQ ID NO: 29)) containing an avian antibody heavy chain variable region sequence
- an antisense primer (primer 4) containing an avian antibody heavy chain variable region region and a mouse IgG2a antibody constant region.
- the heavy chain variable region gene was amplified by PCR using CGATGGGGCTGTTGTTTTGGCGGAGGAGACGATGAACTTC (SEQ ID NO: 30)).
- a sense primer (primer 5: AAGTCTCCGCATCATGCTCCCCTGTTCCA (SEQ ID NO: 31) containing the sense primer sequence of the avian antibody ⁇ light chain variable region and the sequence of the mouse antibody ⁇ light chain constant region. )
- an antisense primer (primer 6: TATGCGGCCGCTTACTAGGAACAGTCA (SEQ ID NO: 32) was used to amplify the mouse light chain constant region gene.
- a sense primer sequence containing the sense antibody sequence of the tri-antibody heavy chain variable region and the mouse IgG2a antibody heavy chain constant region sequence (Primer 7: GCCAAAAACAAGCCCCCATCGTCTCATCCACTGGGCCCCT (SEQ ID NO: 33))
- the mouse heavy chain constant region gene was amplified by PCR using an antisense primer (Primer 8: AGATAGCGGCCGCTTATCATTTACCCGG (SEQ ID NO: 34)).
- the tri-mouse chimeric antibody light chain gene was amplified by PCR using the amplified light chain variable region fragment and the light chain constant region fragment as a template and primers 1 and 6. Further, the tri-mouse chimeric antibody heavy chain gene was amplified by PCR using the amplified heavy chain variable region fragment and heavy chain constant region fragment as a template and primers 3 and 8.
- the tri-mouse chimeric antibody light chain gene and the tri-mouse chimeric antibody heavy chain gene were treated with restriction enzymes HindIII and NotI, respectively, and then cloned into the HindIII-NotI site of the mammalian cell expression plasmid pCEP4 (Life Technologies).
- the cloned antibody gene sequence was confirmed by sequencing using a DNA sequencer. Based on the determined nucleotide sequence, the amino acid sequences of the tri-mouse chimeric antibody light chain and the tri-mouse chimeric antibody heavy chain were translated.
- the amino acid sequence of the finally determined tri-mouse chimeric antibody heavy chain is shown in SEQ ID NO: 35, and the amino acid sequence of the finally determined tri-mouse chimeric antibody light chain is shown in SEQ ID NO: 36.
- the above-prepared plasmid was introduced into FreeStyle293 cell line (Life Technologies) by transfection using polyethyleneimine, and cultured for 7 days at 37 ° C., 8% CO 2 , shaking at 135 rpm. The supernatant was collected.
- the concentration of anti-Sema3A tri-mouse chimeric antibody (IgG) contained in the culture supernatant was quantified by ELISA. From this culture supernatant, anti-Sema3A tri-mouse chimeric antibody was purified using a column prepared by filling Protein G Sepharose 4 Fast Flow (GE Healthcare) into Poly-Prep Chromatography Columns (BIO-RAD). did. The solvent was replaced with PBS buffer using a PD-10 column (GE Healthcare).
- the specificity of the purified antibody for the antigen was measured by ELISA using PBS buffer. 2.5 ⁇ g / mL of the antigen was dispensed into 96 Well Maxi Sorp Plate (Nunc) and reacted at 4 ° C. overnight, and the plate was coated with the antigen (mouse Sema3A protein). In order to examine the specificity of the antibody, ovalbumin, rabbit IgG, and skim milk were also immobilized on the plate in the same manner as a negative control.
- the plate was washed 3 times with 200 ⁇ L of a washing solution (PBS containing 0.05% Tween 20), 200 ⁇ L of a blocking solution (PBS containing 0.5% skim milk) was added, and the mixture was reacted for 30 minutes.
- the plate was washed 3 times with 200 ⁇ L of the washing solution, and 100 ⁇ L of the culture supernatant containing the antibody was added and allowed to react for 1 hour.
- the plate was washed 5 times with 200 ⁇ L of the washing solution, and 100 ⁇ L of horseradish peroxidase (HRP) -labeled anti-mouse IgG2a goat antibody (BETHYL) diluted 2000-fold with PBS buffer was added and reacted for 1 hour.
- HRP horseradish peroxidase
- BETHYL horseradish peroxidase
- the plate was washed 5 times with 200 ⁇ L of the washing solution, 100 ⁇ L of TMB + (Dako) was added, color reaction was performed for 5 minutes, and 100 ⁇ L of 1M sulfuric acid was added to stop the reaction. Absorbance at 450 nm was measured using a ⁇ Quant Biomolecular Spectrometer (Bio-Tek Instruments). The results are shown in FIG.
- CDR sequence of the anti-Sema3A tri-mouse chimeric antibody obtained above was determined.
- the CDR sequence was determined according to the method by Kabat et al. (Sequences of Proteins of Immunological Interest, NIH publication, 91-3242). As a result, the following sequences were identified as the amino acid sequences of CDRs 1 to 3 of the heavy chain and the light chain.
- Example 2 Preparation of anti-Sema3A antibody (humanized antibody, tri-human chimeric antibody) 1) Construction of humanized antibody (Humanized-1, Humanized-2) gene Clone No. 1 obtained in 4) of Example 1 above.
- the design of the two humanized antibodies (Humanized-1 and Humanized-2) was made by changing the framework region of the variable region of the anti-Sema3A antibody produced by strain 4-2 to the amino acid of the framework region of the human antibody. I did it.
- the genes encoding the designed heavy chain (Humanized-1 and 2) and the light chain amino acid sequence of Humanized-1 were synthesized in consideration of codon optimization for expression in human cells.
- the light chain of Humanized-2 is a sense primer (primer A ⁇ : GAAGATCTAAGCTTCCCACTCATGGCAT (SEQ ID NO: 37)) and an antisense primer (primer B ⁇ : TGTTAATAGATCACTGTCACGGGGA (SEQ ID NO: 38)) using the light chain gene sequence of Humanized-1 as a template.
- the product amplified by PCR with the sense primer (primer C ⁇ : TCCCGTGACAGTGATCTTATACAA (SEQ ID NO: 39)) and the antisense primer (primer D ⁇ : AGATAGCGGCCGCTTAGGAACATTC (SEQ ID NO: 40)) were used as the sense primer A ⁇ and the antisense primer D ⁇ . It was synthesized by assembling PCR.
- a tri-human chimeric antibody gene was constructed as follows. Clone No. obtained in 4) of Example 1 above. 4-2 Using the cDNA of strain 4-2 as a template, a sense primer (primer E ⁇ : GAAGATCTAAGCTTCCCACCCATGCCCTGGCTCCTCT (SEQ ID NO: 41)) containing the sequence of the avian antibody ⁇ light chain variable region and the sequence of the avian antibody ⁇ light chain variable region and human antibody ⁇ light chain constant The light chain variable region gene was amplified by PCR using an antisense primer containing a normal region sequence (primer F ⁇ : CTTTGGGCTGGCCTAGGACGGGTCAGGGTTGT (SEQ ID NO: 42)).
- a sense primer (primer G ⁇ : GAAGATCTAAGCTTCCCACCATGAGCCCACTCG (SEQ ID NO: 43)) containing an avian antibody heavy chain variable region sequence
- an antisense primer (primer H ⁇ : GCCCCTTTGTTACTAGCGGAGAGAGACGATGAACTTC) containing a human antibody IgG1 constant region.
- the heavy chain variable region gene was amplified by PCR using (SEQ ID NO: 44).
- a sense primer (primer I ⁇ : GGCCAGCCCAAAGCCACACCTACCGTG (SEQ ID NO: 45)) containing the sequence of the human antibody ⁇ light chain constant region, using the synthesized humanized ⁇ light chain constant region DNA sequence as a template, and antisense
- the human ⁇ light chain constant region gene was amplified by PCR using a primer (primer J ⁇ : AGATAGCGGCCGCTTATAGGAACATTCGGTT (SEQ ID NO: 46)).
- a sense primer (primer K ⁇ : GCTAGTACAAAGGGGCCCCTCAGTGTTCCCACTTG (SEQ ID NO: 47)) containing a human IgG1 antibody heavy chain constant region DNA sequence as a template and a synthesized humanized human IgG1 antibody heavy chain constant region DNA sequence
- the human IgG1 heavy chain constant region gene was amplified by PCR using (primer L ⁇ : AGATAGCGGCCGCTTTATTTTTCCAGGTGAGAG (SEQ ID NO: 48)).
- the Trie human chimeric antibody light chain gene was amplified by PCR using primer E ⁇ and primer J ⁇ . Further, the trie human chimeric antibody heavy chain gene was amplified by PCR using the amplified heavy chain variable region fragment and heavy chain constant region fragment as a template and using the primer G ⁇ and the primer L ⁇ .
- the amino acid sequence of the heavy chain of the finally determined humanized antibodies (Humanized-1 and Humanized-2) is shown in SEQ ID NO: 49, and the base sequence encoding the amino acid sequence is shown in SEQ ID NO: 50.
- the amino acid sequence of the heavy chain (SEQ ID NO: 49) includes the amino acid sequence of the variable region shown in SEQ ID NO: 11.
- the amino acid sequence of the light chain of the finally determined humanized antibody (Humanized-1) is shown in SEQ ID NO: 51
- the base sequence encoding the amino acid sequence is shown in SEQ ID NO: 52.
- the amino acid sequence of the light chain (SEQ ID NO: 51) includes the amino acid sequence of the variable region shown in SEQ ID NO: 12.
- amino acid sequence of the light chain of the finally determined humanized antibody (Humanized-2) is shown in SEQ ID NO: 53, and the base sequence encoding the amino acid sequence is shown in SEQ ID NO: 54.
- the amino acid sequence of the light chain (SEQ ID NO: 53) includes the amino acid sequence of the variable region shown in SEQ ID NO: 13.
- amino acid sequence of the heavy chain of the finally determined tri-human chimeric antibody is shown in SEQ ID NO: 55, and the base sequence encoding the amino acid sequence is shown in SEQ ID NO: 56.
- the amino acid sequence of the heavy chain includes the amino acid sequence of the variable region shown in SEQ ID NO: 8.
- amino acid sequence of the light chain of the finally determined tri-human chimeric antibody is shown in SEQ ID NO: 57, and the base sequence encoding the amino acid sequence is shown in SEQ ID NO: 58.
- the amino acid sequence of the light chain includes the amino acid sequence of the variable region shown in SEQ ID NO: 10.
- the expression plasmid prepared above was introduced into the FreeStyle293 cell line (Life Technologies) by transfection using polyethyleneimine, and cultured for 7 days at 37 ° C., 8% CO 2 , shaking culture at 135 rpm. Thereafter, the culture supernatant was collected.
- An anti-Sema3A humanized antibody (Humanized-1) was prepared from a column prepared by filling Poly-Prep Chromatography Columns (BIO-RAD) with Protein A Sepharose 4 Fast Flow (GE Healthcare) from this culture supernatant. And Humanized-2) and Trihuman chimeric antibodies were purified. The solvent was replaced with PBS buffer using a PD-10 column (GE Healthcare).
- the molecular extinction coefficient was calculated from the amino acid composition of the purified antibody, and the concentration was determined by the ultraviolet absorption method. 500 ng of the purified antibody was electrophoresed in a reduced or non-reduced state, and the gel was stained with CBB (Coomassie Brilliant Blue). The results are shown in FIG. FIG. 3 also shows the results of electrophoresis and CBB staining of the tri-mouse antibody obtained in Example 1 and the CL18M (+) tri-mouse chimeric antibody (control) in the same manner.
- Example 3 Production of Anti-Sema3A Antibody (Tri-Mouse Chimeric Antibody) 1 described in Example 1 except that DT40 cells in which human Sema3A protein was used as an antigen and chicken IgM constant region was substituted with mouse IgG were used. ) And 2), antibody screening was performed to obtain 8 clones showing positive anti-Sema3A antibody production.
- antibody screening was performed to obtain 8 clones showing positive anti-Sema3A antibody production.
- anti-Sema3A tri-mouse chimeric antibody IgG
- the heavy chain and light chain variable regions and CDR sequences of each anti-Sema3A tri-mouse chimeric antibody were determined.
- the CDR sequence was determined according to the method by Kabat et al. (Sequences of Proteins of Immunological Interest, NIH publication, 91-3242). The obtained results are shown in Tables 2 and 3.
- Example 4 Production of anti-Sema3A antibody (avian antibody, tri-human chimeric antibody) 1) Production of avian antibody (IgM) Using mouse Sema3A protein as an antigen in the same manner as in 1) and 2) described in Example 1
- Anti-Sema3A avian antibody is obtained by screening for antibodies, obtaining clones showing positive anti-Sema3A antibody production, using 240-40 and 255-72 strains among them, and producing and purifying anti-Sema3A antibodies (From clone No. 240-40 and clone No. 255-72).
- the heavy and light chain variable regions and CDR sequences of each anti-Sema3A avian antibody were determined.
- the CDR sequence was determined according to the method by Kabat et al. (Sequences of Proteins of Immunological Interest, NIH publication, 91-3242). The results obtained are shown in Tables 4 and 5.
- RNA was extracted from anti-Sema3A antibody-producing clones 240-40 and 255-72, and cDNA was obtained by reverse transcription using reverse transcriptase (SuperScriptIII, Life Technologies). Was synthesized.
- a sense primer (primer M ⁇ : AGCTGTCTAGCGGCCCACCCATGGCCTGGGCTCCTCTC (SEQ ID NO: 91)
- an antisense primer (primer N ⁇ : TCTGCGCGCGCGTAGACTCACCTAGGGGTTGTC) (sequence number using PCR)
- PCR using a sense primer (primer O ⁇ : AGCTTGCTAGCGGCCCACCATGAGCCCACTCGTCCTCC (SEQ ID NO: 93)
- an antisense primer (primer P ⁇ : TCTGGCGGCCCGTAGTAGACTCACCGAGGGAGACGATGAACTTC (SEQ ID NO: 94)) containing the sequence of the avian antibody heavy chain variable region was used.
- the chain variable region gene was amplified.
- SEQ ID NO: 95 for the heavy chain constant region and SEQ ID NO: 96 for the light chain constant region was prepared by total gene synthesis, and both were subjected to NotI treatment and EagI treatment, followed by vector pCEP4 (Life Technologies) ) To the NotI site.
- variable region amplified above was cloned into the vector containing the constant region prepared as described above. Specifically, both the heavy chain constant region vector and the light chain constant region vector were treated with NotI, and the heavy chain variable region cDNA and the light chain variable region cDNA were respectively obtained using In-fusion HD Cloning Kit (Takara Bio Inc.). Connected.
- the plasmid prepared above was introduced into the FreeStyle293 cell line (Life Technologies) by transfection using polyethyleneimine, and cultured with shaking at 37 ° C., 8% CO 2 , 135 rpm / min.
- Protein A anti-Sema3A tri-human chimeric antibody (IgG1) (derived from clone No. 240-40 and clone No. 255-72) was obtained.
- Example 5 Measurement of specificity for antigen About the tri-mouse chimeric antibody obtained in Example 1, and the humanized antibodies (Humanized-1 and Humanized-2) and the tri-human chimeric antibody obtained in Example 2. Specificity for the antigen was determined by antigen solid phase ELISA using PBS buffer. In addition, the antigen-specificity of the tri-mouse chimeric antibody obtained in Example 1 labeled with biotin was also measured. Further, as a negative control, the specificity to the antigen was similarly measured for CL18M (+) tri-mouse chimeric antibody. Specific measurement conditions are as follows.
- the plate was washed 3 times with 50 ⁇ L of the washing solution, 20 ⁇ L of each antibody was added, and the mixture was reacted for 1 hour. Washed 5 times with 50 ⁇ L of the washing solution, horseradish peroxidase (HRP) -labeled anti-human ⁇ light chain goat antibody (SouthernBiotech) diluted 4000 times with PBS buffer, or horseradish peroxidase (HRP) -labeled 20 ⁇ L of anti-mouse IgG2a goat antibody (BETHYL) diluted 2000 times with PBS buffer, horseradish peroxidase (HRP) labeled streptavidin (Thermo) diluted 10,000 times with PBS buffer For 1 hour.
- HRP horseradish peroxidase
- BETHYL horseradish peroxidase labeled streptavidin
- the plate was washed 5 times with 50 ⁇ L of the washing solution, 20 ⁇ L of TMB + (Dako) was added, and the color reaction was carried out for 20 minutes. Absorbance at 450 nm was measured using Infinite M1000 (TECAN).
- Example 6 Measurement of affinity for antigen-1 Clone No. obtained in Example 1
- the affinity for the antigen was evaluated by competitive ELISA.
- CL18M (+) tri-mouse antibody was similarly measured for affinity for the antigen. Specific measurement conditions are as follows.
- Humanized antibodies (Humanized-1 and Humanized-2), tri-human chimera antibody, and tri-mouse chimera antibody were each serially diluted 2-fold to obtain a biotin-labeled anti-Sema3A tri-mouse chimera antibody at a final concentration of 60 ng / mL. Then, the mixture was reacted with an antigen solid phase plate washed 3 times with 50 ⁇ L of washing solution for 1 hour. The plate was washed 5 times with 50 ⁇ L of the washing solution, 20 ⁇ L of horseradish peroxidase (HRP) -labeled streptavidin (Thermo) diluted 10000-fold with PBS buffer was added, and reacted for 30 minutes.
- HRP horseradish peroxidase
- streptavidin Thermo
- the plate was washed 5 times with 50 ⁇ L of the washing solution, 20 ⁇ L of TMB + (Dako) was added, and the color reaction was carried out for 20 minutes. Absorbance at 450 nm was measured using Infinite M1000 (TECAN).
- Table 6 shows the results of calculating the 50% inhibitory concentration IC 50 ( ⁇ g / mL) for the antigen-antibody reaction of the anti-Sema3A tri-mouse chimeric antibody for each antibody.
- IC 50 50% inhibitory concentration
- Example 7 Measurement of affinity for antigen-2 Clone No. obtained in Example 1 4-2 derived tri-mouse chimeric antibody, and humanized antibody (Humanized-1 and Humanized-2) and tri-human chimeric antibody obtained in Example 2 were tested against the antigen using BIACORE (GE Healthcare). Affinity was evaluated. Specific conditions are as follows.
- the binding time was 3 minutes, the divergence time was 4 minutes, and the sensorgram obtained from the reaction was analyzed with Langmuir binding model (BIAevaluation Version 4.1) (GE Healthcare) to calculate the KD value (dissociation constant). .
- the KD value for human Sema3A was 3 to 4 nM for humanized antibodies (Humanized-1 and Humanized-2), and 14 to 15 nM for tri-human chimeric antibodies and tri-mouse chimeric antibodies.
- the KD values for mouse Sema3A were 4-5 nM for humanized antibodies (Humanized-1 and Humanized-2), and 17-18 nM for tri-human chimeric antibody and tri-mouse chimeric antibody. Both humanized-1 and humanized-2 humanized antibodies had lower KD values for Sema3A than the chimeric antibodies, indicating that the affinity was improved by humanization.
- Example 8 Measurement of Human Sema3A by Sandwich ELISA Using the anti-Sema3A antibody obtained in Examples 2 to 4, human Sema3A was measured by sandwich ELISA. Specific conditions are as follows.
- the capture antibodies shown in Table 7 were diluted to 1/2000, dispensed into 384 Well Maxi Sorp Plate (Nunc) and reacted overnight at 4 ° C., and the plates were coated with the capture antibodies. After the reaction, each well was washed 3 times with a washing solution (PBS containing 0.05% Tween 20, pH 7.2), 45 ⁇ L of blocking solution (PBS containing 1% bovine serum albumin, pH 7.2) was added, and 2 ml at room temperature was added. Reacted for hours. Subsequently, it was washed 3 times with a washing solution (PBS containing 0.05% Tween 20, pH 7.2), and 20 ⁇ L of each antibody was added and allowed to react for 1 hour.
