# Patent

## Title
Anti-semaphorin 3A antibody and treatment of Alzheimer's disease and inflammatory immune diseases using same

## Abstract

The present invention mainly addresses the problem of providing an antibody against semaphorin 3A protein, said antibody enabling effective prevention and/or treatment of a disease, in which Sema 3A protein participates, such as a neurodegenerative disease, autoimmune disease, inflammatory disease, cancer, infectious disease, etc. or disseminated intravascular coagulation syndrome. An anti-Sema 3A antibody comprising CDRs having specific amino acid sequences (SEQ ID NOS: 1-6, 60-62, 64-66, 68-70, 72-74, 76-78, 80-82, 84-86 and 88-90) enables effective prevention and/or treatment of a disease, in which Sema 3A protein participates, such as a neurodegenerative disease, autoimmune disease, inflammatory disease, cancer, infectious disease, etc. or disseminated intravascular coagulation syndrome and, therefore, remarkably ameliorates symptoms associated with such a disease.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a U.S. National Phase Application of PCT International Application Number PCT/JP2014/052758, filed on Feb. 6, 2014, designating the United States of America and published in the English language, which is an International Application of and claims the benefit of priority to Japanese Patent Application No. 2013-021309, filed on Feb. 6, 2013. The disclosures of the above-referenced applications are hereby expressly incorporated by reference in their entireties.
REFERENCE TO SEQUENCE LISTING
A Sequence Listing submitted as an ASCII text file via EFS-Web is hereby incorporated by reference in accordance with 35 U.S.C. § 1.52(e). The name of the ASCII text file for the Sequence Listing is SeqList-LEX010-001APC.txt, the date of creation of the ASCII text file is Jul. 23, 2015, and the size of the ASCII text file is 71 KB.
TECHNICAL FIELD
The present invention relates to anti-semaphorin 3A antibody or its antibody fragment effective for prophylaxis and/or treatment of diseases involving semaphorin 3A protein. The present invention also relates to a pharmaceutical composition containing the anti-semaphorin 3A antibody or antibody fragment thereof. Furthermore, the present invention relates to a method for measuring semaphorin 3A protein using anti-semaphorin 3A antibody or its antibody fragment.
BACKGROUND ART
Alzheimer's disease (AD) is the most common form of dementia (loss of memory) in the elderly. Currently used therapeutic agents for Alzheimer's disease have any action mechanism which allows to improve cholinergic neurotransmission in the central nerve system with a cholinesterase inhibitor or suppress excitotoxicity with a N-methyl-D-aspartate (NMDA) type glutamate receptor inhibitor, however, all of the agents only provide mild improvement of the related symptoms. A radical treatment method has been expected to develop which suppresses or ameliorates progression of neurodegeneration responsible for Alzheimer's disease.
Principal pathological damage of AD, which is found in brain, is caused by extracellular deposits of β amyloid protein in the forms of plaques and vascular clogs, and intracellular neurofibrillary tangle of τ protein which is clumped and hyper-phosphorylated. Recent evidences demonstrate that an increased level of β amyloid in brain appears in advance of pathological changes of τ protein and correlates with cognitive decline. Recent studies also suggest that β amyloid is ascribed as the etiology of AD and reveal that clumped β amyloid has toxicity to neurons in cell culture.