- a washing solution PBS containing 0.05% Tween 20, pH 7.2
- test samples recombinant human Sema3A-Fc chimeric protein (R & D systems, # 1250-S3-02J) serially diluted with PBS, or recombinant human Sema3A-Fc chimeric protein (R & D systems, # 1250- S3-02J) was serially diluted with human serum type AB (Sigma, # H4522) diluted 5-fold with PBS.
- HRP horseradish peroxidase
- GE # NA931 horseradish peroxidase
- PBS pH 7.2
- bovine serum albumin a washing solution
- 20 ⁇ L of TMB Dako, # S1599 was added, and a color reaction was performed for 20 minutes.
- the reaction was stopped by adding 20 ⁇ L of 1N sulfuric acid. Absorbance at 450 nm was measured using Infinite M1000 (TECAN).
- the plate was washed 5 times with a washing solution (PBS containing 0.05% Tween 20, pH 7.2). Thereafter, 20 ⁇ L of TMB (Dako, # S1599) was added, and a color reaction was performed for 20 minutes. The reaction was stopped by adding 20 ⁇ L of 1N sulfuric acid. Absorbance at 450 nm was measured using Infinite M1000 (TECAN).
- FIG. 6 shows the relationship between the antigen (Sema3A) and OD450 (absorbance at 450 nm) as a result of measuring test samples that do not contain serum under conditions 1 to 4 and 8.
- FIG. 7 shows the relationship between the antigen (Sema3A) and OD450 (absorbance at 450 nm) as a result of measuring test samples containing serum under conditions 1 and 2. From these results, it was revealed that Sema3A can be measured by ELISA using the anti-Sema3A antibody obtained in Examples 1 to 4.
- Example 2 humanized antibody (Humanized-2) obtained in Example 2 was used as a capture antibody, and the tri-mouse chimeric antibody obtained in Example 3 (derived from clone No. 165 and clone No. 165) was used as a primary antibody. 582 strain derived) (Conditions 1 and 2), it was also confirmed that Sema3A in serum could be measured with high accuracy and that Sema3A in biological samples could be measured. On the other hand, when a commercially available anti-Sema3A antibody was used (condition 9), Sema3A could not be detected.
- Example 9 Collapse assay-1 1) Test method The dorsal root ganglion (DRG) of a chicken 7-day embryo was removed. The excised DRG was statically cultured at 37 ° C. for 16-20 hours in a PLL / laminin-coated 24-well dish containing 250 ⁇ L of NGF-containing medium, and it was confirmed that NGF-sensitive DRG neurons were extended. Separately, chicken-derived Sema3A protein (3 nM) and the anti-Sema3A avian antibody (culture supernatant) obtained in Example 1 (antibody concentration 10 ⁇ g / mL) or anti-Sema3A avian-mouse chimeric antibody (10 ⁇ g / mL).
- DRG dorsal root ganglion
- the containing mixture was preincubated on ice for 30 minutes. This mixed solution was added to the 24-well dish so that the final concentrations of chicken-derived Sema3A protein were 0, 0.1, and 0.3 nM, followed by static culture at 37 ° C. for 30 minutes. Next, after fixing with a 3.7% formalin solution, the growth cone formed at the tip of the axon of cultured neurons was visualized with Alexa488-conjugated Phalloidin. Under the fluorescence microscope, the ratio (%) of neurons in which the growth cone was collapsed among all NGF-sensitive DRG neurons was determined.
- anti-Sema3A avian antibody or anti-Sema3A tri-mouse chimeric antibody an anti-rabbit IgG antibody or mouse IgG was used, and a regression assay was performed in the same manner as described above.
- Example 10 Collapse assay-2 1) Test method A regression assay was performed in the same manner as in Example 9 except that human-derived Sema3A protein was used instead of chicken-derived Sema3A protein.
- Example 11 Collapse assay-3 1) Test method The dorsal root ganglion (DRG) of a chicken 7-day embryo was removed. The excised DRG was statically cultured at 37 ° C. for 16-20 hours in a PLL / laminin-coated 24-well dish containing 250 ⁇ L of NGF-containing medium, and it was confirmed that NGF-sensitive DRG neurons were extended. Separately, a mixture containing human-derived Sema3A protein (3 nM) and anti-Sema3A humanized antibody (Humanized-2) (culture supernatant) obtained in Example 2 (100 ⁇ g / mL as the antibody concentration) on ice was mixed with 30 ml. Pre-incubated for minutes.
- DRG dorsal root ganglion
- This mixed solution was added to the 24-well dish so that the final concentration of the human-derived Sema3A protein was 0, 0.05, 0.1, and 0.3 nM, followed by static culture at 37 ° C. for 30 minutes.
- the growth cone formed at the tip of the axon of cultured neurons was visualized with Alexa488-conjugated Phalloidin. Under the fluorescence microscope, the ratio (%) of neurons in which the growth cone was collapsed among all NGF-sensitive DRG neurons was determined.
- a regression assay was performed in the same manner as described above using Human IgG-lambda instead of the anti-Sema3A humanized antibody.
- FIG. 11 shows the results of the regression assay using the anti-Sema3A humanized antibody.
- the anti-Sema3A humanized antibody has an action of suppressing the regression of the growth cone by the Sema3A protein. That is, it has been clarified that an anti-Sema3A antibody having CDRs 1 to 3 having a specific amino acid sequence can effectively suppress the ability of Sema3A protein to induce growth cone retraction even when humanized.
- Example 12 Binding Assay for Human Sema3A 1) Test Method COS-7 cells transformed to express wild-type COS-7 cells or EGFP (Enhanced Green Fluorescent Protein) -fused NRP1 (Neuropilin-1) (NRP1) Expression COS-7 cells) were prepared.
- EGFP Enhanced Green Fluorescent Protein
- NRP1 Neuroropilin-1
- alkaline phosphatase-conjugated human-derived Sema3A protein 0.1 nM
- avian anti-Sema3A antibody culture supernatant obtained in Example 1
- antibody concentration 10 ⁇ g / mL or anti-Sema3A tri-mouse chimeric antibody (10 ⁇ g) / Ml
- 500 ⁇ L of this medium is preliminarily containing HBH buffer (20 mM sodium HEPES, pH 7.00, 0.05% by volume BSA, and 10% by volume FBS).
- the anti-Sema3A antibody having CDRs 1 to 3 having a specific amino acid sequence can bind to the human-derived Sema3A protein and inhibit the binding between the human-derived Sema3A protein and NRP1.
- Example 13 Binding assay for human Sema3F 1) Test method COS-7 cells transformed to express EGFP-fused NRP2 (Neuropilin-2) instead of NRP1-expressing COS-7 cells (NRP2-expressing COS-7 cells) ) And the alkaline phosphatase-conjugated human-derived Sema3A protein was used in place of the alkaline phosphatase-conjugated human-derived Sema3A protein, and a binding assay was performed in the same manner as in Example 12 above.
- Example 14 Analysis of effect of anti-Sema3A antibody on cognitive function 1) Test material 1-1) A ⁇ (25-35) and A ⁇ (35-25) A ⁇ (25-35) (Amyloid ⁇ -Protein (25-35)) (Bachem, # H-1192) and A ⁇ (35-25) (Amyloid ⁇ -Protein (35-25)) (Bachem, # H-2964 ) was adjusted to 1 mg / mL with distilled water and incubated at 37 ° C. for 4 days to prepare an A ⁇ (25-35) solution and an A ⁇ (35-25) solution. By this operation, A ⁇ (25-35) aggregates and acquires cytotoxicity.
- a ⁇ (35-25) is an inactive A ⁇ obtained by reversing the amino acid sequence of the active A ⁇ 25-35 and does not exhibit cytotoxicity even after the above operation. A ⁇ (35-25) was used as a negative control.
- Anti-Sema3A Antibody The anti-Sema3A tri-mouse chimeric antibody prepared in Example 1 was prepared at 1 mg / mL with physiological saline to prepare an anti-Sema3A antibody solution.
- normal IgG 1 mg / mL normal mouse IgG obtained from Calbiochem was used as the normal IgG solution.
- mice Three-week-old mice (C57BL / 6J) purchased from Charles River Japan were used.
- NERT novel object recognition task
- a 3-week-old mouse was prepared, and handling work was carried out by placing the mouse on the experimenter's hand for 10 minutes a day for 2 weeks so that the mouse would get used to the experimenter.
- the mice were divided into 5 groups shown in Table 8, and A ⁇ and antibody were administered into the ventricle under the conditions shown in Table 8. These solutions were administered 1 mm below the right from bregma.
- a Teflon tube (Eicom) was connected to the tip of a microsyringe, and a 27 G injection needle (Terumo) with a 3.3 mm needle tip was inserted at the tip was used.
- mice Three days after intraventricular administration, the mice were transferred to a 35 cm ⁇ 35 cm ⁇ 35 cm test cage and habituated to the test cage for 10 minutes.
- the objects A and B were placed at predetermined positions in the test cage, and the time for accessing each object during 10 minutes was measured (acquisition trial). At this time, the time when the distance between the object and the nose tip was 1 cm or less was measured as the access time. Further, on the next day (5 days after administration), the object B was replaced with the new object C, and the access time during the 10-minute trial was measured (test trial).
- the objects A, B, and C were different in shape and color. Specific shapes and colors of the objects A, B, and C are as follows.
- Object A A base portion (green) extending in the horizontal direction and an extension portion (green) extending vertically from the center thereof are formed in a T-shape when viewed from the front.
- the shapes of the base and the extension are both cuboids.
- Object B Same as Object A except that the base is yellow and the extension is cylindrical and red.
- Object C Same as Object A, except that the base is yellow and the extension is yellow with the shape cut out in a circular arc when viewed from the side.
- the bases of the objects A, B, and C were placed on the bottom surface of the test cage, and the extensions were installed so as to extend upward.
- the access rate to the object B of all groups was approximately 50%, and no significant difference was recognized between any groups. Therefore, it was shown that no impairment such as cognitive function was observed, and evaluation for memory acquisition in this experiment was possible.
- the access rate to the object C was about 75% in the Intact group and the A ⁇ (35-25) administration group (negative control).
- the access rate to the object C was limited to about 50%, and a decrease in the preference for the object C was recognized with a significant difference compared to the intact group (p ⁇ 0. 0001, vs. Intact group). This result indicates that the memory for object A was not acquired due to memory impairment due to administration of A ⁇ (25-35).
- the access rate to the object C is about 75%
- the A ⁇ (25-35) administration group and A ⁇ (25 ⁇ 35) Significant recovery compared to the + normal IgG administration group (p ⁇ 0.0001 vs. A ⁇ (25-35) administration group or A ⁇ (25-35) + normal IgG administration group).
- the anti-Sema3A antibody having a CDR of a specific amino acid sequence has an effect of inhibiting the induction of memory impairment caused by A ⁇ .
- An increase in Sema3A protein expression has already been observed in the postmortem brain of Alzheimer's disease patients, and Collaspin Response Mediator Protein (CRMP), which undergoes phosphorylation modification in the Sema3A signaling pathway, is one of Alzheimer's lesions. It is known that the fiber is highly phosphorylated in CRMP. Therefore, such a pharmacological effect is considered that the anti-Sema3A antibody blocks the Sema3A-CRMP signal.
- CRMP Collaspin Response Mediator Protein
- Example 15 Analysis of the effect of anti-Sema3A tri-mouse chimeric antibody on immune / inflammatory diseases
- a Lipopolysaccharide inoculation model which is a pseudo-pathological model of sepsis
- Test material 1-1) Lipopolysaccharide Lipopolysaccharide (hereinafter LPS, Sigma, Lot number: 032M4082V) was prepared with physiological saline to a concentration of 6 mg / mL.
- Tri-mouse anti-Sema3A antibody The anti-Sema3A tri-mouse chimeric antibody prepared in Example 2 was diluted to 5 mg / mL with physiological saline to obtain an anti-Sema3A antibody solution.
- Non-specific tri-mouse chimeric antibody (IgG) (negative control chimeric antibody) prepared using an antibody library constructed from DT40 cells derived from chicken B cells, The solution was diluted to 5 mg / mL with saline to obtain a negative control chimeric antibody solution.
- mice Male 6-week-old mice (C57BL / 6J) purchased from Charles River Japan were used.
- Results Table 10 shows the results obtained.
- Example 16 Analysis of the effect of an anti-Sema3A humanized antibody on immune / inflammatory diseases The effect of the anti-Sema3A humanized antibody obtained in Example 2 on an LPS-induced inflammatory disease model was analyzed in the same manner as in Example 12. did.
- Test material 1-1) LPS LPS was prepared in the same manner as in Example 15.
- Anti-Sema3A humanized antibody The anti-Sema3A humanized antibody (Humanized-2) prepared in Example 2 was diluted with physiological saline to obtain an anti-Sema3A humanized antibody solution.
- an anti-Sema3A humanized antibody (Humanized-2) prepared to a concentration of 5 mg / mL was used, and in the anti-Sema3A humanized antibody 100 ⁇ g administration group, the anti-Sema3A humanized antibody (Humanized antibody) was used.
- -2) prepared to a concentration of 1 mg / mL was used.
- Human antibody for negative control group A non-specific human polyclonal antibody (human IgG POLYCLONAL Isotype Control; BioXCell, # BE0092) was diluted to 5 mg / mL with physiological saline, and a human antibody solution for negative control It was.
- mice Male 6-week-old mice (C57BL / 6J) purchased from Charles River Japan were used.
- Example 3 Statistical analysis Similar to Example 12, the unilateral Cochran-Armitage test was used for the ineffective and effective binarized survival rates, and three possible dose response types, namely linear increase, low dose saturation The dose response of the effect of improving the survival rate was determined by setting the contrast of the high dose rising type. In addition, the survival time extension effect was determined by applying a log-rank test.
- Results Table 12 shows the survival rate of each group.
- the survival rate 60%) was twice that of the negative control group (30%), and in the 500 ⁇ g group, the survival rate (80%) exceeded that of the 100 ⁇ g group. Observed. Survival was improved at both doses of anti-Sema3A humanized antibody and a dose-dependent trend was observed.
- the anti-Sema3A humanized antibody also has the effect of suppressing the LPS-induced lethal inflammatory pathology and improving the survival rate, like the anti-Sema3A tri-mouse chimeric antibody shown in Example 15. Is clearly shown.
- FIG. 17 shows the survival rate curve of the 500 ⁇ g administration group in which a clear survival rate improving effect was observed.
- p 0.0303
- Example 17 Analysis of the effect of anti-Sema3A humanized antibody on lethal inflammatory disease after induction of pathology Based on the results obtained in Example 16, the effect of anti-Sema3A humanized antibody was compared with that of LPS. The effect of treatment after inoculation and induction of pathology was determined.
- Test material 1-1) LPS LPS was prepared in the same manner as in Example 15.
- Anti-Sema3A humanized antibody The anti-Sema3A humanized antibody (Humanized-2) prepared in Example 2 was diluted with physiological saline to obtain an anti-Sema3A humanized antibody solution.
- Anti-Sema3A humanized antibody 500 ⁇ g, 250 ⁇ g, and 125 ⁇ g administration groups were prepared using anti-Sema3A humanized antibody (Humanized-2) prepared at concentrations of 5, 2.5, and 1.25 mg / mL, respectively.
- mice Male 6-week-old mice (C57BL / 6J) purchased from Charles River Japan were used.
- Dose response types that can be taken by the four groups including the negative control group are as follows: (1) linear regression of practical dose, (2) linear increase, (3) medium dose rise, (4) high dose rise, (5 Seven types are envisaged: medium dose saturation, (6) low dose saturation, and (7) medium dose rising saturation.
- the survival time extension effect was determined by applying a log-rank test.
- Results Table 14 shows the survival rate of each group 4 days after administration.
- the survival rate of the negative control group was 10%, but the survival rate of each group administered with anti-Sema3A humanized antibodies 125, 250, and 500 ⁇ g 1 hour after LPS inoculation was 30, 40, and 100%, respectively. A tendency to increase the survival rate with increasing dose was observed.
- the anti-Sema3A humanized antibody 500 ⁇ g administration group a remarkable effect was confirmed that all 10 mice survived.
- Table 15 shows the calculation result of the p value. Note that the number of display digits is unnecessarily increased in order to indicate the order of the p values.
- FIG. 18 shows the survival rate curve of the anti-Sema3A humanized antibody 500 ⁇ g administration group in which all cases survived and a clear survival rate improvement effect was observed.
- p ⁇ 0.0001 and the result satisfying the significance level was obtained. It became clear that the anti-Sema3A humanized antibody controls the pathological condition and exhibits a survival time-extending effect even after the onset of the lethal inflammatory pathological condition.
- Example 18 Effect of anti-Sema3A humanized antibody on disseminated intravascular coagulation syndrome model mice
- DIC disseminated intravascular coagulation
- PAI-1 plasma Plasmogen activator inhibitor- 1
- PAI-1 increases and suppresses the fibrinolytic system, causing multiple organ failure and becoming serious. Since the progression of disseminated intravascular coagulation syndrome can be prevented if the increase in blood PAI-1 level can be suppressed, the effect of anti-Sema3A humanized antibody on blood PAI-1 level fluctuation was examined.
- Test material 1-1) LPS LPS (Sigma, Lot number: 102M4017V) was prepared in physiological saline to a concentration of 1.5 mg / mL.
- Anti-Sema3A humanized antibody The anti-Sema3A humanized antibody (Humanized-2) prepared in Example 2 was prepared in physiological saline to a concentration of 5 mg / mL.
- mice Male 6-week-old mice (C57BL / 6J) purchased from Charles River Japan were used.
- Test method 2-1 Group configuration After arrival, C57BL / 6J mice acclimated in the breeding room were divided into 5 mice per group, and the test groups shown in Table 16 were established.
- LPS inoculation and antibody administration LPS was inoculated intraperitoneally to a dose of 15 mg / kg. The 5 mg / mL solution was administered from the 0.1 mL tail vein so that the antibody solution had an inoculum of 500 ⁇ g / mouse. In addition, physiological saline was inoculated intraperitoneally into the condition 1 group without LPS inoculation.
- FIG. 19 shows the measurement results of blood PAI-1 concentration.
- the blood PAI-1 level was very small, but the blood PAI-1 level after LPS inoculation showed a trend of increase from 1.5 hours, 3 hours and 9 hours later Later it was significantly increased compared to non-LPS treated animals (conditions 2, 5 and 8).
- the anti-Sema3A humanized antibody (humanized-2) prepared in Example 2 has an action of suppressing an increase in blood PAI-1 level, which is one of the exacerbating factors of DIC. Became clear.
- Example 19 Effect of anti-Sema3A humanized antibody on cancer cell migration ability It is known that the survival rate of cancer patients is greatly reduced by disseminated metastasis or distant metastasis that occurs after surgical treatment or chemotherapy remission is introduced. . Activation of cancer cell migration ability leads to disseminated metastasis and distant metastasis from cancer cells, confirming the effect of anti-Sema3A humanized antibodies on cancer malignant transformation induced by Sema3A did.
- Example 2 Test method The anti-Sema3A humanized antibody (Humanized-2) prepared in Example 2 was used to evaluate the effect on cancer cell migration ability induced by Sema3A. Here, the effect was examined using cells of pancreatic cancer, for which the prognosis of patients with high Sema3A expression was poor.
- the specific experimental method is as follows.
- Fibronectin was diluted to 0.1 mg / mL with PBS buffer solution to prepare a fibronectin diluted solution, and 10 ⁇ L of this fibronectin diluted solution was applied to the lower surface of the filter of a 24-well chamber (Kurabo Chemotaxel chamber, 8 ⁇ m hole).
- the migration chamber was produced by allowing to stand at room temperature for about 1 hour and drying.
- the migration chamber prepared above was set in a 24-well plate, and 600 ⁇ L of DMEM medium containing 0.1% fetal bovine serum was added to the outer layer.
- 2 ⁇ 10 5 cells / mL human pancreatic cancer cell line (MIAPaCa-2) and 200 ⁇ L of serum-free DMEM medium containing each additive component shown in Table 17 were added to the inner layer of the chamber at 37 ° C., 5% CO 2. Culture was performed in the presence of 2 for 4 hours. Thereafter, the chamber was taken out, the cancer cells in the inner layer of the chamber were removed by suction, and the cells remaining in the chamber were removed with a cotton swab moistened with PBS buffer.
- the chamber was immersed in a cell staining solution (Diff-Quick, International Reagent) for 10 minutes or more, and then washed twice with ultrapure water and dried. After drying, the number of cells that migrated to the lower surface of the filter in the chamber was counted with a microscope.
- a cell staining solution Diff-Quick, International Reagent
- Example 20 Effect of anti-Sema3A humanized antibody on cancer cell invasion ability Cancer cells with activated migration ability melt and infiltrate the surrounding extracellular matrix, destroy the basement membrane, and migrate to blood vessels and lymphatic vessels Eventually, it will spread to distant organs. Suppression of the ability of cancer cells to infiltrate and metastasize leads to suppression of metastatic recurrence, and is thought to be useful for improving the survival rate of cancer patients. In addition to the inhibitory effect on Sema3A-induced increased migration ability examined in Example 19, the effect of anti-Sema3A humanized antibody on cancer cell invasion ability was also examined.
- Test method Sema3A-inducible using an invasion chamber (BD, BioCoat TM 8 ⁇ m pore, # 354443) filled with an inner layer of an extracellular matrix (BD, Matrigel TM Growth Factor Reduced) from which growth factors have been removed.
- BD BioCoat TM 8 ⁇ m pore, # 354443
- BD Matrigel TM Growth Factor Reduced
- human pancreatic cancer cell line MIAPaCa-2
- human glioblastoma cell line U87MG
- mouse-derived lung cancer cell line 3LL
- the invasion chamber was set in a 24-well plate, and 0.1% fetal bovine serum (in the case of 3LL cells in the case of 3LL cells) was set on the outer layer.
- 0.75 mL of DMEM medium containing 1% fetal bovine serum 125 ⁇ L of serum-free DMEM medium containing 2 ⁇ 10 5 cells / mL of cancer cells and additional components of each concentration shown in Table 18 are added to the inner layer,
- the culture was performed at 37 ° C.
- Example 19 the number of infiltrating cells that had moved to the lower surface of the filter was measured with a microscope.
- FIG. 21 shows the results of measurement of infiltrating cells in each condition when using MIAPaCa-2 cells, FIG. 22 using U87MG cells, and FIG. 23 using 3LL cells.
- Sema3A was added (condition 5)
- the invasive ability of cancer cells was clearly enhanced in all cell lines compared to the Sema3A non-stimulated group (condition 1).
- anti-Sema3A humanized antibody was added together with human Sema3A, invasion of cancer cells was suppressed to the same extent as Sema3A unstimulated in any cancer cell line (conditions 6-8). Under Sema3A non-stimulation conditions, no remarkable invasion suppressing action was observed except for 3LL (conditions 1 to 4).
- FIG. 24 shows a microscopic image of infiltrated 3LL cells. Cancer cell invasion is marked in condition 5 where Sema3A is added compared to condition 1 where Sema3A is not added, and cancer induced by Sema3A in conditions 7 and 8 where anti-Sema3A humanized antibody is treated in the presence of Sema3A It can be visually confirmed that the invasive activity of the cells is clearly suppressed.
- the anti-Sema3A antibody having a CDR of a specific amino acid sequence has an action of suppressing the invasive activity of cancer cells induced by Sema3A to the same extent as Sema3A non-stimulation. became.
- Example 21 Effect of anti-Sema3A humanized antibody on anticancer agent unresponsiveness induced by Sema3A
- pancreatic cancer has a very low 5-year survival rate among cancers. The cause of this is that pancreatic cancer tissue is poorly vascularized, but cancer cells proliferate and progress even under such nutrient-starvation conditions, often against anticancer drugs such as gemcitabine hydrochloride (GEM). It is mentioned that resistance is shown. That is, since overcoming anti-cancer drug unresponsiveness has become an important issue in the treatment of pancreatic cancer, Sema3A drug resistance induction under nutrient starvation conditions and its release action by anti-Sema3A humanized antibodies were evaluated.
- GEM gemcitabine hydrochloride
- a cell suspension obtained by suspending 4 ⁇ 10 4 cells / mL human pancreatic cancer cells (MIAPaCa-2) in DMEM medium containing 10% fetal bovine serum was seeded in each well of a 96-well plate. After culturing at 37 ° C. in the presence of 5% CO 2 for 24 hours, the medium was replaced with 100 ⁇ L of 0.1% fetal bovine serum-containing DMEM medium, and further cultured for 24 hours. Next, 100 ⁇ L of 0.1% fetal bovine serum-containing DMEM medium containing a predetermined amount of each of the components shown in Table 20 was added to each well and cultured for 2 days.
- FIG. 25 shows the results of a pancreatic cancer cell proliferation determination test.
- GEM condition 2 for condition 1
- human Sema3A attenuated the sensitivity to GEM and induced drug unresponsiveness
- This Sema3A-induced GEM resistance was relieved by treatment with anti-Sema3A humanized antibody 1-10 ⁇ g / mL and was shown to restore GEM sensitivity to the same extent as in the absence of Sema3A (as opposed to condition 3).
- Conditions 4-6 On the other hand, in the negative control human antibody addition group, GEM sensitivity could not be recovered to the same extent as the Sema3A absence condition (conditions 7 to 9).
- the humanized anti-Sema3A antibody prepared in Example 2 also has an action of canceling anticancer drug resistance induced by Sema3A under nutrient starvation conditions such as pancreatic cancer tissue. It became clear.
- SEQ ID NO: 1 is the amino acid sequence of heavy chain CDR1 of a tri-mouse chimeric antibody (derived from clone No. 4-2).
- SEQ ID NO: 2 is the amino acid sequence of heavy chain CDR2 of a tri-mouse chimeric antibody (derived from clone No. 4-2 strain).
- SEQ ID NO: 3 is the amino acid sequence of heavy chain CDR3 of a tri-mouse chimeric antibody (derived from clone No. 4-2 strain).
- SEQ ID NO: 4 is the amino acid sequence of the light chain CDR1 of the tri-mouse chimeric antibody (derived from clone No. 4-2).
- SEQ ID NO: 5 is the amino acid sequence of light chain CDR2 of a tri-mouse chimeric antibody (derived from clone No. 4-2).
- SEQ ID NO: 6 is the amino acid sequence of the light chain CDR3 of the tri-mouse chimeric antibody (derived from clone No. 4-2 strain).
- SEQ ID NO: 7 is the amino acid sequence of the heavy chain variable region of the tri-human chimeric antibody (derived from clone No. 4-2 strain).
- SEQ ID NO: 8 is the amino acid sequence of the heavy chain variable region of the tri-human chimeric antibody (derived from clone No. 4-2 strain).
- SEQ ID NO: 9 is the amino acid sequence of the light chain variable region of the tri-human chimeric antibody (derived from clone No. 4-2 strain).
- SEQ ID NO: 10 is the amino acid sequence of the light chain variable region of the tri-human chimeric antibody (derived from clone No. 4-2 strain).
- SEQ ID NO: 11 is the amino acid sequence of the heavy chain variable region of the humanized antibody (derived from clone No. 4-2).
- SEQ ID NO: 12 is the amino acid sequence of the light chain variable region of a humanized antibody (Humanized-1) (derived from clone No. 4-2).
- SEQ ID NO: 13 is the amino acid sequence of the light chain variable region of a humanized antibody (Humanized-2) (derived from clone No. 4-2).
- SEQ ID NO: 14 is the base sequence encoding the amino acid sequence (SEQ ID NO: 7) of the heavy chain variable region of the tri-human chimeric antibody (derived from clone No. 4-2).
- SEQ ID NO: 15 is the base sequence encoding the amino acid sequence (SEQ ID NO: 8) of the heavy chain variable region of the tri-human chimeric antibody (derived from clone No. 4-2).
- SEQ ID NO: 16 is the base sequence encoding the amino acid sequence (SEQ ID NO: 9) of the light chain variable region of the tri-human chimeric antibody (derived from clone No. 4-2).
- SEQ ID NO: 17 is the base sequence encoding the amino acid sequence (SEQ ID NO: 10) of the light chain variable region of the tri-human chimeric antibody (derived from clone No. 4-2).
- SEQ ID NO: 18 is a base sequence encoding the amino acid sequence of heavy chain CDR1 of a tri-mouse chimeric antibody (derived from clone No. 4-2 strain).
- SEQ ID NO: 19 is a nucleotide sequence encoding the amino acid sequence of heavy chain CDR2 of a tri-mouse chimeric antibody (derived from clone No. 4-2 strain).
- SEQ ID NO: 20 is a base sequence encoding the amino acid sequence of heavy chain CDR3 of a tri-mouse chimeric antibody (derived from clone No. 4-2 strain).
- SEQ ID NO: 21 is the base sequence encoding the amino acid sequence of light chain CDR1 of the tri-mouse chimeric antibody (derived from clone No. 4-2 strain).
- SEQ ID NO: 22 is a nucleotide sequence encoding the amino acid sequence of light chain CDR2 of a tri-mouse chimeric antibody (derived from clone No. 4-2).
- SEQ ID NO: 23 is a base sequence encoding the amino acid sequence of light chain CDR3 of a tri-mouse chimeric antibody (derived from clone No.
- SEQ ID NO: 24 is the base sequence encoding the amino acid sequence (SEQ ID NO: 11) of the heavy chain variable region of the humanized antibody (from clone No. 4-2 strain).
- SEQ ID NO: 25 is a base sequence encoding the amino acid sequence (SEQ ID NO: 12) of the light chain variable region of a humanized antibody (Humanized-1) (derived from clone No. 4-2).
- SEQ ID NO: 26 is a base sequence encoding the amino acid sequence (SEQ ID NO: 13) of the light chain variable region of a humanized antibody (Humanized-2) (derived from clone No. 4-2).
- SEQ ID NO: 27 is the base sequence of primer 1.
- SEQ ID NO: 28 is the base sequence of primer 2.
- SEQ ID NO: 29 is the base sequence of primer 3.
- SEQ ID NO: 30 is the base sequence of the primer 4.
- SEQ ID NO: 31 is the base sequence of the primer 5.
- SEQ ID NO: 32 is the base sequence of the primer 6.
- SEQ ID NO: 33 is the base sequence of the primer 7.
- SEQ ID NO: 34 is the base sequence of the primer 8.
- SEQ ID NO: 35 is the amino acid sequence of the heavy chain of a tri-mouse chimeric antibody (derived from clone No. 4-2 strain).
- SEQ ID NO: 36 is the amino acid sequence of the light chain of a tri-mouse chimeric antibody (derived from clone No. 4-2).
- SEQ ID NO: 37 is the base sequence of the primer A ⁇ .
- SEQ ID NO: 38 is the base sequence of the primer B ⁇ .
- SEQ ID NO: 39 is the base sequence of the primer C ⁇ .
- SEQ ID NO: 40 is the base sequence of the primer D ⁇ .
- SEQ ID NO: 41 is the base sequence of the primer E ⁇ .
- SEQ ID NO: 42 is the base sequence of the primer F ⁇ .
- SEQ ID NO: 43 is the base sequence of the primer G ⁇ .
- SEQ ID NO: 44 is the base sequence of the primer H ⁇ .
- SEQ ID NO: 45 is the base sequence of the primer I ⁇ .
- SEQ ID NO: 46 is the base sequence of the primer J ⁇ .
- SEQ ID NO: 47 is the base sequence of the primer K ⁇ .
- SEQ ID NO: 48 is the base sequence of the primer L ⁇ .
- SEQ ID NO: 49 is the amino acid sequence of the heavy chain of a humanized antibody (Humanized-1 and Humanized-2) (from clone No. 4-2 strain).
- SEQ ID NO: 50 is the base sequence encoding the amino acid sequence of the heavy chain of the humanized antibody (Humanized-1 and Humanized-2) (from clone No. 4-2 strain).
- SEQ ID NO: 51 is the amino acid sequence of the light chain of a humanized antibody (Humanized-1) (from clone No. 4-2 strain).
- SEQ ID NO: 52 is a nucleotide sequence encoding the amino acid sequence of the light chain of a humanized antibody (Humanized-1) (derived from clone No. 4-2).
- SEQ ID NO: 53 is the amino acid sequence of the light chain of a humanized antibody (Humanized-2) (derived from clone No. 4-2).
- SEQ ID NO: 54 is the base sequence encoding the amino acid sequence of the light chain of humanized antibody (Humanized-2) (from clone No. 4-2 strain).
- SEQ ID NO: 55 is the amino acid sequence of the heavy chain of a tri-human chimeric antibody (derived from clone No. 4-2).
- SEQ ID NO: 56 is the base sequence encoding the amino acid sequence of the heavy chain of the tri-human chimeric antibody (derived from clone No. 4-2 strain).
- SEQ ID NO: 57 is the amino acid sequence of the light chain of the tri-human chimeric antibody (derived from clone No. 4-2 strain).
- SEQ ID NO: 58 is the base sequence encoding the amino acid sequence of the light chain of the tri-human chimeric antibody (derived from clone No. 4-2 strain).
- SEQ ID NO: 59 is the amino acid sequence of the heavy chain variable region of the tri-mouse chimeric antibody (derived from clone No. 165 strain).
- SEQ ID NO: 60 is the amino acid sequence of heavy chain CDR1 of the tri-mouse chimeric antibody (derived from clone No. 165 strain).
- SEQ ID NO: 61 is the amino acid sequence of heavy chain CDR2 of a tri-mouse chimeric antibody (derived from clone No. 165 strain).
- SEQ ID NO: 62 is the amino acid sequence of heavy chain CDR3 of the tri-mouse chimeric antibody (derived from clone No. 165 strain).
- SEQ ID NO: 63 is the amino acid sequence of the light chain variable region of the tri-mouse chimeric antibody (derived from clone No. 165 strain).
- SEQ ID NO: 64 is the amino acid sequence of light chain CDR1 of the tri-mouse chimeric antibody (derived from clone No. 165 strain).
- SEQ ID NO: 65 is the amino acid sequence of light chain CDR2 of a tri-mouse chimeric antibody (derived from clone No. 165 strain).
- SEQ ID NO: 66 is the amino acid sequence of light chain CDR3 of the tri-mouse chimeric antibody (derived from clone No. 165 strain).
- SEQ ID NO: 67 is the amino acid sequence of the heavy chain variable region of the tri-mouse chimeric antibody (derived from clone No. 582).
- SEQ ID NO: 68 is the amino acid sequence of heavy chain CDR1 of the tri-mouse chimeric antibody (derived from clone No. 165 strain).
- SEQ ID NO: 69 is the amino acid sequence of heavy chain CDR2 of a tri-mouse chimeric antibody (derived from clone No. 165 strain).
- SEQ ID NO: 70 is the amino acid sequence of heavy chain CDR3 of the tri-mouse chimeric antibody (derived from clone No. 165 strain).
- SEQ ID NO: 71 is the amino acid sequence of the light chain variable region of the tri-mouse chimeric antibody (derived from clone No. 582 strain).
- SEQ ID NO: 72 is the amino acid sequence of light chain CDR1 of the tri-mouse chimeric antibody (derived from clone No. 582).
- SEQ ID NO: 73 is the amino acid sequence of light chain CDR2 of a tri-mouse chimeric antibody (derived from clone No. 582).
- SEQ ID NO: 74 is the amino acid sequence of light chain CDR3 of the tri-mouse chimeric antibody (derived from clone No. 582).
- SEQ ID NO: 75 is the amino acid sequence of the heavy chain variable region of the avian antibody (derived from clone No. 240-40).
- SEQ ID NO: 76 is the amino acid sequence of heavy chain CDR1 of the avian antibody (from clone No. 240-40 strain).
- SEQ ID NO: 77 is the amino acid sequence of heavy chain CDR2 of the avian antibody (from clone No.
- SEQ ID NO: 78 is the amino acid sequence of heavy chain CDR3 of the avian antibody (derived from clone No. 240-40 strain).
- SEQ ID NO: 79 is the amino acid sequence of the light chain variable region of the avian antibody (derived from clone No. 240-40).
- SEQ ID NO: 80 is the amino acid sequence of light chain CDR1 of the avian antibody (derived from clone No. 240-40).
- SEQ ID NO: 81 is the amino acid sequence of light chain CDR2 of the avian antibody (derived from clone No. 240-40).
- SEQ ID NO: 82 is the amino acid sequence of light chain CDR3 of the avian antibody (derived from clone No. 240-40).
- SEQ ID NO: 83 is the amino acid sequence of the heavy chain variable region of the avian antibody (derived from clone No. 255-72).
- SEQ ID NO: 84 is the amino acid sequence of heavy chain CDR1 of the avian antibody (derived from clone No. 255-72).
- SEQ ID NO: 85 is the amino acid sequence of heavy chain CDR2 of the avian antibody (derived from clone No. 255-72).
- SEQ ID NO: 86 is the amino acid sequence of heavy chain CDR3 of the avian antibody (derived from clone No. 255-72).
- SEQ ID NO: 87 is the amino acid sequence of the light chain variable region of the avian antibody (derived from clone No. 255-72).
- SEQ ID NO: 88 is the amino acid sequence of light chain CDR1 of the avian antibody (derived from clone No. 255-72).
- SEQ ID NO: 89 is the amino acid sequence of light chain CDR2 of the avian antibody (derived from clone No. 255-72).
- SEQ ID NO: 90 is the amino acid sequence of light chain CDR3 of the avian antibody (derived from clone No. 255-72).
- SEQ ID NO: 91 is the base sequence of the primer M ⁇ .
- SEQ ID NO: 92 is the base sequence of the primer N ⁇ .
- SEQ ID NO: 93 is the base sequence of the primer O ⁇ .
- SEQ ID NO: 94 is the base sequence of the primer P ⁇ .
- SEQ ID NO: 95 is the base sequence encoding the amino acid sequence of the heavy chain constant region of the humanized antibody.
- SEQ ID NO: 96 is the base sequence encoding the amino acid sequence of the light chain constant region of the humanized antibody.
- SEQ ID NO: 97 is the base sequence encoding the amino acid sequence (SEQ ID NO: 60) of the heavy chain CDR1 of the tri-mouse chimeric antibody (derived from clone No. 165 strain).
- SEQ ID NO: 98 is the base sequence encoding the amino acid sequence (SEQ ID NO: 61) of the heavy chain CDR2 of the tri-mouse chimeric antibody (derived from clone No. 165 strain).
- SEQ ID NO: 99 is the base sequence encoding the amino acid sequence (SEQ ID NO: 62) of the heavy chain CDR3 of the tri-mouse chimeric antibody (derived from clone No. 165 strain).
- SEQ ID NO: 100 is the base sequence encoding the amino acid sequence (SEQ ID NO: 64) of the light chain CDR1 of the tri-mouse chimeric antibody (derived from clone No. 165 strain).
- SEQ ID NO: 101 is the base sequence encoding the amino acid sequence (SEQ ID NO: 65) of the light chain CDR2 of the tri-mouse chimeric antibody (derived from clone No. 165 strain).