β amyloid protein is mainly composed of peptides in the length of 39 to 42 amino acids and is produced from a large protein precursor called as amyloid precursor protein (APP) through consecutive action of protease and β- and γ-secretases. Rare but early onset AD cases are attributed to genetic mutation of APP which causes overproduction of any one of total β amyloid protein and isoforms thereof. Individuals with Down's syndrome have an extra chromosome containing an APP-encoding gene and they have an increased level of β amyloid and inevitably develop AD with age.
Semaphorins are endogenous proteins identified as factors which allow to degenerate nerve growth cone and suppress axon elongation. Semaphorin genes are categorized into eight gene subfamilies and classes based on their structures and are previously known to have approximately 20 molecular species (Non-patent document 1). However, little is known about main functions of most semaphorin families. More frequently studied one is a subfamily gene cluster called as Class III and among them, most frequently studied one is semaphorin 3A (Sema 3A) (Non-patent documents 2 and 3). Sema 3A protein is known to induce degeneration of growth cones in cultured nerve cells and inhibit regeneration and elongation of nerves at a low level of 10 pM within a short time.
In recent years, one research has been reported that dendritic cells detect Sema 3A protein, subsequently enter a lymph channel and transfer into a lymph node, suggesting that Sema 3A protein is involved in autoimmune disease (See e.g. Non-patent document 4), and other research has revealed that signals mediated by Sema 3A protein and its receptor, plexin-A4, have an important role in the onset of sepsis (See e.g. Non-patent document 6), and from these findings, Sema 3A protein is also known to be deeply involved in formations of pathological conditions of immune and infectious diseases, etc. Additionally, another researches have been reported that Sema 3A protein is secreted by cancer cells and tissues and blocks a signal pathway of mitogen-activated protein kinase (MAP) to suppress activation of T cells, and that pancreatic cancer patients with high expression level of Sema 3A in the cancer tissue have poor outcome, revealing that Sema 3A protein is also involved in malignant alteration of cancer (See, e.g. Non-patent documents 5 and 7).
Disseminated intravascular coagulation (DIC) is a serious pathological condition in which coagulation significantly activates in a systemic and persistent manner along with deteriorated underlying disease. DIC is known that its main symptoms are episodes of bleeding and organ disordering and the occurrence of these clinical symptoms leads to extremely poor outcome. Examples of underlying diseases inductive of DIC include sepsis, acute leukemia, solid cancer, premature separation of normally implanted placenta, amniotic fluid embolism, trauma, burn injury, connective tissue disease, shock, aortic aneurysm, fulminant hepatitis, liver cirrhosis, acute pancreatitis, rhabdomyolysis, thrombosis, severe infectious disease, etc., however, a relationship of the diseases and Sema 3A is not clear.
Previously an antibody to Sema 3A protein has been also reported. For example, researches have been reported that Sema 3A protein activity inhibitor such as anti-Sema 3A antibody is effective for treatment of Alzheimer's disease and Parkinson's disease (See Patent document 1), and also effective for treatment of immune disease and inflammatory disease (See Patent document 2). However, when a disease involving Sema 3A protein is prevented and/or treated with anti-Sema 3A antibody, it is not known a structure of anti-Sema 3A antibody used in order to effectively achieve the high drug efficacy.
As described above, Sema 3A protein is known to be involved in various pathological conditions and a technique for measuring Sema 3A protein in a high accurate manner is essential to elucidate the pathological conditions and develop a therapeutic agent for the conditions.
PRIOR ART DOCUMENTS
Non-Patent Document