- SEQ ID NO: 102 is the base sequence encoding the amino acid sequence (SEQ ID NO: 66) of the light chain CDR3 of the tri-mouse chimeric antibody (derived from clone No. 165 strain).
- SEQ ID NO: 103 is the base sequence encoding the amino acid sequence (SEQ ID NO: 59) of the heavy chain variable region of the tri-mouse chimeric antibody (derived from clone No. 165 strain).
- SEQ ID NO: 104 is the base sequence encoding the amino acid sequence (SEQ ID NO: 63) of the light chain variable region of the tri-mouse chimeric antibody (derived from clone No. 165 strain).
- SEQ ID NO: 105 is the base sequence encoding the amino acid sequence (SEQ ID NO: 68) of the heavy chain CDR1 of the tri-mouse chimeric antibody (derived from clone No. 582).
- SEQ ID NO: 106 is a base sequence encoding the amino acid sequence (SEQ ID NO: 69) of the heavy chain CDR2 of a tri-mouse chimeric antibody (derived from clone No. 582).
- SEQ ID NO: 107 is the base sequence encoding the amino acid sequence (SEQ ID NO: 70) of the heavy chain CDR3 of the tri-mouse chimeric antibody (derived from clone No. 582 strain).
- SEQ ID NO: 108 is the base sequence encoding the amino acid sequence (SEQ ID NO: 72) of the light chain CDR1 of the tri-mouse chimeric antibody (derived from clone No. 582).
- SEQ ID NO: 109 is the base sequence encoding the amino acid sequence (SEQ ID NO: 73) of the light chain CDR2 of the tri-mouse chimeric antibody (derived from clone No. 582).
- SEQ ID NO: 110 is the base sequence encoding the amino acid sequence (SEQ ID NO: 74) of the light chain CDR3 of the tri-mouse chimeric antibody (derived from clone No. 582).
- SEQ ID NO: 111 is the base sequence encoding the amino acid sequence (SEQ ID NO: 67) of the heavy chain variable region of the tri-mouse chimeric antibody (derived from clone No. 582).
- SEQ ID NO: 112 is the base sequence encoding the amino acid sequence (SEQ ID NO: 71) of the light chain variable region of the tri-mouse chimeric antibody (derived from clone No. 582).
- SEQ ID NO: 113 is the base sequence encoding the amino acid sequence (SEQ ID NO: 76) of the heavy chain CDR1 of the avian antibody (derived from clone No. 240-40 strain).
- SEQ ID NO: 114 is the base sequence encoding the amino acid sequence (SEQ ID NO: 77) of the heavy chain CDR2 of the avian antibody (from clone No. 240-40 strain).
- SEQ ID NO: 115 is the base sequence encoding the amino acid sequence (SEQ ID NO: 78) of the heavy chain CDR3 of the avian antibody (derived from clone No. 240-40 strain).
- SEQ ID NO: 116 is the base sequence encoding the amino acid sequence (SEQ ID NO: 80) of the light chain CDR1 of the avian antibody (derived from clone No. 240-40).
- SEQ ID NO: 117 is the base sequence encoding the amino acid sequence (SEQ ID NO: 81) of the light chain CDR2 of the avian antibody (derived from clone No. 240-40 strain).
- SEQ ID NO: 118 is the base sequence encoding the amino acid sequence of the light chain CDR3 (SEQ ID NO: 82) of the avian antibody (derived from clone No. 240-40 strain).
- SEQ ID NO: 119 is a base sequence encoding the amino acid sequence (SEQ ID NO: 75) of the heavy chain variable region of the avian antibody (derived from clone No. 240-40 strain).
- SEQ ID NO: 120 is the base sequence encoding the amino acid sequence (SEQ ID NO: 79) of the light chain variable region of the avian antibody (from clone No. 240-40 strain).
- SEQ ID NO: 121 is the base sequence encoding the amino acid sequence (SEQ ID NO: 84) of the heavy chain CDR1 of the avian antibody (derived from clone No. 255-72).
- SEQ ID NO: 122 is the base sequence encoding the amino acid sequence (SEQ ID NO: 85) of the heavy chain CDR2 of the avian antibody (derived from clone No. 255-72).
- SEQ ID NO: D123 is a base sequence encoding the amino acid sequence (SEQ ID NO: 86) of the heavy chain CDR3 of the avian antibody (derived from clone No. 255-72).
- SEQ ID NO: 124 is the base sequence encoding the amino acid sequence (SEQ ID NO: 88) of the light chain CDR1 of the avian antibody (derived from clone No. 255-72).
- SEQ ID NO: 125 is the base sequence encoding the amino acid sequence (SEQ ID NO: 89) of the light chain CDR2 of the avian antibody (derived from clone No. 255-72).
- SEQ ID NO: 126 is the base sequence encoding the amino acid sequence (SEQ ID NO: 90) of the light chain CDR3 of the avian antibody (derived from clone No. 255-72).
- SEQ ID NO: 127 is the base sequence encoding the amino acid sequence (SEQ ID NO: 83) of the heavy chain variable region of the avian antibody (derived from clone No. 255-72).
- SEQ ID NO: 128 is the base sequence encoding the amino acid sequence (SEQ ID NO: 87) of the light chain variable region of the avian antibody (derived from clone No. 255-72).
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Abstract
Description
項1. 下記(A)~(E)のいずれか示される重鎖可変領域及び軽鎖可変領域を含む、抗セマフォリン3A抗体、又はその抗原結合領域を含む抗体断片:
(A) 配列番号1に示すアミノ酸配列、或いは配列番号1に示すアミノ酸配列において1~数個のアミノ酸が置換、欠失、付加又は挿入したアミノ酸配列を含むCDR1;配列番号2に示すアミノ酸配列、或いは配列番号2に示すアミノ酸配列において1~数個のアミノ酸が置換、欠失、付加又は挿入したアミノ酸配列に示すアミノ酸配列を含むCDR2;及び配列番号3に示すアミノ酸配列、或いは配列番号3に示すアミノ酸配列において1~数個のアミノ酸が置換、欠失、付加又は挿入したアミノ酸配列を含むCDR3を有する重鎖可変領域、並びに
配列番号4に示すアミノ酸配列、或いは配列番号4に示すアミノ酸配列において1~数個のアミノ酸が置換、欠失、付加又は挿入したアミノ酸配列を含むCDR1;配列番号5に示すアミノ酸配列、或いは配列番号5に示すアミノ酸配列において1~数個のアミノ酸が置換、欠失、付加又は挿入したアミノ酸配列を含むCDR2;及び配列番号6に示すアミノ酸配列、或いは配列番号6に示すアミノ酸配列において1~数個のアミノ酸が置換、欠失、付加又は挿入したアミノ酸配列を含むCDR3を有する軽鎖可変領域;
(B) 配列番号60に示すアミノ酸配列、或いは配列番号60に示すアミノ酸配列において1~数個のアミノ酸が置換、欠失、付加又は挿入したアミノ酸配列を含むCDR1;配列番号61に示すアミノ酸配列、或いは配列番号61に示すアミノ酸配列において1~数個のアミノ酸が置換、欠失、付加又は挿入したアミノ酸配列に示すアミノ酸配列を含むCDR2;及び配列番号62に示すアミノ酸配列、或いは配列番号62に示すアミノ酸配列において1~数個のアミノ酸が置換、欠失、付加又は挿入したアミノ酸配列を含むCDR3を有する重鎖可変領域、並びに
配列番号64に示すアミノ酸配列、或いは配列番号64に示すアミノ酸配列において1~数個のアミノ酸が置換、欠失、付加又は挿入したアミノ酸配列を含むCDR1;配列番号65に示すアミノ酸配列、或いは配列番号65に示すアミノ酸配列において1~数個のアミノ酸が置換、欠失、付加又は挿入したアミノ酸配列を含むCDR2;及び配列番号66に示すアミノ酸配列、或いは配列番号66に示すアミノ酸配列において1~数個のアミノ酸が置換、欠失、付加又は挿入したアミノ酸配列を含むCDR3を有する軽鎖可変領域;
(C) 配列番号68に示すアミノ酸配列、或いは配列番号68に示すアミノ酸配列において1~数個のアミノ酸が置換、欠失、付加又は挿入したアミノ酸配列を含むCDR1;配列番号69に示すアミノ酸配列、或いは配列番号69に示すアミノ酸配列において1~数個のアミノ酸が置換、欠失、付加又は挿入したアミノ酸配列に示すアミノ酸配列を含むCDR2;及び配列番号70に示すアミノ酸配列、或いは配列番号70に示すアミノ酸配列において1~数個のアミノ酸が置換、欠失、付加又は挿入したアミノ酸配列を含むCDR3を有する重鎖可変領域、並びに
配列番号72に示すアミノ酸配列、或いは配列番号72に示すアミノ酸配列において1~数個のアミノ酸が置換、欠失、付加又は挿入したアミノ酸配列を含むCDR1;配列番号73に示すアミノ酸配列、或いは配列番号73に示すアミノ酸配列において1~数個のアミノ酸が置換、欠失、付加又は挿入したアミノ酸配列を含むCDR2;及び配列番号74に示すアミノ酸配列、或いは配列番号74に示すアミノ酸配列において1~数個のアミノ酸が置換、欠失、付加又は挿入したアミノ酸配列を含むCDR3を有する軽鎖可変領域;
(D) 配列番号76に示すアミノ酸配列、或いは配列番号76に示すアミノ酸配列において1~数個のアミノ酸が置換、欠失、付加又は挿入したアミノ酸配列を含むCDR1;配列番号77に示すアミノ酸配列、或いは配列番号77に示すアミノ酸配列において1~数個のアミノ酸が置換、欠失、付加又は挿入したアミノ酸配列に示すアミノ酸配列を含むCDR2;及び配列番号78に示すアミノ酸配列、或いは配列番号78に示すアミノ酸配列において1~数個のアミノ酸が置換、欠失、付加又は挿入したアミノ酸配列を含むCDR3を有する重鎖可変領域、並びに
配列番号80に示すアミノ酸配列、或いは配列番号80に示すアミノ酸配列において1~数個のアミノ酸が置換、欠失、付加又は挿入したアミノ酸配列を含むCDR1;配列番号81に示すアミノ酸配列、或いは配列番号81に示すアミノ酸配列において1~数個のアミノ酸が置換、欠失、付加又は挿入したアミノ酸配列を含むCDR2;及び配列番号82に示すアミノ酸配列、或いは配列番号82に示すアミノ酸配列において1~数個のアミノ酸が置換、欠失、付加又は挿入したアミノ酸配列を含むCDR3を有する軽鎖可変領域;
(E) 配列番号84に示すアミノ酸配列、或いは配列番号84に示すアミノ酸配列において1~数個のアミノ酸が置換、欠失、付加又は挿入したアミノ酸配列を含むCDR1;配列番号85に示すアミノ酸配列、或いは配列番号85に示すアミノ酸配列において1~数個のアミノ酸が置換、欠失、付加又は挿入したアミノ酸配列に示すアミノ酸配列を含むCDR2;及び配列番号86に示すアミノ酸配列、或いは配列番号86に示すアミノ酸配列において1~数個のアミノ酸が置換、欠失、付加又は挿入したアミノ酸配列を含むCDR3を有する重鎖可変領域、並びに
配列番号88に示すアミノ酸配列、或いは配列番号88に示すアミノ酸配列において1~数個のアミノ酸が置換、欠失、付加又は挿入したアミノ酸配列を含むCDR1;配列番号89に示すアミノ酸配列、或いは配列番号89に示すアミノ酸配列において1~数個のアミノ酸が置換、欠失、付加又は挿入したアミノ酸配列を含むCDR2;及び配列番号90に示すアミノ酸配列、或いは配列番号90に示すアミノ酸配列において1~数個のアミノ酸が置換、欠失、付加又は挿入したアミノ酸配列を含むCDR3を有する軽鎖可変領域。
項2. キメラ抗体、ヒト化抗体、又はこれらの抗体断片である、項1に記載の抗セマフォリン3A抗体又は抗体断片。
項3. 神経再生促進のために使用される、項1又は2に記載の抗セマフォリン3A抗体又は抗体断片。
項4. アルツハイマー病の予防及び/又は治療のために使用される、項1又は2に記載の抗セマフォリン3A抗体又は抗体断片。
項5. 敗血症の予防及び/又は治療のために使用される、項1又は2に記載の抗セマフォリン3A抗体又は抗体断片。
項6. がんの予防及び/又は治療のために使用される、項1又は2に記載の抗セマフォリン3A抗体又は抗体断片。
項7. 播種性血管内凝固症候群の予防及び/又は治療剤の製造のための、項1又は2に記載の抗セマフォリン3A抗体又は抗体断片の使用。
項8. 中枢又は末梢神経系疾患、自己免疫疾患、炎症性疾患、感染症、及びアレルギー性疾患よりなる群から選択される少なくとも1種の疾患の予防及び/又は治療のために使用される、項1又は2に記載の抗セマフォリン3A抗体又は抗体断片。
項9. 項1~8のいずれかに記載の抗セマフォリン3A抗体又は抗体断片を含有する、医薬組成物。
項10. 神経再生促進用である、項9に記載の医薬組成物。
項11. アルツハイマー病の予防及び/又は治療用である、項9に記載の医薬組成物。
項12. 敗血症の予防及び/又は治療用である、項9に記載の医薬組成物。
項13. がんの予防及び/又は治療用である、項9に記載の医薬組成物。
項14. がんが、大腸癌、結腸直腸癌、肺癌、乳癌、脳腫瘍、黒色腫、腎細胞癌、白血病、リンパ腫、T細胞リンパ腫、胃癌、膵臓癌、子宮頚癌、子宮内膜癌、卵巣癌、食道癌、肝臓癌、頭頚部扁平上皮癌、皮膚癌、尿路癌、前立腺癌、絨毛癌、咽頭癌、又は喉頭癌である、項13に記載の医薬組成物。
項15. 播種性血管内凝固症候群の予防及び/又は治療用である、項9に記載の医薬組成物。
項16. 播種性血管内凝固症候群が、敗血症、急性白血病、固形がん、常位胎盤早期剥離、羊水塞栓、外傷、熱傷、膠原病、ショック、大動脈瘤、劇症肝炎、肝硬変、急性膵炎、横紋筋融解、血栓症、及び重症感染症よりなる群から選択される少なくとも1種を伴っている、項15に記載の医薬組成物。
項17. 中枢又は末梢神経系疾患、自己免疫疾患、炎症性疾患、感染症、及びアレルギー性疾患よりなる群から選択される少なくとも1種の疾患の予防及び/又は治療用である、項9に記載の医薬組成物。
項18. 中枢又は末梢神経系疾患が、神経障害性の疼痛、脊髄損傷、又は神経変性疾患である、項17に記載の医薬組成物。
項19. 神経変性疾患が、アルツハイマー病、パーキンソン病、ハンチントン病、筋萎縮性側索硬化症、進行性核上性麻痺、黒質線状体変性症、シャイ・ドレーガー症候群、オリーブ橋小脳萎縮症、又は脊髄小脳変性症である、項18に記載の医薬組成物。
項20. 自己免疫疾患が、慢性関節リウマチ、I型糖尿病、炎症性腸疾患、クローン病、全身性エリテマトーデス、又は多発性硬化症である、項17に記載の医薬組成物。
項21. 炎症性疾患が、敗血症、慢性閉塞性肺疾患、喘息、関節炎、肝炎、脊椎関節炎、又はシェーグレン症候群である、項17に記載の医薬組成物。
項22. 感染症が、細菌感染症、脳炎、髄膜炎、心内膜炎、C型肝炎、インフルエンザ、重症急性呼吸器症候群、肺炎、敗血症、火傷性、又は外傷性感染症である、項17に記載の医薬組成物。
項23. アレルギー性疾患が、アレルギー性鼻炎、アレルギー性結膜炎、アトピー性皮膚炎、気管支喘息、又は食物アレルギーである、項17に記載の医薬組成物。
項24. 神経再生が求められる患者に対して、項1又は2に記載の抗セマフォリン3A抗体又は抗体断片を投与する工程を含む、神経再生方法。
項25. アルツハイマー病を罹患している患者に対して、項1又は2に記載の抗セマフォリン3A抗体又は抗体断片を投与する工程を含む、アルツハイマー病の治療方法。