    Non-Patent Document 1: Cell, 97, 551, 1999
    Non-Patent Document 2: Cell, 75, 217, 1993
    Non-Patent Document 3: Cell, 75, 1389, 1993
    Non-Patent Document 4: Nature Immunology 11, 594-600, 2010
    Non-Patent Document 5: Blood 107, 3321-3329, 2006
    Non-Patent Document 6: Journal of Experimental Medicine 207, 2943, 2010
    Non-Patent Document 7: International Journal of Cancer 121, 2421-2433, 2007


Patent Documents


    Patent Document 1: WO 03/007803
    Patent Document 2: WO 2011/066284


SUMMARY OF THE INVENTION
Problems to be Solved by the Invention
An object of the present invention is to provide an antibody to Sema 3A protein wherein the antibody can effectively prevent and/or treat diseases involving Sema 3A protein such as neurodegenerative disease, autoimmune disease, inflammatory disease, cancer, infectious disease, etc. and disseminated intravascular coagulation syndrome. Another object of the present invention is to provide a pharmaceutical composition capable of prophylaxis and/or treatment of diseases involving Sema 3A protein as well as amelioration of symptoms associated with such diseases. Still another object of the present invention is to provide an antibody to Sema 3A effective for measuring Sema 3A protein and a method for measuring Sema 3A protein using said antibody.
Means for Solving the Problem
The present inventors have intensively studied to solve the above mentioned problems, and have consequently found that anti-Sema 3A antibody containing a complementary determining region (CDR) having a specific amino sequence can effectively prevent and/or treat neurodegenerative disease such as Alzheimer's disease and significantly ameliorate symptoms associated with the neurodegenerative disease. The anti-Sema 3A antibody also led to drastic improvement of the survival rate and extension of the survival period in a fatal sepsis pathological model. Furthermore, the present inventors have found that the anti-Sema 3A antibody allows to decrease blood level of plasminogen activator inhibitor-1 (PAI-1), an aggravating factor of disseminated intravascular coagulation. Sema 3A induces migration/invasion and drug tolerance of cancer cells. The present inventors have also found that anti-Sema 3A antibody can suppress migration/invasion and drug tolerance of cancer cells and therefore the antibody can minimize malignant alteration of cancer.
Additionally, the present inventors have also found that Sema 3A protein can be measured by ELISA using anti-Sema 3A antibody containing a CDR having a specific amino acid sequence.
The present inventors have further studied based on such findings and completed the present invention. That is, the present invention provides the invention of the following aspects:
Item 1. Anti-semaphorin 3A antibody containing a heavy chain variable region and a light chain variable region shown in any one of the following (A) to (E) or antibody fragment thereof containing an antigen binding region thereof:
(A) a heavy chain variable region containing CDR1 having the amino acid sequence shown in SEQ ID NO:1 or an amino acid sequence in which one or a few amino acids are substituted, deleted, added or inserted in the amino acid sequence shown in SEQ ID NO:1; CDR2 having the amino acid sequence shown in SEQ ID NO:2 or an amino acid sequence in which one or a few amino acids are substituted, deleted, added or inserted in the amino acid sequence shown in SEQ ID NO:2; and CDR3 having the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence in which one or a few amino acids are substituted, deleted, added or inserted in the amino acid sequence shown in SEQ ID NO:3, and
a light chain variable region containing CDR1 having the amino acid sequence shown in SEQ ID NO:4 or an amino acid sequence in which one or a few amino acids are substituted, deleted, added or inserted in the amino acid sequence shown in SEQ ID NO:4; CDR2 having the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence in which one or a few amino acids are substituted, deleted, added or inserted in the amino acid sequence shown in SEQ ID NO:5; and CDR3 having the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence in which one or a few amino acids are substituted, deleted, added or inserted in the amino acid sequence shown in SEQ ID NO:6;
(B) a heavy chain variable region containing CDR1 having the amino acid sequence shown in SEQ ID NO:60 or an amino acid sequence in which one or a few amino acids are substituted, deleted, added or inserted in the amino acid sequence shown in SEQ ID NO:60; CDR2 having the amino acid sequence shown in SEQ ID NO:61 or an amino acid sequence in which one or a few amino acids are substituted, deleted, added or inserted in the amino acid sequence shown in SEQ ID NO:61; and CDR3 having the amino acid sequence shown in SEQ ID NO:62 or an amino acid sequence in which one or a few amino acids are substituted, deleted, added or inserted in the amino acid sequence shown in SEQ ID NO:62, and