項26. 敗血症を罹患している患者に対して、項1又は2に記載の抗セマフォリン3A抗体又は抗体断片を投与する工程を含む、敗血症の治療方法。
項27. がんを罹患している患者に対して、項1又は2に記載の抗セマフォリン3A抗体又は抗体断片を投与する工程を含む、癌の治療方法。
項28. がんが、大腸癌、結腸直腸癌、肺癌、乳癌、脳腫瘍、黒色腫、腎細胞癌、白血病、リンパ腫、T細胞リンパ腫、胃癌、膵臓癌、子宮頚癌、子宮内膜癌、卵巣癌、食道癌、肝臓癌、頭頚部扁平上皮癌、皮膚癌、尿路癌、前立腺癌、絨毛癌、咽頭癌、又は喉頭癌である、項27に記載の治療方法。
項29. 播種性血管内凝固症候群を罹患している患者に対して、項1又は2に記載の抗セマフォリン3A抗体又は抗体断片を投与する工程を含む、播種性血管内凝固症候群の治療方法。
項30. 播種性血管内凝固症候群が、敗血症、急性白血病、固形がん、常位胎盤早期剥離、羊水塞栓、外傷、熱傷、膠原病、ショック、大動脈瘤、劇症肝炎、肝硬変、急性膵炎、横紋筋融解、血栓症、及び重症感染症よりなる群から選択される少なくとも1種を伴っている、項29に記載の播種性血管内凝固症候群の治療方法。
項30. 中枢又は末梢神経系疾患、自己免疫疾患、炎症性疾患、感染症、及びアレルギー性疾患よりなる群から選択される少なくとも1種の疾患を罹患している患者に対して、項1又は2に記載の抗セマフォリン3A抗体又は抗体断片を投与する工程を含む、前記疾患の治療方法。
項31. 神経再生用の医薬の製造のための、項1又は2に記載の抗セマフォリン3A抗体又は抗体断片の使用。
項32. アルツハイマー病の予防及び/又は治療剤の製造のための、項1又は2に記載の抗セマフォリン3A抗体又は抗体断片の使用。
項33. 敗血症の予防及び/又は治療剤の製造のための、項1又は2に記載の抗セマフォリン3A抗体又は抗体断片の使用。
項
項34. がんの予防及び/又は治療剤の製造のための、項1又は2に記載の抗セマフォリン3A抗体又は抗体断片の使用。
項35. 播種性血管内凝固症候群の予防及び/又は治療剤の製造のための、項1又は2に記載の抗セマフォリン3A抗体又は抗体断片の使用。
項36. 中枢又は末梢神経系疾患、自己免疫疾患、炎症性疾患、感染症、及びアレルギー性疾患よりなる群から選択される少なくとも1種の疾患の予防及び/又は治療剤の製造のための、項1又は2に記載の抗セマフォリン3A抗体又は抗体断片の使用。
項37.項1又は2に記載の抗Sema3A抗体又は抗体断片を用いて免疫測定により検体中のSema3Aタンパク質を測定する工程を含む、Sema3Aタンパク質の測定方法。
項38.項1又は2に記載の抗Sema3A抗体又は抗体断片を含む、Sema3Aタンパク質の測定用キット。
本発明の抗Sema3A抗体は、CDR1~CDR3が特定のアミノ酸配列を有する重鎖可変領域、及びCDR1~CDR3が特定のアミノ酸配列を有する軽鎖可変領域を含むことを特徴とする。
(A) 配列番号1に示すアミノ酸配列を含むCDR1、配列番号2に示すアミノ酸配列を含むCDR2、及び配列番号3に示すアミノ酸配列を含むCDR3を有する重鎖可変領域;並びに配列番号4に示すアミノ酸配列を含むCDR1、配列番号5に示すアミノ酸配列を含むCDR2、及び配列番号6に示すアミノ酸配列を含むCDR3を有する軽鎖可変領域。
(B) 配列番号60に示すアミノ酸配列を含むCDR1、配列番号61に示すアミノ酸配列を含むCDR2、及び配列番号62に示すアミノ酸配列を含むCDR3を有する重鎖可変領域;並びに配列番号64に示すアミノ酸配列を含むCDR1、配列番号65に示すアミノ酸配列を含むCDR2、及び配列番号66に示すアミノ酸配列を含むCDR3を有する軽鎖可変領域。
(C) 配列番号68に示すアミノ酸配列を含むCDR1、配列番号69に示すアミノ酸配列を含むCDR2、及び配列番号70に示すアミノ酸配列を含むCDR3を有する重鎖可変領域;並びに配列番号72に示すアミノ酸配列を含むCDR1;配列番号73に示すアミノ酸配列を含むCDR2、及び配列番号74に示すアミノ酸配列を含むCDR3を有する軽鎖可変領域。
(D) 配列番号76に示すアミノ酸配列を含むCDR1、配列番号77に示すアミノ酸配列を含むCDR2、及び配列番号78に示すアミノ酸配列を含むCDR3を有する重鎖可変領域;並びに配列番号80に示すアミノ酸配列を含むCDR1;配列番号81に示すアミノ酸配列を含むCDR2、及び配列番号82に示すアミノ酸配列を含むCDR3を有する軽鎖可変領域。
(E) 配列番号84に示すアミノ酸配列を含むCDR1、配列番号85に示すアミノ酸配列を含むCDR2、及び配列番号86に示すアミノ酸配列を含むCDR3を有する重鎖可変領域;並びに配列番号88に示すアミノ酸配列を含むCDR1、配列番号89に示すアミノ酸配列を含むCDR2、及び配列番号90に示すアミノ酸配列を含むCDR3を有する軽鎖可変領域。
塩基性アミノ酸:リジン、アルギニン、ヒスチジン
酸性アミノ酸:グルタミン酸、アスパラギン酸
中性アミノ酸:グリシン、アラニン、セリン、トレオニン、メチオニン、システイン、フェニルアラニン、トリプトファン、チロシン、ロイシン、イソロイシン、バリン、グルタミン、アスパラギン、プロリン
更に、前記中性アミノ酸は、極性側鎖を有するもの(アスパラギン、グルタミン、セリン、トレオニン、チロシン、システイン)、非極性側鎖を有するもの(グリシン、アラニン、バリン、ロイシン、イソロイシン、プロリン、フェニルアラニン、メチオニン、トリプトファン)、アミド含有側鎖を有するもの(アスパラギン、グルタミン)、硫黄含有側鎖を有するもの(メチオニン、システイン)、芳香族側鎖を有するもの(フェニルアラニン、トリプトファン、チロシン)、水酸基含有側鎖を有するもの(セリン、トレオニン、チロシン)、脂肪族側鎖を有するもの(アラニン、ロイシン、イソロイシン、バリン)等に分類することもできる。
本発明は、前記抗Sema3A抗体又は抗体断片を含む医薬組成物を提供する。本発明の医薬組成物は、抗Sema3A抗体又は抗体断片によって、Sema3Aタンパク質の機能を効果的に阻害することができ、各種薬理効果を奏することができる。
本発明は、前記抗Sema3A抗体又は抗体断片を利用したSema3Aタンパク質の測定方法を提供する。本発明の測定方法では、前記抗Sema3A抗体又は抗体断片と、検体中のSema3Aとの抗原抗体反応を利用して検体中のSema3Aを免疫測定する。
1)細胞培養
ニワトリB細胞由来のDT40細胞の細胞培養を以下の方法で行った。培養機はCO2恒温槽を用い、5%のCO2存在下で39.5℃で培養した。培地は、IMDM培地(Life Technoligies社)を用い、10容量%FBS、1容量%ニワトリ血清、ペニシリン100単位/mL、ストレプトマイシン100μg/mL、2-メルカプトエタノール55μMを加えて使用した。また、トリコスタチンA(和光純薬)は、DMSOに5mg/mLに溶解したものをストックとし、最終濃度が1.25ng/mL又は2.5ng/mLとなるように、適宜培地で希釈して用いた。
横浜市立大学医学部分子薬理神経生物学教室で所有しているマウスSema3Aタンパク質発現株を用いて作製したマウスSema3Aタンパク質を抗原として、Autonomously Diversifying Library(ADLib)システム(カイオム・バイオサイエンス社)を用いて抗体産生細胞を得た。具体的には以下の実験プロセスを経ている。
His-tag用磁気ビーズへのマウスSema3Aタンパク質の固定は、以下の手順に従って実施した。磁気ビーズはDynabeads TALON (Life Technologies社)を、磁気スタンドはDynal MPC(Life Technologies社)を用いた。
トリコスタチンA1.25ng/mL又は2.5ng/mLを含むIMDM培地で5週間以上培養した野生型DT40細胞約8×108個を1×108個ずつ8分割し、それぞれを洗浄液(1重量%BSAを含むPBS緩衝液)10mLで1回、更に同洗浄液1mLで1回洗浄した後、1mLの洗浄液中において2-1)で作製した抗原固定磁気ビーズ5×106個とそれぞれ混合し、4℃で30分間、穏やかに回転させつつインキュベートした。その後KingFisher mL(サーモフィッシャーサイエンティフィック社)を使用して、1.7mLの洗浄液で3分、3回洗浄した。最後に、抗原固定磁気ビーズに結合した細胞を500μL培地に懸濁し、これを20mLの培地に加えたのち、96穴プレートに200μLずつ分注し、39.5℃で培養した。この作業以降の培地は、IMDM培地(Life Technologies社)に、10容量%FBS、ペニシリン100単位/mL、ストレプトマイシン100μg/mL,2-メルカプトエタノール55μMを加えたものを使用した。
ELISAは直接固相法で、以下の手順に従って行った。前記2-2)のステップの6日後、マウスSema3Aタンパク質を2.5μg/mLで384well Maxisorp(Nunc社)に20μLずつ分注し一晩放置した。なお、抗体の特異性を検討するために、ネガティブコントロールとして、オボアルブミン(OA)、ウサギIgG(rIgG)も同様にプレートに固定した。翌日プレートの中身を捨て、ブロッキング液(1%BSAを含むPBS緩衝液)45μLを入れ、室温で1時間インキュベートした。ELISA洗浄バッファー(0.05重量% Tween20を含むPBS)120μLで5回洗浄し、前記2-2)で選別して生じたコロニー由来の培養上清それぞれ25μLを入れ、室温で1時間インキュベートした。ELISA洗浄液120μLで5回洗浄したのち、二次抗体をブロッキングバッファーで2000倍に希釈したものを25μL入れ、室温で45分インキュベートした。なお、二次抗体はanti-chicken IgM-HRP(BETHYL社)を使用した。ELISA洗浄液120μLで5回洗浄したのち、TMB+(Dako社)を25μL入れ、30分インキュベートした。その後反応を1Nの硫酸25μLで停止し、450nmの吸光度を測定した。
ELISAにより力価を解析した培養上清は、血清由来のIgM等を除去するため、以下のようにして調製した。ニワトリ血清成分をAIM-V無血清培地(Life Technoligies社)に3%の濃度で加えた。ここに約1×106個/mLの濃度になるように細胞を加え、2日培養して、培養上清を取得した。
前記3)で得られたELISA用培養上清について、ELISAにより抗Sema3A抗体濃度の測定を行い、抗Sema3A抗体産生能が高いクローンNo.4-2株を得た。
前記4)で得られた抗Sema3Aトリ抗体産生クローンNo.4-2株を用いて、IMDM培地で5%CO2存在下、39.5℃でCO2恒温槽を用いて培養し、その培養上清を回収した。
前記4)で得た抗Sema3A抗体産生クローンNo.4-2株から全RNAを抽出し、逆転写酵素(SuperScriptIII、Life Technoligies社)及びoligo(dT)プライマーを用いた逆転写反応によりcDNAの合成を行った。このcDNAを鋳型とし、トリ抗体λ軽鎖可変領域の配列を含むセンスプライマー(プライマー1:GAAGATCTAAGCTTGCCATGGCCTGGGCTCCTCTCCTCCT(配列番号27))、トリ抗体λ軽鎖可変領域の配列とマウス抗体軽鎖定常領域の配列を含むアンチセンスプライマー(プライマー2:TGGCGAAGACTTCGGCTGGCCTAGGAC(配列番号28))を用いたPCRにより、軽鎖可変領域遺伝子を増幅した。その一方で、トリ抗体重鎖可変領域の配列を含むセンスプライマー(プライマー3:GAAGATCTAAGCTTACCATGAGCCCACTCG(配列番号29))、トリ抗体重鎖可変領域の領域とマウスIgG2a抗体定常領域を含むアンチセンスプライマー(プライマー4:CGATGGGGCTGTTGTTTTGGCGGAGGAGACGATGACTTC(配列番号30))を用いたPCRにより、重鎖可変領域遺伝子を増幅した。一方、マウス抗体λ軽鎖定常領域のDNA配列を鋳型とし、トリ抗体λ軽鎖可変領域のセンスプライマー配列とマウス抗体λ軽鎖定常領域の配列を含むセンスプライマー(プライマー5:AAGTCTTCGCCATCAGTCACCCTGTTTCCA(配列番号31))、及びアンチセンスプライマー(プライマー6:TATGCGGCCGCTTACTAGGAACAGTCA(配列番号32))を用いたPCRにより、マウス軽鎖定常領域遺伝子を増幅した。さらにマウスIgG2a抗体重鎖定常領域のcDNA配列を鋳型とし、トリ抗体重鎖可変領域のセンスプライマー配列とマウスIgG2a抗体重鎖定常領域の配列を含むセンスプライマー(プライマー7:GCCAAAACAACAGCCCCATCGGTCTATCCACTGGCCCCT(配列番号33))、及びアンチセンスプライマー(プライマー8:AGATAGCGGCCGCTTATCATTTACCCGG(配列番号34))を用いたPCRにより、マウス重鎖定常領域遺伝子を増幅した。
上記で得られた抗Sema3Aトリ-マウスキメラ抗体について、CDR配列の決定を行った。CDR配列の決定は、Kabatらによる方法(Sequences of Proteins of Immunological Interest, NIH pubrication,91-3242)に従って行った。その結果、重鎖及び軽鎖のCDR1~3のアミノ酸配列として、下記配列が特定された。
1)ヒト化抗体(Humanized-1、Humanized-2)遺伝子の構築
前記実施例1の4)で得たクローンNo.4-2株が産生する抗Sema3A抗体の可変領域のフレームワーク領域を、ヒト抗体のフレームワーク領域のアミノ酸に変更することにより2種のヒト化抗体(Humanized-1及びHumanized-2)の設計を行なった。
トリ‐ヒトキメラ抗体遺伝子を次のように構築した。前記実施例1の4)で得たクローンNo.4-2株のcDNAを鋳型とし、トリ抗体λ軽鎖可変領域の配列を含むセンスプライマー(プライマーEγ:GAAGATCTAAGCTTCCACCATGGCCTGGGCTCCTCT(配列番号41))及びトリ抗体λ軽鎖可変領域の配列とヒト抗体λ軽鎖定常領域の配列を含むアンチセンスプライマー(プライマーFγ: CTTTGGGCTGGCCTAGGACGGTCAGGGTTGT(配列番号42))を用いたPCRにより、軽鎖可変領域遺伝子を増幅した。また、トリ抗体重鎖可変領域の配列を含むセンスプライマー(プライマーGγ:GAAGATCTAAGCTTCCACCATGAGCCCACTCG(配列番号43))、トリ抗体重鎖可変領域の領域とヒト抗体IgG1定常領域を含むアンチセンスプライマー(プライマーHγ: GCCCCTTTGTACTAGCGGAGGAGACGATGACTTC(配列番号44))を用いたPCRにより、重鎖可変領域遺伝子を増幅した。一方、合成したHumanized-1のヒト抗体λ軽鎖定常領域のDNA配列を鋳型とし、ヒト抗体λ軽鎖定常領域の配列を含むセンスプライマー(プライマーIγ: GGCCAGCCCAAAGCCAACCCTACCGTG(配列番号45))、及びアンチセンスプライマー(プライマーJγ: AGATAGCGGCCGCTTATTAGGAACATTCGGTT(配列番号46))を用いたPCRにより、ヒトλ軽鎖定常領域遺伝子を増幅した。さらに合成したHumanized-1のヒトIgG1抗体重鎖定常領域のDNA配列を鋳型とし、ヒトIgG1抗体重鎖定常領域の配列を含むセンスプライマー(プライマーKγ:GCTAGTACAAAGGGGCCCTCAGTGTTCCCACTG(配列番号47))、及びアンチセンスプライマー(プライマーLγ:AGATAGCGGCCGCTTATTATTTTCCAGGTGACAG(配列番号48))を用いたPCRにより、ヒトIgG1重鎖定常領域遺伝子を増幅した。
合成したHumanized-1の軽鎖遺伝子及び重鎖遺伝子、Humanized-2の軽鎖遺伝子及び重鎖遺伝子、並びにトリ‐ヒトキメラ抗体の軽鎖遺伝子及び重鎖遺伝子を、制限酵素HindIII及びNotIで処理した後、哺乳細胞発現用プラスミドpCEP4(Life Technoligies社)のHindIII-NotIサイトにそれぞれクローニングした。クローニングされた抗体遺伝子配列はDNAシークエンサーを用いたシークエンシングによって確認した。決定された塩基配列をもとに、ヒト化抗体(Humanized-1及びHumanized-2)とトリ‐ヒトキメラ抗体の重鎖と軽鎖のアミノ酸配列を翻訳した。
ヒトSema3Aタンパク質を抗原とし、ニワトリIgMの定常領域をマウスIgGに置換したDT40細胞を用いたこと以外は、実施例1に記載の1)及び2)と同様の手法で抗体のスクリーニングを行い、抗Sema3A抗体産生陽性を示すクローン8株を得た。抗Sema3A抗体産生陽性を示すクローンの中の165株及び582株を使用し、抗Sema3A抗体の製造及び精製を行うことにより、抗Sema3Aトリ-マウスキメラ抗体(IgG)(クローンNo.165株由来及びクローンNo.582株由来)を得た。各抗Sema3Aトリ-マウスキメラ抗体の重鎖及び軽鎖の可変領域及びCDR配列の決定を行った。CDR配列の決定は、Kabatらによる方法(Sequences of Proteins of Immunological Interest, NIH pubrication,91-3242)に従って行った。得られた結果を表2及び3に示す。
1)トリ抗体(IgM)の作製
マウスSema3Aタンパク質を抗原として、実施例1に記載の1)及び2)と同様の手法で抗体のスクリーニングを行い、抗Sema3A抗体産生陽性を示すクローンを得て、その中の240-40株及び255-72株を使用し、抗Sema3A抗体の製造及び精製を行うことにより、抗Sema3Aトリ抗体(クローンNo.240-40株由来及びクローンNo.255-72株由来)を得た。各抗Sema3A トリ抗体の重鎖及び軽鎖の可変領域及びCDR配列の決定を行った。CDR配列の決定は、Kabatらによる方法(Sequences of Proteins of Immunological Interest, NIH pubrication,91-3242)に従って行った。得られた結果を表4及び5に示す。
抗Sema3A抗体産生クローン240-40株及び255-72株から全RNAを抽出し、逆転写酵素(SuperScriptIII、Life Technologies社)を用いた逆転写反応によりcDNAの合成を行った。このcDNAを鋳型とし、トリ抗体λ軽鎖可変領域の配列を含むセンスプライマー(プライマーMγ:AGCTTGCTAGCGGCCACCATGGCCTGGGCTCCTCTC(配列番号91))、及びアンチセンスプライマー(プライマーNγ:TCTGGCGGCCGCTAGACTCACCTAGGACGGTCAGGGTTGTC(配列番号92))を用いたPCRにより、軽鎖可変領域遺伝子を増幅した。その一方で、トリ抗体重鎖可変領域の配列を含むセンスプライマー(プライマーOγ:AGCTTGCTAGCGGCCACCATGAGCCCACTCGTCTCC(配列番号93))、及びアンチセンスプライマー(プライマーPγ:TCTGGCGGCCGCTAGACTCACCGGAGGAGACGATGACTTC(配列番号94))を用いたPCRにより、重鎖可変領域遺伝子を増幅した。
実施例1で得られたトリ-マウスキメラ抗体、並びに実施例2で得られたヒト化抗体(Humanized-1及びHumanized-2)及びトリ-ヒトキメラ抗体について、抗原に対する特異性をPBS緩衝液を用いた抗原固相ELISAによって測定した。また、実施例1で得られたトリ-マウスキメラ抗体をビオチン標識したものについても、同様に、抗原に対する特異性を測定した。更に、ネガティブコントロールとしてCL18M(+)トリ-マウスキメラ抗体についても、同様に、抗原に対する特異性を測定した。具体的測定条件は、以下の通りである。
実施例1で得られたクローンNo.4-2由来のトリ-マウスキメラ抗体、並びに実施例2で得られたヒト化抗体(Humanized-1及びHumanized-2)及びトリ-ヒトキメラ抗体について、抗原に対する親和性の評価を競合ELISAにより行った。また、ネガティブコントロールとしてCL18M(+)トリ-マウス抗体についても、同様に、抗原に対する親和性を測定した。具体的測定条件は、以下の通りである。
実施例1で得られたクローンNo.4-2由来のトリ-マウスキメラ抗体、並びに実施例2で得られたヒト化抗体(Humanized-1及びHumanized-2)及びトリ-ヒトキメラ抗体について、BIACORE(GEヘルスケア社)を用いて抗原に対する親和性の評価を行った。具体的条件は、以下の通りである。
実施例2~4で得られた抗Sema3A抗体を用いて、サンドイッチELISAによるヒトSema3Aの測定を行った。具体的条件は、以下の通りである。
西洋ワサビペルオキシダーゼ(HRP)標識された抗マウスIgG抗体(GE #NA931;1%牛血清アルブミンを含むPBS(pH7.2)で1/1000に希釈)を各ウェルに25μL添加し、室温で1時間反応させた後に、洗浄液(0.05%Tween20を含むPBS、pH7.2)で5回洗浄した。その後、TMB(Dako、#S1599)20μLを加え、20分発色反応させ、1N硫酸20μLを加えて反応を停止させた。Infinite M1000(TECAN社)を用いて、450nmの吸光度を測定した。
ビオチン標識されたマウス抗トリIgM抗体(BeckmanCoulter #733087;1%牛血清アルブミンを含むPBS(pH7.2)で1/20000に希釈)を各ウェルに25μL添加し、室温で1時間反応させた後に、洗浄液(0.05%Tween20を含むPBS、pH7.2)で5回洗浄した。その後、西洋ワサビペルオキシダーゼ(HRP)標識されたストレプトアビジン(Thermo 21130;1%牛血清アルブミンを含むPBS(pH7.2)で10000倍希釈)を各ウェルに25μL添加し、室温で1時間反応させた後に、洗浄液(0.05%Tween20を含むPBS、pH7.2)で5回洗浄した。その後、TMB(Dako、#S1599)20μLを加え、20分発色反応させ、1N硫酸20μLを加えて反応を停止させた。Infinite M1000(TECAN社)を用いて、450nmの吸光度を測定した。
西洋ワサビペルオキシダーゼ(HRP)標識された抗ヤギIgG抗体(BETHYL社 #A50;1%牛血清アルブミンを含むPBS(pH7.2)で1/10000に希釈)を各ウェルに25μL添加し、室温で1時間反応させた後に、洗浄液(0.05%Tween20を含むPBS、pH7.2)で5回洗浄した。その後、TMB(Dako、#S1599)20μLを加え、20分発色反応させ、1N硫酸20μLを加えて反応を停止させた。Infinite M1000(TECAN社)を用いて、450nmの吸光度を測定した。
1)試験方法
ニワトリ7日胚の後根神経節(DRG)を摘出した。摘出したDRGをNGF含有培地250μL入りのPLL・ラミニンコートした24ウェルディッシュにて、37℃で16~20時間静置培養し、NGF感受性DRGニューロンが伸長しているのを確認した。別途、ニワトリ由来Sema3Aタンパク質(3nM)と、実施例1で得られた抗Sema3Aトリ抗体(培養上清)(抗体濃度として10μg/mL)又は抗Sema3Aトリ-マウスキメラ抗体(10μg/mL)とを含む混合液を氷上で30分間プレインキュベートした。この混合液を、ニワトリ由来Sema3Aタンパク質の最終濃度が、0、0.1、0.3nMになるように、前記24ウェルディッシュに添加し、37℃で30分間静置培養した。次いで、3.7%ホルマリン溶液で固定後、Alexa488-conjugated Phalloidinにて、培養ニューロンの軸索先端に形成される成長円錐の可視化を行った。蛍光顕微鏡下にて、全NGF感受性DRGニューロンのうち、成長円錐が退縮(collapse)しているニューロンの割合(%)を求めた。また、コントロールとして、抗Sema3Aトリ抗体又は抗Sema3Aトリ-マウスキメラ抗体の代わりに、抗ウサギIgG抗体又はマウスIgGを使用して、上記と同様に退縮アッセイを行った。
抗Sema3Aトリ抗体を使用した退縮アッセイの結果を図8に、抗Sema3Aトリ-マウスキメラ抗体を使用した退縮アッセイの結果を図9に示す。図8及び9から明らかなように、抗Sema3Aトリ抗体及び抗Sema3Aトリ-マウスキメラ抗体には、Sema3Aタンパク質によって成長円錐が退縮するのを抑制する作用があることが認められた。即ち、特定のアミノ酸配列のCDR1~3を有する抗Sema3A抗体は、Sema3Aタンパク質による成長円錐の退縮誘導能を効果的に抑制できることが明らかとなった。
1)試験方法
ニワトリ由来Sema3Aタンパク質の代わりにヒト由来Sema3Aタンパク質を使用したこと以外は、上記実施例9と同様の手法で、退縮アッセイを行った。
抗Sema3Aトリ抗体を使用した退縮アッセイの結果を図10に示す。図10から分かるように、抗Sema3Aトリ抗体がヒト由来Sema3Aタンパク質の機能を効果的に抑制し、成長円錐の退縮を抑制できていた。即ち、特定のアミノ酸配列のCDR1~3を有する抗Sema3A抗体は、ヒトにおいても、Sema3Aタンパク質による成長円錐の退縮誘導能を効果的に抑制できることが明らかとなった。
1)試験方法
ニワトリ7日胚の後根神経節(DRG)を摘出した。摘出したDRGをNGF含有培地250μL入りのPLL・ラミニンコートした24ウェルディッシュにて、37℃で16~20時間静置培養し、NGF感受性DRGニューロンが伸長しているのを確認した。別途、ヒト由来Sema3Aタンパク質(3nM)と、実施例2で得られた抗Sema3Aヒト化抗体(Humanized-2)(培養上清)(抗体濃度として100μg/mL)とを含む混合液を氷上で30分間プレインキュベートした。この混合液を、ヒト由来Sema3Aタンパク質の最終濃度が、0、0.05、0.1、0.3nMになるように、前記24ウェルディッシュに添加し、37℃で30分間静置培養した。次いで、3.7%ホルマリン溶液で固定後、Alexa488-conjugated Phalloidinにて、培養ニューロンの軸索先端に形成される成長円錐の可視化を行った。蛍光顕微鏡下にて、全NGF感受性DRGニューロンのうち、成長円錐が退縮(collapse)しているニューロンの割合(%)を求めた。また、コントロールとして、抗Sema3Aヒト化抗体の代わりに、Human IgG-lambdaを使用して、上記と同様に退縮アッセイを行った。
抗Sema3Aヒト化抗体を使用した退縮アッセイの結果を図11に示す。図11から明らかなように、抗Sema3Aヒト化抗体には、Sema3Aタンパク質によって成長円錐が退縮するのを抑制する作用があることが認められた。即ち、特定のアミノ酸配列のCDR1~3を有する抗Sema3A抗体は、ヒト化した場合にもSema3Aタンパク質による成長円錐の退縮誘導能を効果的に抑制できることが明らかとなった。
1)試験方法
野生型COS-7細胞、又はEGFP(Enhanced Green Fluorescent Protein)融合NRP1(Neuropilin-1)を発現するように形質転換させたCOS-7細胞(NRP1発現COS-7細胞)を準備した。別途、アルカリフォスファターゼ結合ヒト由来Sema3Aタンパク質(0.1nM)と、実施例1で得られたトリ抗Sema3A抗体(培養上清)(抗体濃度として10μg/mL)又は抗Sema3Aトリ-マウスキメラ抗体(10μg/mL)とを含むDMEM培地を氷上で30分間プレインキュベートしておき、この培地500μLを、予めHBHバッファー(20mM sodium HEPES,pH7.00,0.05容量%BSA,及び10容量%FBSを含むハンクス平衡塩類溶液)でブロッキングしておいた野生型COS-7細胞又はNRP1発現COS-7細胞に添加して、氷上で1時間静置した。次いで、HBHバッファーを用いて細胞を4回洗浄した後、4%ホルムアルデヒドを用いて細胞の固定化を行った。固定化された細胞をHHバッファー(20mM sodium HEPES,pH7.00を含むハンクス平衡塩類溶液)で1回洗浄した後、500μLのアルカリフォスファターゼ基質(NBT/BCIP:nitro-blue tetrazolium chloride/ 5-bromo-4-chloro-3’-indolyphosphate p-toluidine salt)を添加して、室温で一晩静置した。その後、顕微鏡にて細胞の発色の程度を観察した。また、コントロールとして、アルカリフォスファターゼ結合ヒト由来Sema3Aタンパク質を添加しなかったこと、或いは抗Sema3Aトリ抗体又は抗Sema3Aトリ-マウスキメラ抗体の代わりにウサギIgGを使用したこと以外は、上記と同様の手法で、結合アッセイを行った。なお、本試験に供したNRP1発現COS-7細胞において、NRP1発現量に差がないことは、細胞から発せられるEGFPの蛍光強度から確認している。
抗Sema3Aトリ抗体を使用した結合アッセイの結果を図12に、抗Sema3Aトリ-マウスキメラ抗体を使用した結合アッセイの結果を図13に示す。図12及び13から明らかなように、トリ抗Sema3A抗体又は抗Sema3Aトリ-マウスキメラ抗体と、ヒト由来Sema3Aタンパク質との混合物を添加した場合には、NRP1発現COS-7細胞へのヒト由来Sema3Aタンパク質の結合が阻害された。即ち、本結果から、特定のアミノ酸配列のCDR1~3を有する抗Sema3A抗体は、ヒト由来Sema3Aタンパク質に結合し、ヒト由来Sema3Aタンパク質とNRP1との結合を阻害できることが明らかとなった。
1)試験方法
NRP1発現COS-7細胞の代わりに、EGFP融合NRP2(Neuropilin-2)を発現するように形質転換したCOS-7細胞(NRP2発現COS-7細胞)を使用し、且つアルカリフォスファターゼ結合ヒト由来Sema3Aタンパク質の代わりにアルカリフォスファターゼ結合ヒト由来Sema3Fタンパク質を使用したこと以外は、上記実施例12と同様の手法で、結合アッセイを行った。
抗Sema3Aトリ抗体を使用した結合アッセイの結果を図14に示す。図14から分かるように、抗Sema3Aトリ抗体と、ヒト由来Sema3Fタンパク質との混合物を添加した場合でも、ヒト由来Sema3Fタンパク質のNRP2発現COS-7細胞への結合は阻害されなかった。即ち、本結果から、特定のアミノ酸配列のCDR1~3を有する抗Sema3A抗体は、Sema3Aタンパク質と相同性の高いサブファミリー分子であるSema3Fタンパク質には結合せず、Sema3Aタンパク質に対して特異的に結合する可能性が示唆された。
1)試験材料
1-1)Aβ(25-35)及びAβ(35-25)
Aβ(25-35)(Amyloid β-Protein(25-35))(Bachem,#H-1192)及びAβ(35-25)(Amyloid β-Protein(35-25))(Bachem,#H-2964)を蒸留水にて1mg/mLに調製した後、37℃にて4日間インキュベートし、Aβ(25-35)溶液及びAβ(35-25)溶液を調製した。この操作により、Aβ(25-35)は凝集し、細胞毒性を獲得するようになる。また、Aβ(35-25)は、活性型であるAβ25-35のアミノ酸配列を逆転させた不活性型のAβであり、上記操作後でも細胞毒性を発揮しない。Aβ(35-25)は、ネガティブコントロールとして使用した。
前記実施例1で作製した抗Sema3Aトリ-マウスキメラ抗体を生理食塩水にて1mg/mLに調製し、抗Sema3A抗体溶液を調製した。
1mg/mLのnormal mouse IgG(Calbiochemより入手)をnormal IgG溶液として用いた。
日本チャールス・リバー社より購入した3週齢のマウス(C57BL/6J)を使用した。
Novel Object Recognition Task (NORT)を以下の方法に従い行った。
まず、3週齢のマウスを用意し、マウスが実験者に慣れるよう、2週間の間、一日10分間、実験者の手の上にマウスをのせるという、ハンドリング作業を行った。次に、マウスを表8に示す5群に分けて、表8に示す条件でAβ及び抗体を脳室内に投与した。これらの溶液は、bregmaから右下1mmに投与した。投与には、マイクロシリンジの針先にテフロンチューブ(エイコム)を連結させ、その先端に針先3.3mmの部分を直角にした27Gの注射針(テルモ)を挿入したものを用いた。
物体A:水平方向に延びる基部(緑色)と、その中央から垂直に伸びる延長部(緑色)とで構成され、正面視T字型に形成されている。当該基部と延長部の形状は、共に直方体である。
物体B:基部が黄色、延長部が円柱形状で赤色であること以外は、上記物体Aと同様である。
物体C:基部が黄色、延長部が側面の正面視で円弧状にくり抜かれた形状で黄色であること以外は、上記物体Aと同様である。
物体A、B、及びCの基部をテストケージの底面に配置し、延長部が上方に延びるように設置した。
獲得試行において、物体A及び物体Bへの合計アクセス時間に対する、物体Bへのアクセス時間の割合(物体Bに対するアクセス率)を算出した。獲得試行時は物体A及び物体B共に、新規物体であるので、どちらも同程度にアクセスすることが見込まれる。この時どちらかの物体に偏ったアクセスを示した場合は、薬物投与によって、認知機能等が障害された可能性があるので、本実験では評価できないということになる。更に、テスト試行において、物体A及び物体Cへの合計アクセス時間に対する、物体Cへのアクセス時間の割合(物体Cに対するアクセス率)を算出した。テスト試行時は物体Cのみが新規物体であるので、もし物体Aについての記憶を獲得していれば、物体Cに偏ったアクセスが見込まれる。以上から算出した物体Bに対するアクセス率、物体Cに対するアクセス率を、それぞれ一元配置分散分析を用いて統計解析を行った。群間比較はTukey-KramerのHSD検定により行った。
得られた結果を図15A(獲得試行)及び15B(テスト試行)に示す。
実施例2で得られたトリ-マウスキメラ抗体について、敗血症の疑似病態モデルとされるLipopolysaccharide 接種モデルを用い、致死的炎症病態に対する影響を分析した。
1-1)Lipopolysaccharide
Lipopolysaccharide(以下LPS、Sigma社、Lot番号:032M4082V)を生理食塩水にて6mg/mLの濃度に調製した。
実施例2で作製した抗Sema3Aトリ-マウスキメラ抗体を生理食塩水にて5mg/mLに希釈し、抗Sema3A抗体溶液とした。
ニワトリB細胞由来のDT40細胞によって構築される抗体ライブラリーを用いて作製された非特異的トリ‐マウスキメラ抗体(IgG)(陰性対照用キメラ抗体)を、生理食塩水にて5mg/mLに希釈し、陰性対照用キメラ抗体溶液とした。
日本チャールス・リバー社より購入した雄性6週齢のマウス(C57BL/6J)を使用した。
入荷後、飼育室で馴化させたC57BL/6Jマウスを1群5匹に分けて、表9に示す試験群を設定した。各抗体を尾静脈より投与した後、30分後にLPSを腹腔内に接種した。これらの処置を施した日をDay0とし、Day4までの各個体の生死を観察し、効力判定の指標とした。同一の試験を計3回実施し再現性の確認を行った。
投与4日以前の死亡例を生存期間の延長なし(無効)とし、4日まで生存した例を生存期間の延長有り(有効)と判定した。これらの2値化データについて、Breslow-Day検定を用いて3試験間の同一性を判定した上で、Cochran-Mantel-Haenzel検定により3試験を併合した有効率の差を判定した。生存期間延長効果はlog-rank検定を適用し判定した。
得られた結果を表10に示す。表10から明らかなように、3試験すべてにおいて抗Sema3Aトリ-マウスキメラ抗体投与群の生存率が、陰性対照である非特異配列キメラ抗体投与群に比べ優れていた。これらの試験間での非乖離性を確認するため実施したBresslow-Day検定が有意ではないことから3試験間に乖離はないと判断した(p=0.6202)。そのうえで3試験の結果を併合し、Cochran-Mantel-Haenzel検定を実施したところ、p=0.0364となり、陰性対照群と抗Sema3Aトリ-マウスキメラ抗体群間の生存率には有意水準5%で統計的有意差が認められた。これらの結果から抗Sema3Aトリ-マウスキメラ抗体は、致死的炎症病態を有するモデル動物において生存率を改善する効果を示し、かつその効果には再現性のあることが明らかとなった。
実施例2で得られた抗Sema3Aヒト化抗体についても、実施例12と同様にLPS誘発炎症疾患モデルに対する影響を分析した。
1-1)LPS
LPSの調製は実施例15と同様に行った。
実施例2で作製した抗Sema3Aヒト化抗体(Humanized-2)を生理食塩水にて希釈し、抗Sema3Aヒト化抗体溶液とした。抗Sema3Aヒト化抗体500μg投与群では、抗Sema3Aヒト化抗体(Humanized-2)を5mg/mLの濃度に調製したものを用い、抗Sema3Aヒト化抗体100μg投与群では、抗Sema3Aヒト化抗体(Humanized-2)を1mg/mLの濃度に調製したものを用いた。
非特異的なヒトポリクローナル抗体(human IgG POLYCLONAL Isotype Control; BioXCell社、#BE0092)を、生理食塩水にて5mg/mLに希釈し、陰性対照用ヒト抗体溶液とした。
日本チャールス・リバー社より購入した雄性6週齢のマウス(C57BL/6J)を使用した。
入荷後、飼育室で馴化させたC57BL/6Jマウスを1群10匹に分けて、表11に示す試験群を設定した。各抗体を尾静脈より投与した後、30分後にLPSを腹腔内に接種した。これらの処置を施した日をDay0とし、Day4までの各個体の生死を観察し、効力判定の指標とした。
実施例12と同様に無効及び有効の2値化した生存割合に対し、片側によるCochran-Armitage検定を用い、想定される3種類の用量反応型、即ち直線的増加、低用量飽和、及び高用量立ち上がり型の対比を設定して生存率改善効果の用量反応性を判定した。また、生存期間延長効果はlog-rank検定を適用し判定した。
表12に各群の生存率を示す。抗Sema3Aヒト化抗体投与100μg投与群では、陰性対照群(30%)の2倍の生存率(60%)となり、更に500μg投与群は、100μg投与群の結果を上回る生存率(80%)が観察された。抗Sema3Aヒト化抗体両用量において生存率が改善され、かつ用量依存傾向が認められた。
実施例16で得られた結果をもとに、抗Sema3Aヒト化抗体の効果について、LPSを先に接種し、病態を誘発した後からの処置による影響を判定した。
1-1)LPS
LPSの調製は実施例15と同様に行った。
実施例2で作製した抗Sema3Aヒト化抗体(Humanized-2)を生理食塩水にて希釈し、抗Sema3Aヒト化抗体溶液とした。抗Sema3Aヒト化抗体500μg、250μg、125μg投与群では、抗Sema3Aヒト化抗体(Humanized-2)をそれぞれ5、2.5、1.25mg/mLの濃度に調製したものを用いた。
実施例16と同様の方法で、陰性対照用ヒト抗体溶液を調製した。
日本チャールス・リバー社より購入した雄性6週齢のマウス(C57BL/6J)を使用した。
入荷後、飼育室で馴化させたC57BL/6Jマウスを1群10匹に分けて、表13に示す試験群を設定した。本試験では実施例15及び16と異なり、実医療に則した使用を想定し、既に炎症が惹起された後からの投与による効力判定を行った。まずLPSを腹腔内に接種し炎症反応を惹起した。各抗体はLPS腹腔内接種1時間後に尾静脈より投与した。これらの処置を施した日をDay0とし、Day4までの各個体の生死を観察し、効力判定の指標とした。
陰性対照群を含む4群がとり得る用量反応型として、(1)実用量の直線回帰、(2)直線増加、(3)中用量立上り、(4)高用量立上り、(5)中用量飽和、(6)低用量飽和、及び(7)中用量立上り飽和型、の7つのタイプが想定される。この計7種の反応パターンに対しCochran-Armitage検定を用い、並べ替え再抽出法で多重調整したp値を算出したうえで、そのp値が最小となる対比反応型を適切な用量反応型として採択することとした。また、生存期間延長効果はlog-rank検定を適用し判定した。
表14に投与4日後の各群の生存率を示す。陰性対照群の生存率は10%であったが、LPS接種後1時間に抗Sema3Aヒト化抗体125、250、及び500μgを投与した各群の生存率はそれぞれ、30、40、100%であり、用量増加に伴い生存率も上がる傾向が観察された。特に、抗Sema3Aヒト化抗体500μg投与群ではマウス10匹全個体が生存するという特筆すべき効果が確認された。
敗血症や固形腫瘍等に伴う播種性血管内凝固症候群(Dissemminated Intravascular Coagulation、以下DIC)では、血中Plasminogen activator inhibitor-1(以下PAI-1)が増加し、線溶系を抑制することから多臓器不全を起こし重篤化する。血中PAI-1量の増加を抑制できれば播種性血管内凝固症候群の進行を阻止できると考えられることから、抗Sema3Aヒト化抗体の血中PAI-1量変動への影響を検討した。
1-1)LPS
LPS(Sigma社、Lot番号:102M4017V)を生理食塩水にて1.5mg/mLの濃度に調製した。
実施例2で作製した抗Sema3Aヒト化抗体(Humanized-2)を生理食塩水にて5mg/mLの濃度に調製した。
日本チャールス・リバー社より購入した雄性6週齢のマウス(C57BL/6J)を使用した。
2-1)群構成
入荷後、飼育室で馴化させたC57BL/6Jマウスを1群5匹に分けて、表16に示す試験群を設定した。
LPSは15mg/kgの接種量となるよう腹腔内に接種した。抗体溶液は500μg/マウスの接種量となるよう、5mg/mL溶液を0.1mL尾静脈より投与した。なお、LPS非接種の条件1群には生理食塩水を腹腔内接種した。
LPS接種から1.5、3、及び9時間後に血漿を採取し、マウスPAI-1 ELISA kit(Innovative Research社)を用い処置動物の血中PAI-1濃度を測定した。
抗体非投与群に対する抗体投与群について両側Studentのt検定によりp値を算出し、有意水準5%以内の場合、統計有意性があるものと判断した。
図19に血中PAI-1濃度の測定結果を示す。LPS非処置群(条件1)では血中PAI-1量はごくわずかであったが、LPS接種後の血中PAI-1量は1.5時間から増加傾向を示し、3時間後及び9時間後ではLPS非処置動物に比べ著しく増加していた(条件2、5及び8)。
外科治療、又は化学療法寛解導入後に起こる播種性転移や遠隔転移により、癌患者の生存率が大きく低下することが知られている。癌細胞の遊走能の活性化は、癌細胞が原発巣から離脱し、播種性転移や遠隔転移につながることから、Sema3Aによって誘導されるがんの悪性化に対する抗Sema3Aヒト化抗体の影響を確認した。
実施例2で作製した抗Sema3Aヒト化抗体(Humanized-2)を用い、Sema3Aにより誘導される癌細胞遊走能に与える影響について評価した。ここでは、Sema3A高発現患者の予後が不良とされている膵癌の細胞を用いてその効果を検討した。具体的な実験手法は以下の通りである。
多重性を考慮し、条件2に対する条件3~5、及び条件6~8について両側Dunnett検定によりp値を算出し、有意水準5%以内の場合、統計有意性があるものと判断した。
得られた結果を図20に示す。ヒトSema3Aの刺激により膵癌細胞MIAPaCa-2の遊走活性は2倍以上亢進した(条件2)。しかし、抗Sema3Aヒト化抗体存在下では、Sema3Aにより誘導される膵癌細胞の遊走活性が抑制された(条件3~5)。抗Sema3A抗体1μg/mL処置群(P=0.0031)と10μg/mL処置群(P<0.001)では、Sema3A非処置群(条件1)と同程度にまで癌細胞の遊走が抑制されていた。一方、陰性対照群では、Sema3Aによる癌細胞遊走を抑制しなかった(条件6~8)。即ち、本結果から、特定のアミノ酸配列のCDRを有する抗Sema3A抗体は、Sema3Aにより誘導される膵癌細胞の遊走を特異的に抑制することが明らかとなった。
遊走能が活性化された癌細胞は周辺の細胞外基質を融解・浸潤し、基底膜を破壊して血管やリンパ管に移行し、やがて遠隔臓器へと転移する。癌細胞の浸潤・転移能の抑制は、転移性再発の抑止につながり、癌患者の生存率を改善する上で有用と考えられている。実施例19で検討したSema3A誘導性遊走能亢進に対する抑制効果に加え、癌細胞浸潤能に対する抗Sema3Aヒト化抗体の効果についても検討した。
増殖因子を除去した細胞外基質(BD社、MatrigelTM Growth Factor Redused)を内層に充填したインベージョンチャンバー(BD社、BioCoatTM 8μm孔、#354483)を用いてSema3A誘導性の癌細胞浸潤能に対する抗Sema3Aヒト化抗体が与える影響について評価した。具体的な実験手法は以下の通りである。
Sema3A誘導性の浸潤活性に対する阻害作用について、多重性を考慮し、条件5に対する条件6~8および条件9~10についてそれぞれ両側Dunnett検定によりp値を算出し、有意水準5%以内の場合、統計有意性があるものと判断した。p値一覧を表19に示す。
図21にMIAPaCa-2細胞を用いた場合、図22にU87MG細胞を用いた場合、及び図23に3LL細胞を用いた場合について、各条件での浸潤細胞を計測した結果を示す。Sema3Aを添加した場合(条件5)、いずれの細胞株においてもSema3A非刺激群(条件1)に比べ癌細胞の浸潤能が明らかに亢進していた。そして、ヒトSema3Aと共に抗Sema3Aヒト化抗体を添加した場合、いずれの癌細胞株においてもSema3A非刺激と同程度にまで癌細胞の浸潤が抑制されていた(条件6~8)。Sema3A非刺激条件下では、3LLを除いて特筆すべき浸潤抑制作用は観察されなかった(条件1~4)。一方、陰性対照抗体添加群ではSema3Aによる癌細胞浸潤能の亢進をほとんど抑制しなかった(条件9~11)。また、図24には浸潤した3LL細胞の顕微鏡下撮影像を示す。Sema3A非添加の条件1に対し、Sema3Aを添加した条件5において癌細胞浸潤が顕著であること、Sema3A存在下で抗Sema3Aヒト化抗体を処置した条件7および条件8において、Sema3Aによって誘導される癌細胞の浸潤活性が明らかに抑制されていることが可視的にも確認できる。これらの結果から明らかなように、特定のアミノ酸配列のCDRを有する抗Sema3A抗体は、Sema3Aによって誘導される癌細胞の浸潤活性をSema3A非刺激と同程度にまで抑制する作用を有することが明らかとなった。
膵癌は、がんの中でも5年生存率が非常に低いことが知られている。その原因として、膵癌組織は乏血管性の状態となっているが、このような栄養飢餓状態でも癌細胞の増殖・進展が起こり、しばしばゲムシタビン塩酸塩(以下GEM)等の抗がん剤に対し耐性化を示すことが挙げられる。即ち、膵癌治療において、抗がん剤不応答性の克服は重要な課題となっていることから、栄養飢餓条件におけるSema3Aの薬剤耐性誘導と抗Sema3Aヒト化抗体によるその解除作用について評価した。
膵癌の特徴である栄養飢餓条件において、Sema3Aにより誘導されるGEM不応答性に対するヒト化抗Sema3A抗体(実施例2で作製したhumanized-2)が与える影響について評価した。具体的な実験手法は以下の通りである。
条件3に対する条件4~6、及び条件7~9について多重性を考慮し、両側Dunnett検定を実施しによりp値を算出し、有意水準5%以内の場合、統計有意性があるものと判断した。p値を表21に示す。
図25に膵癌細胞増殖判定試験の結果を示す。GEMにより膵癌細胞の増殖は抑制されたが(条件1に対し条件2)、ヒトSema3Aは、GEMに対する感受性を減弱させ薬剤不応答性を誘導した(条件2に対し条件3)。このSema3Aにより誘導されるGEM抵抗性は、抗Sema3Aヒト化抗体1ないし10μg/mL処置により解除され、Sema3A非存在条件と同程度にまでGEM感受性を回復させることが示された(条件3に対し条件4~6)。一方、陰性対照ヒト抗体添加群では、Sema3A非存在条件と同程度までのGEM感受性を回復することはできなかった(条件7~9)。本結果から、実施例2によって作製したヒト化抗Sema3A抗体には、膵癌組織のような栄養飢餓条件において、Sema3Aにより誘導される抗がん剤抵抗性を解除する作用も有していることが明らかとなった。
配列番号2は、トリ-マウスキメラ抗体(クローンNo.4-2株由来)の重鎖CDR2のアミノ酸配列である。
配列番号3は、トリ-マウスキメラ抗体(クローンNo.4-2株由来)の重鎖CDR3のアミノ酸配列である。
配列番号4は、トリ-マウスキメラ抗体(クローンNo.4-2株由来)の軽鎖CDR1のアミノ酸配列である。
配列番号5は、トリ-マウスキメラ抗体(クローンNo.4-2株由来)の軽鎖CDR2のアミノ酸配列である。
配列番号6は、トリ-マウスキメラ抗体(クローンNo.4-2株由来)の軽鎖CDR3のアミノ酸配列である。
配列番号7は、トリ-ヒトキメラ抗体(クローンNo.4-2株由来)の重鎖可変領域のアミノ酸配列である。
配列番号8は、トリ-ヒトキメラ抗体(クローンNo.4-2株由来)の重鎖可変領域のアミノ酸配列である。
配列番号9は、トリ-ヒトキメラ抗体(クローンNo.4-2株由来)の軽鎖可変領域のアミノ酸配列である。
配列番号10は、トリ-ヒトキメラ抗体(クローンNo.4-2株由来)の軽鎖可変領域のアミノ酸配列である。
配列番号11は、ヒト化抗体(クローンNo.4-2株由来)の重鎖可変領域のアミノ酸配列である。
配列番号12は、ヒト化抗体(Humanized -1)(クローンNo.4-2株由来)の軽鎖可変領域のアミノ酸配列である。
配列番号13は、ヒト化抗体(Humanized -2)(クローンNo.4-2株由来)の軽鎖可変領域のアミノ酸配列である。
配列番号14は、トリ-ヒトキメラ抗体(クローンNo.4-2株由来)の重鎖可変領域のアミノ酸配列(配列番号7)をコードしている塩基配列である。
配列番号15は、トリ-ヒトキメラ抗体(クローンNo.4-2株由来)の重鎖可変領域のアミノ酸配列(配列番号8)をコードしている塩基配列である。
配列番号16は、トリ-ヒトキメラ抗体(クローンNo.4-2株由来)の軽鎖可変領域のアミノ酸配列(配列番号9)をコードしている塩基配列である。
配列番号17は、トリ-ヒトキメラ抗体(クローンNo.4-2株由来)の軽鎖可変領域のアミノ酸配列(配列番号10)をコードしている塩基配列である。
配列番号18は、トリ-マウスキメラ抗体(クローンNo.4-2株由来)の重鎖CDR1のアミノ酸配列をコードしている塩基配列である。
配列番号19は、トリ-マウスキメラ抗体(クローンNo.4-2株由来)の重鎖CDR2のアミノ酸配列をコードしている塩基配列である。
配列番号20は、トリ-マウスキメラ抗体(クローンNo.4-2株由来)の重鎖CDR3のアミノ酸配列をコードしている塩基配列である。
配列番号21は、トリ-マウスキメラ抗体(クローンNo.4-2株由来)の軽鎖CDR1のアミノ酸配列をコードしている塩基配列である。
配列番号22は、トリ-マウスキメラ抗体(クローンNo.4-2株由来)の軽鎖CDR2のアミノ酸配列をコードしている塩基配列である。
配列番号23は、トリ-マウスキメラ抗体(クローンNo.4-2株由来)の軽鎖CDR3のアミノ酸配列をコードしている塩基配列である。
配列番号24は、ヒト化抗体(クローンNo.4-2株由来)の重鎖可変領域のアミノ酸配列(配列番号11)をコードしている塩基配列である。
配列番号25は、ヒト化抗体(Humanized -1)(クローンNo.4-2株由来)の軽鎖可変領域のアミノ酸配列(配列番号12)をコードしている塩基配列である。
配列番号26は、ヒト化抗体(Humanized -2)(クローンNo.4-2株由来)の軽鎖可変領域のアミノ酸配列(配列番号13)をコードしている塩基配列である。
配列番号27は、前記プライマー1の塩基配列である。
配列番号28は、前記プライマー2の塩基配列である。
配列番号29は、前記プライマー3の塩基配列である。
配列番号30は、前記プライマー4の塩基配列である。
配列番号31は、前記プライマー5の塩基配列である。
配列番号32は、前記プライマー6の塩基配列である。
配列番号33は、前記プライマー7の塩基配列である。
配列番号34は、前記プライマー8の塩基配列である。
配列番号35は、トリ-マウスキメラ抗体(クローンNo.4-2株由来)の重鎖のアミノ酸配列である。
配列番号36は、トリ-マウスキメラ抗体(クローンNo.4-2株由来)の軽鎖のアミノ酸配列である。
配列番号37は、前記プライマーAγの塩基配列である。
配列番号38は、前記プライマーBγの塩基配列である。
配列番号39は、前記プライマーCγの塩基配列である。
配列番号40は、前記プライマーDγの塩基配列である。
配列番号41は、前記プライマーEγの塩基配列である。
配列番号42は、前記プライマーFγの塩基配列である。
配列番号43は、前記プライマーGγの塩基配列である。
配列番号44は、前記プライマーHγの塩基配列である。
配列番号45は、前記プライマーIγの塩基配列である。
配列番号46は、前記プライマーJγの塩基配列である。
配列番号47は、前記プライマーKγの塩基配列である。
配列番号48は、前記プライマーLγの塩基配列である。
配列番号49は、ヒト化抗体(Humanized -1及びHumanized -2)(クローンNo.4-2株由来)の重鎖のアミノ酸配列である。
配列番号50は、ヒト化抗体(Humanized -1及びHumanized -2)(クローンNo.4-2株由来)の重鎖のアミノ酸配列をコードしている塩基配列である。
配列番号51は、ヒト化抗体(Humanized -1)(クローンNo.4-2株由来)の軽鎖のアミノ酸配列である。
配列番号52は、ヒト化抗体(Humanized -1)(クローンNo.4-2株由来)の軽鎖のアミノ酸配列をコードしている塩基配列である。
配列番号53は、ヒト化抗体(Humanized -2)(クローンNo.4-2株由来)の軽鎖のアミノ酸配列である。
配列番号54は、ヒト化抗体(Humanized -2)(クローンNo.4-2株由来)の軽鎖のアミノ酸配列をコードしている塩基配列である。
配列番号55は、トリ-ヒトキメラ抗体(クローンNo.4-2株由来)の重鎖のアミノ酸配列である。
配列番号56は、トリ-ヒトキメラ抗体(クローンNo.4-2株由来)の重鎖のアミノ酸配列をコードしている塩基配列である。
配列番号57は、トリ-ヒトキメラ抗体(クローンNo.4-2株由来)の軽鎖のアミノ酸配列である。
配列番号58は、トリ-ヒトキメラ抗体(クローンNo.4-2株由来)の軽鎖のアミノ酸配列をコードしている塩基配列である。
配列番号59は、トリ-マウスキメラ抗体(クローンNo.165株由来)の重鎖可変領域のアミノ酸配列である。
配列番号60は、トリ-マウスキメラ抗体(クローンNo.165株由来)の重鎖CDR1のアミノ酸配列である。
配列番号61は、トリ-マウスキメラ抗体(クローンNo.165株由来)の重鎖CDR2のアミノ酸配列である。
配列番号62は、トリ-マウスキメラ抗体(クローンNo.165株由来)の重鎖CDR3のアミノ酸配列である。
配列番号63は、トリ-マウスキメラ抗体(クローンNo.165株由来)の軽鎖可変領域のアミノ酸配列である。
配列番号64は、トリ-マウスキメラ抗体(クローンNo.165株由来)の軽鎖CDR1のアミノ酸配列である。
配列番号65は、トリ-マウスキメラ抗体(クローンNo.165株由来)の軽鎖CDR2のアミノ酸配列である。
配列番号66は、トリ-マウスキメラ抗体(クローンNo.165株由来)の軽鎖CDR3のアミノ酸配列である。
配列番号67は、トリ-マウスキメラ抗体(クローンNo.582株由来)の重鎖可変領域のアミノ酸配列である。
配列番号68は、トリ-マウスキメラ抗体(クローンNo.165株由来)の重鎖CDR1のアミノ酸配列である。
配列番号69は、トリ-マウスキメラ抗体(クローンNo.165株由来)の重鎖CDR2のアミノ酸配列である。
配列番号70は、トリ-マウスキメラ抗体(クローンNo.165株由来)の重鎖CDR3のアミノ酸配列である。
配列番号71は、トリ-マウスキメラ抗体(クローンNo.582株由来)の軽鎖可変領域のアミノ酸配列である。
配列番号72は、トリ-マウスキメラ抗体(クローンNo.582株由来)の軽鎖CDR1のアミノ酸配列である。
配列番号73は、トリ-マウスキメラ抗体(クローンNo.582株由来)の軽鎖CDR2のアミノ酸配列である。
配列番号74は、トリ-マウスキメラ抗体(クローンNo.582株由来)の軽鎖CDR3のアミノ酸配列である。
配列番号75は、トリ抗体(クローンNo.240-40株由来)の重鎖可変領域のアミノ酸配列である。
配列番号76は、トリ抗体(クローンNo.240-40株由来)の重鎖CDR1のアミノ酸配列である。
配列番号77は、トリ抗体(クローンNo.240-40株由来)の重鎖CDR2のアミノ酸配列である。
配列番号78は、トリ抗体(クローンNo.240-40株由来)の重鎖CDR3のアミノ酸配列である。
配列番号79は、トリ抗体(クローンNo.240-40株由来)の軽鎖可変領域のアミノ酸配列である。
配列番号80は、トリ抗体(クローンNo.240-40株由来)の軽鎖CDR1のアミノ酸配列である。
配列番号81は、トリ抗体(クローンNo.240-40株由来)の軽鎖CDR2のアミノ酸配列である。
配列番号82は、トリ抗体(クローンNo.240-40株由来)の軽鎖CDR3のアミノ酸配列である。
配列番号83は、トリ抗体(クローンNo.255-72株由来)の重鎖可変領域のアミノ酸配列である。
配列番号84は、トリ抗体(クローンNo.255-72株由来)の重鎖CDR1のアミノ酸配列である。
配列番号85は、トリ抗体(クローンNo.255-72株由来)の重鎖CDR2のアミノ酸配列である。
配列番号86は、トリ抗体(クローンNo.255-72株由来)の重鎖CDR3のアミノ酸配列である。
配列番号87は、トリ抗体(クローンNo.255-72株由来)の軽鎖可変領域のアミノ酸配列である。
配列番号88は、トリ抗体(クローンNo.255-72株由来)の軽鎖CDR1のアミノ酸配列である。
配列番号89は、トリ抗体(クローンNo.255-72株由来)の軽鎖CDR2のアミノ酸配列である。
配列番号90は、トリ抗体(クローンNo.255-72株由来)の軽鎖CDR3のアミノ酸配列である。
配列番号91は、前記プライマーMγの塩基配列である。
配列番号92は、前記プライマーNγの塩基配列である。
配列番号93は、前記プライマーOγの塩基配列である。
配列番号94は、前記プライマーPγの塩基配列である。
配列番号95は、ヒト化抗体の重鎖定常領域のアミノ酸配列をコードしている塩基配列である。
配列番号96は、ヒト化抗体の軽鎖定常領域のアミノ酸配列をコードしている塩基配列である。
配列番号97は、トリ-マウスキメラ抗体(クローンNo.165株由来)の重鎖CDR1のアミノ酸配列(配列番号60)をコードしている塩基配列である。
配列番号98は、トリ-マウスキメラ抗体(クローンNo.165株由来)の重鎖CDR2のアミノ酸配列(配列番号61)をコードしている塩基配列である。
配列番号99は、トリ-マウスキメラ抗体(クローンNo.165株由来)の重鎖CDR3のアミノ酸配列(配列番号62)をコードしている塩基配列である。
配列番号100は、トリ-マウスキメラ抗体(クローンNo.165株由来)の軽鎖CDR1のアミノ酸配列(配列番号64)をコードしている塩基配列である。
配列番号101は、トリ-マウスキメラ抗体(クローンNo.165株由来)の軽鎖CDR2のアミノ酸配列(配列番号65)をコードしている塩基配列である。
配列番号102は、トリ-マウスキメラ抗体(クローンNo.165株由来)の軽鎖CDR3のアミノ酸配列(配列番号66)をコードしている塩基配列である。
配列番号103は、トリ-マウスキメラ抗体(クローンNo.165株由来)の重鎖可変領域のアミノ酸配列(配列番号59)をコードしている塩基配列である。
配列番号104は、トリ-マウスキメラ抗体(クローンNo.165株由来)の軽鎖可変領域のアミノ酸配列(配列番号63)をコードしている塩基配列である。
配列番号105は、トリ-マウスキメラ抗体(クローンNo.582株由来)の重鎖CDR1のアミノ酸配列(配列番号68)をコードしている塩基配列である。
配列番号106は、トリ-マウスキメラ抗体(クローンNo.582株由来)の重鎖CDR2のアミノ酸配列(配列番号69)をコードしている塩基配列である。
配列番号107は、トリ-マウスキメラ抗体(クローンNo.582株由来)の重鎖CDR3のアミノ酸配列(配列番号70)をコードしている塩基配列である。
配列番号108は、トリ-マウスキメラ抗体(クローンNo.582株由来)の軽鎖CDR1のアミノ酸配列(配列番号72)をコードしている塩基配列である。
配列番号109は、トリ-マウスキメラ抗体(クローンNo.582株由来)の軽鎖CDR2のアミノ酸配列(配列番号73)をコードしている塩基配列である。
配列番号110は、トリ-マウスキメラ抗体(クローンNo.582株由来)の軽鎖CDR3のアミノ酸配列(配列番号74)をコードしている塩基配列である。
配列番号111は、トリ-マウスキメラ抗体(クローンNo.582株由来)の重鎖可変領域のアミノ酸配列(配列番号67)をコードしている塩基配列である。
配列番号112は、トリ-マウスキメラ抗体(クローンNo.582株由来)の軽鎖可変領域のアミノ酸配列(配列番号71)をコードしている塩基配列である。
配列番号113は、トリ抗体(クローンNo.240-40株由来)の重鎖CDR1のアミノ酸配列(配列番号76)をコードしている塩基配列である。
配列番号114は、トリ抗体(クローンNo.240-40株由来)の重鎖CDR2のアミノ酸配列(配列番号77)をコードしている塩基配列である。
配列番号115は、トリ抗体(クローンNo.240-40株由来)の重鎖CDR3のアミノ酸配列(配列番号78)をコードしている塩基配列である。
配列番号116は、トリ抗体(クローンNo.240-40株由来)の軽鎖CDR1のアミノ酸配列(配列番号80)をコードしている塩基配列である。
配列番号117は、トリ抗体(クローンNo.240-40株由来)の軽鎖CDR2のアミノ酸配列(配列番号81)をコードしている塩基配列である。
配列番号118は、トリ抗体(クローンNo.240-40株由来)の軽鎖CDR3のアミノ酸配列(配列番号82)をコードしている塩基配列である。
配列番号119は、トリ抗体(クローンNo.240-40株由来)の重鎖可変領域のアミノ酸配列(配列番号75)をコードしている塩基配列である。
配列番号120は、トリ抗体(クローンNo.240-40株由来)の軽鎖可変領域のアミノ酸配列(配列番号79)をコードしている塩基配列である。
配列番号121は、トリ抗体(クローンNo.255-72株由来)の重鎖CDR1のアミノ酸配列(配列番号84)をコードしている塩基配列である。
配列番号122は、トリ抗体(クローンNo.255-72株由来)の重鎖CDR2のアミノ酸配列(配列番号85)をコードしている塩基配列である。
配列番号D123は、トリ抗体(クローンNo.255-72株由来)の重鎖CDR3のアミノ酸配列(配列番号86)をコードしている塩基配列である。
配列番号124は、トリ抗体(クローンNo.255-72株由来)の軽鎖CDR1のアミノ酸配列(配列番号88)をコードしている塩基配列である。
配列番号125は、トリ抗体(クローンNo.255-72株由来)の軽鎖CDR2のアミノ酸配列(配列番号89)をコードしている塩基配列である。
配列番号126は、トリ抗体(クローンNo.255-72株由来)の軽鎖CDR3のアミノ酸配列(配列番号90)をコードしている塩基配列である。
配列番号127は、トリ抗体(クローンNo.255-72株由来)の重鎖可変領域のアミノ酸配列(配列番号83)をコードしている塩基配列である。
配列番号128は、トリ抗体(クローンNo.255-72株由来)の軽鎖可変領域のアミノ酸配列(配列番号87)をコードしている塩基配列である。
Claims (31)
- 下記(A)~(E)のいずれか示される重鎖可変領域及び軽鎖可変領域を含む、抗セマフォリン3A抗体、又はその抗原結合領域を含む抗体断片:
(A) 配列番号1に示すアミノ酸配列、或いは配列番号1に示すアミノ酸配列において1~数個のアミノ酸が置換、欠失、付加又は挿入したアミノ酸配列を含むCDR1;配列番号2に示すアミノ酸配列、或いは配列番号2に示すアミノ酸配列において1~数個のアミノ酸が置換、欠失、付加又は挿入したアミノ酸配列に示すアミノ酸配列を含むCDR2;及び配列番号3に示すアミノ酸配列、或いは配列番号3に示すアミノ酸配列において1~数個のアミノ酸が置換、欠失、付加又は挿入したアミノ酸配列を含むCDR3を有する重鎖可変領域、並びに
配列番号4に示すアミノ酸配列、或いは配列番号4に示すアミノ酸配列において1~数個のアミノ酸が置換、欠失、付加又は挿入したアミノ酸配列を含むCDR1;配列番号5に示すアミノ酸配列、或いは配列番号5に示すアミノ酸配列において1~数個のアミノ酸が置換、欠失、付加又は挿入したアミノ酸配列を含むCDR2;及び配列番号6に示すアミノ酸配列、或いは配列番号6に示すアミノ酸配列において1~数個のアミノ酸が置換、欠失、付加又は挿入したアミノ酸配列を含むCDR3を有する軽鎖可変領域;
(B) 配列番号60に示すアミノ酸配列、或いは配列番号60に示すアミノ酸配列において1~数個のアミノ酸が置換、欠失、付加又は挿入したアミノ酸配列を含むCDR1;配列番号61に示すアミノ酸配列、或いは配列番号61に示すアミノ酸配列において1~数個のアミノ酸が置換、欠失、付加又は挿入したアミノ酸配列に示すアミノ酸配列を含むCDR2;及び配列番号62に示すアミノ酸配列、或いは配列番号62に示すアミノ酸配列において1~数個のアミノ酸が置換、欠失、付加又は挿入したアミノ酸配列を含むCDR3を有する重鎖可変領域、並びに
配列番号64に示すアミノ酸配列、或いは配列番号64に示すアミノ酸配列において1~数個のアミノ酸が置換、欠失、付加又は挿入したアミノ酸配列を含むCDR1;配列番号65に示すアミノ酸配列、或いは配列番号65に示すアミノ酸配列において1~数個のアミノ酸が置換、欠失、付加又は挿入したアミノ酸配列を含むCDR2;及び配列番号66に示すアミノ酸配列、或いは配列番号66に示すアミノ酸配列において1~数個のアミノ酸が置換、欠失、付加又は挿入したアミノ酸配列を含むCDR3を有する軽鎖可変領域;
(C) 配列番号68に示すアミノ酸配列、或いは配列番号68に示すアミノ酸配列において1~数個のアミノ酸が置換、欠失、付加又は挿入したアミノ酸配列を含むCDR1;配列番号69に示すアミノ酸配列、或いは配列番号69に示すアミノ酸配列において1~数個のアミノ酸が置換、欠失、付加又は挿入したアミノ酸配列に示すアミノ酸配列を含むCDR2;及び配列番号70に示すアミノ酸配列、或いは配列番号70に示すアミノ酸配列において1~数個のアミノ酸が置換、欠失、付加又は挿入したアミノ酸配列を含むCDR3を有する重鎖可変領域、並びに
配列番号72に示すアミノ酸配列、或いは配列番号72に示すアミノ酸配列において1~数個のアミノ酸が置換、欠失、付加又は挿入したアミノ酸配列を含むCDR1;配列番号73に示すアミノ酸配列、或いは配列番号73に示すアミノ酸配列において1~数個のアミノ酸が置換、欠失、付加又は挿入したアミノ酸配列を含むCDR2;及び配列番号74に示すアミノ酸配列、或いは配列番号74に示すアミノ酸配列において1~数個のアミノ酸が置換、欠失、付加又は挿入したアミノ酸配列を含むCDR3を有する軽鎖可変領域;
(D) 配列番号76に示すアミノ酸配列、或いは配列番号76に示すアミノ酸配列において1~数個のアミノ酸が置換、欠失、付加又は挿入したアミノ酸配列を含むCDR1;配列番号77に示すアミノ酸配列、或いは配列番号77に示すアミノ酸配列において1~数個のアミノ酸が置換、欠失、付加又は挿入したアミノ酸配列に示すアミノ酸配列を含むCDR2;及び配列番号78に示すアミノ酸配列、或いは配列番号78に示すアミノ酸配列において1~数個のアミノ酸が置換、欠失、付加又は挿入したアミノ酸配列を含むCDR3を有する重鎖可変領域、並びに
配列番号80に示すアミノ酸配列、或いは配列番号80に示すアミノ酸配列において1~数個のアミノ酸が置換、欠失、付加又は挿入したアミノ酸配列を含むCDR1;配列番号81に示すアミノ酸配列、或いは配列番号81に示すアミノ酸配列において1~数個のアミノ酸が置換、欠失、付加又は挿入したアミノ酸配列を含むCDR2;及び配列番号82に示すアミノ酸配列、或いは配列番号82に示すアミノ酸配列において1~数個のアミノ酸が置換、欠失、付加又は挿入したアミノ酸配列を含むCDR3を有する軽鎖可変領域;
(E) 配列番号84に示すアミノ酸配列、或いは配列番号84に示すアミノ酸配列において1~数個のアミノ酸が置換、欠失、付加又は挿入したアミノ酸配列を含むCDR1;配列番号85に示すアミノ酸配列、或いは配列番号85に示すアミノ酸配列において1~数個のアミノ酸が置換、欠失、付加又は挿入したアミノ酸配列に示すアミノ酸配列を含むCDR2;及び配列番号86に示すアミノ酸配列、或いは配列番号86に示すアミノ酸配列において1~数個のアミノ酸が置換、欠失、付加又は挿入したアミノ酸配列を含むCDR3を有する重鎖可変領域、並びに
配列番号88に示すアミノ酸配列、或いは配列番号88に示すアミノ酸配列において1~数個のアミノ酸が置換、欠失、付加又は挿入したアミノ酸配列を含むCDR1;配列番号89に示すアミノ酸配列、或いは配列番号89に示すアミノ酸配列において1~数個のアミノ酸が置換、欠失、付加又は挿入したアミノ酸配列を含むCDR2;及び配列番号90に示すアミノ酸配列、或いは配列番号90に示すアミノ酸配列において1~数個のアミノ酸が置換、欠失、付加又は挿入したアミノ酸配列を含むCDR3を有する軽鎖可変領域。 - キメラ抗体、ヒト化抗体、又はこれらの抗体断片である、請求項1に記載の抗セマフォリン3A抗体又は抗体断片。
- 請求項1又は2に記載の抗セマフォリン3A抗体又は抗体断片を含有する、医薬組成物。
- 神経再生促進用である、請求項3に記載の医薬組成物。
- アルツハイマー病の予防及び/又は治療用である、請求項3に記載の医薬組成物。
- 敗血症の予防及び/又は治療用である、請求項3に記載の医薬組成物。
- がんの予防及び/又は治療用である、請求項3に記載の医薬組成物。
- がんが、大腸癌、結腸直腸癌、肺癌、乳癌、脳腫瘍、黒色腫、腎細胞癌、白血病、リンパ腫、T細胞リンパ腫、胃癌、膵臓癌、子宮頚癌、子宮内膜癌、卵巣癌、食道癌、肝臓癌、頭頚部扁平上皮癌、皮膚癌、尿路癌、前立腺癌、絨毛癌、咽頭癌、又は喉頭癌である、請求項7に記載の医薬組成物。
- 播種性血管内凝固症候群の予防及び/又は治療用である、請求項3に記載の医薬組成物。
- 播種性血管内凝固症候群が、敗血症、急性白血病、固形がん、常位胎盤早期剥離、羊水塞栓、外傷、熱傷、膠原病、ショック、大動脈瘤、劇症肝炎、肝硬変、急性膵炎、横紋筋融解、血栓症、及び重症感染症よりなる群から選択される少なくとも1種を伴っている、請求項9に記載の医薬組成物。
- 中枢又は末梢神経系疾患、自己免疫疾患、炎症性疾患、感染症、及びアレルギー性疾患よりなる群から選択される少なくとも1種の疾患の予防及び/又は治療用である、請求項3に記載の医薬組成物。
- 中枢又は末梢神経系疾患が、神経障害性の疼痛、脊髄損傷、又は神経変性疾患である、請求項11に記載の医薬組成物。
- 神経変性疾患が、アルツハイマー病、パーキンソン病、ハンチントン病、筋萎縮性側索硬化症、進行性核上性麻痺、黒質線状体変性症、シャイ・ドレーガー症候群、オリーブ橋小脳萎縮症、又は脊髄小脳変性症である、請求項11に記載の医薬組成物。
- 自己免疫疾患が、慢性関節リウマチ、I型糖尿病、炎症性腸疾患、クローン病、全身性エリテマトーデス、又は多発性硬化症である、請求項11に記載の医薬組成物。
- 炎症性疾患が、敗血症、慢性閉塞性肺疾患、喘息、関節炎、肝炎、脊椎関節炎、又はシェーグレン症候群である、請求項11に記載の医薬組成物。
- 感染症が、細菌感染症、脳炎、髄膜炎、心内膜炎、C型肝炎、インフルエンザ、重症急性呼吸器症候群、肺炎、敗血症、火傷性、又は外傷性感染症である、請求項11に記載の医薬組成物。
- アレルギー性疾患が、アレルギー性鼻炎、アレルギー性結膜炎、アトピー性皮膚炎、気管支喘息、又は食物アレルギーである、請求項11に記載の医薬組成物。
- 神経再生が求められる患者に対して、請求項1又は2に記載の抗セマフォリン3A抗体又は抗体断片を投与する工程を含む、神経再生方法。
- アルツハイマー病を罹患している患者に対して、項1又は2に記載の抗セマフォリン3A抗体又は抗体断片を投与する工程を含む、アルツハイマー病の治療方法。
- 敗血症を罹患している患者に対して、請求項1又は2に記載の抗セマフォリン3A抗体又は抗体断片を投与する工程を含む、敗血症の治療方法。
- がんを罹患している患者に対して、請求項1又は2に記載の抗セマフォリン3A抗体又は抗体断片を投与する工程を含む、がんの治療方法。
- 播種性血管内凝固症候群を罹患している患者に対して、請求項1又は2に記載の抗セマフォリン3A抗体又は抗体断片を投与する工程を含む、播種性血管内凝固症候群の治療方法。
- 中枢又は末梢神経系疾患、自己免疫疾患、炎症性疾患、感染症、及びアレルギー性疾患よりなる群から選択される少なくとも1種の疾患を罹患している患者に対して、請求項1又は2に記載の抗セマフォリン3A抗体又は抗体断片を投与する工程を含む、前記疾患の治療方法。
- 神経再生用の医薬の製造のための、請求項1又は2に記載の抗セマフォリン3A抗体又は抗体断片の使用。
- アルツハイマー病の予防及び/又は治療剤の製造のための、請求項1又は2に記載の抗セマフォリン3A抗体又は抗体断片の使用。
- 敗血症の予防及び/又は治療剤の製造のための、請求項1又は2に記載の抗セマフォリン3A抗体又は抗体断片の使用。
- がんの予防及び/又は治療剤の製造のための、請求項1又は2に記載の抗セマフォリン3A抗体又は抗体断片の使用。
- 播種性血管内凝固症候群の予防及び/又は治療剤の製造のための、請求項1又は2に記載の抗セマフォリン3A抗体又は抗体断片の使用。
- 中枢又は末梢神経系疾患、自己免疫疾患、炎症性疾患、感染症、及びアレルギー性疾患よりなる群から選択される少なくとも1種の疾患の予防及び/又は治療剤の製造のための、請求項1又は2に記載の抗セマフォリン3A抗体又は抗体断片の使用。
- 請求項1又は2に記載の抗Sema3A抗体又は抗体断片を用いて免疫測定により検体中のSema3Aタンパク質を測定する工程を含む、Sema3Aタンパク質の測定方法。
- 請求項1又は2に記載の抗Sema3A抗体又は抗体断片を含む、Sema3Aタンパク質の測定用キット。
Priority Applications (7)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
ES14749177T ES2728854T3 (es) | 2013-02-06 | 2014-02-06 | Anticuerpo anti-semaforina 3A y tratamiento de enfermedad de Alzheimer y enfermedades inmunitarias inflamatorias usando el mismo |
DK14749177.3T DK2955195T3 (da) | 2013-02-06 | 2014-02-06 | Antisemaphorin-3a-antistof og behandling af alzheimers sygdom og inflammatoriske immunsygdomme ved anvendelse af samme |
US14/766,062 US9879075B2 (en) | 2013-02-06 | 2014-02-06 | Anti-semaphorin 3A antibody and treatment of Alzheimer's disease and inflammatory immune diseases using same |
EP14749177.3A EP2955195B1 (en) | 2013-02-06 | 2014-02-06 | Anti-semaphorin 3a antibody and treatment of alzheimer's disease and inflammatory immune diseases using same |
JP2014560799A JP6372040B2 (ja) | 2013-02-06 | 2014-02-06 | 抗セマフォリン3a抗体、並びにこれを用いたアルツハイマー病及び免疫・炎症性疾患の治療 |
US15/847,740 US10836814B2 (en) | 2013-02-06 | 2017-12-19 | Anti-semaphorin 3A antibody and treatment of Alzheimer's disease and inflammatory immune diseases using same |
US17/065,246 US20210095012A1 (en) | 2013-02-06 | 2020-10-07 | Anti-semaphorin 3a antibody and treatment of alzheimer's disease and inflammatory immune diseases using same |
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US15/847,740 Division US10836814B2 (en) | 2013-02-06 | 2017-12-19 | Anti-semaphorin 3A antibody and treatment of Alzheimer's disease and inflammatory immune diseases using same |
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EP (1) | EP2955195B1 (ja) |
JP (1) | JP6372040B2 (ja) |
DK (1) | DK2955195T3 (ja) |
ES (1) | ES2728854T3 (ja) |
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EP2497498A1 (en) * | 2009-11-05 | 2012-09-12 | Osaka University | Therapeutic agent for autoimmune diseases or allergy, and method for screening for the therapeutic agent |
WO2016197009A1 (en) | 2015-06-05 | 2016-12-08 | Vertex Pharmaceuticals Incorporated | Triazoles for the treatment of demyelinating diseases |
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KR101854529B1 (ko) | 2015-10-27 | 2018-05-04 | (주) 팬젠 | 인간 및 마우스 Sema3A에 교차결합하는 항체 및 그의 용도 |
WO2018106641A1 (en) | 2016-12-06 | 2018-06-14 | Vertex Pharmaceuticals Incorporated | Pyrazoles for the treatment of demyelinating diseases |
WO2018106646A1 (en) | 2016-12-06 | 2018-06-14 | Vertex Pharmaceuticals Incorporated | Aminotriazoles for the treatment of demyelinating diseases |
WO2018106643A1 (en) | 2016-12-06 | 2018-06-14 | Vertex Pharmaceuticals Incorporated | Heterocyclic azoles for the treatment of demyelinating diseases |
WO2018147432A1 (ja) | 2017-02-10 | 2018-08-16 | 株式会社カイオム・バイオサイエンス | 抗体可変領域の多様化を促進する方法 |
US10604571B2 (en) | 2015-10-27 | 2020-03-31 | Samsung Life Public Welfare Foundation | Antibody to human and mouse SEMA3A and use thereof |
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US20150368327A1 (en) | 2015-12-24 |
US20210095012A1 (en) | 2021-04-01 |
US20180208644A1 (en) | 2018-07-26 |
DK2955195T3 (da) | 2019-05-06 |
US10836814B2 (en) | 2020-11-17 |
EP2955195A4 (en) | 2016-11-02 |
EP2955195A1 (en) | 2015-12-16 |
ES2728854T3 (es) | 2019-10-29 |
JP6372040B2 (ja) | 2018-08-15 |
EP2955195B1 (en) | 2019-03-27 |
US9879075B2 (en) | 2018-01-30 |
JPWO2014123186A1 (ja) | 2017-02-02 |
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