a light chain variable region containing CDR1 having the amino acid sequence shown in SEQ ID NO:64 or an amino acid sequence in which one or a few amino acids are substituted, deleted, added or inserted in the amino acid sequence shown in SEQ ID NO:64; CDR2 having the amino acid sequence shown in SEQ ID NO:65 or an amino acid sequence in which one or a few amino acids are substituted, deleted, added or inserted in the amino acid sequence shown in SEQ ID NO:65; and CDR3 having the amino acid sequence shown in SEQ ID NO:66 or an amino acid sequence in which one or a few amino acids are substituted, deleted, added or inserted in the amino acid sequence shown in SEQ ID NO:66;
(C) a heavy chain variable region containing CDR1 having the amino acid sequence shown in SEQ ID NO:68 or an amino acid sequence in which one or a few amino acids are substituted, deleted, added or inserted in the amino acid sequence shown in SEQ ID NO:68; CDR2 having the amino acid sequence shown in SEQ ID NO:69 or an amino acid sequence in which one or a few amino acids are substituted, deleted, added or inserted in the amino acid sequence shown in SEQ ID NO:69; and CDR3 having the amino acid sequence shown in SEQ ID NO:70 or an amino acid sequence in which one or a few amino acids are substituted, deleted, added or inserted in the amino acid sequence shown in SEQ ID NO:70, and
a light chain variable region containing CDR1 having the amino acid sequence shown in SEQ ID NO:72 or an amino acid sequence in which one or a few amino acids are substituted, deleted, added or inserted in the amino acid sequence shown in SEQ ID NO:72; CDR2 having the amino acid sequence shown in SEQ ID NO:73 or an amino acid sequence in which one or a few amino acids are substituted, deleted, added or inserted in the amino acid sequence shown in SEQ ID NO:73; and CDR3 having the amino acid sequence shown in SEQ ID NO:74 or an amino acid sequence in which one or a few amino acids are substituted, deleted, added or inserted in the amino acid sequence shown in SEQ ID NO:74;
(D) a heavy chain variable region containing CDR1 having the amino acid sequence shown in SEQ ID NO:76 or an amino acid sequence in which one or a few amino acids are substituted, deleted, added or inserted in the amino acid sequence shown in SEQ ID NO:76; CDR2 having the amino acid sequence shown in SEQ ID NO:77 or an amino acid sequence in which one or a few amino acids are substituted, deleted, added or inserted in the amino acid sequence shown in SEQ ID NO:77; and CDR3 having the amino acid sequence shown in SEQ ID NO:78 or an amino acid sequence in which one or a few amino acids are substituted, deleted, added or inserted in the amino acid sequence shown in SEQ ID NO:78, and
a light chain variable region containing CDR1 having the amino acid sequence shown in SEQ ID NO:80 or an amino acid sequence in which one or a few amino acids are substituted, deleted, added or inserted in the amino acid sequence shown in SEQ ID NO:80; CDR2 having the amino acid sequence shown in SEQ ID NO:81 or an amino acid sequence in which one or a few amino acids are substituted, deleted, added or inserted in the amino acid sequence shown in SEQ ID NO:81; and CDR3 having the amino acid sequence shown in SEQ ID NO:82 or an amino acid sequence in which one or a few amino acids are substituted, deleted, added or inserted in the amino acid sequence shown in SEQ ID NO:82;
(E) a heavy chain variable region containing CDR1 having the amino acid sequence shown in SEQ ID NO:84 or an amino acid sequence in which one or a few amino acids are substituted, deleted, added or inserted in the amino acid sequence shown in SEQ ID NO:84; CDR2 having the amino acid sequence shown in SEQ ID NO:85 or an amino acid sequence in which one or a few amino acids are substituted, deleted, added or inserted in the amino acid sequence shown in SEQ ID NO:85; and CDR3 having the amino acid sequence shown in SEQ ID NO:86 or an amino acid sequence in which one or a few amino acids are substituted, deleted, added or inserted in the amino acid sequence shown in SEQ ID NO:86, and
a light chain variable region containing CDR1 having the amino acid sequence shown in SEQ ID NO:88 or an amino acid sequence in which one or a few amino acids are substituted, deleted, added or inserted in the amino acid sequence shown in SEQ ID NO:88; CDR2 having the amino acid sequence shown in SEQ ID NO:89 or an amino acid sequence in which one or a few amino acids are substituted, deleted, added or inserted in the amino acid sequence shown in SEQ ID NO:89; and CDR3 having the amino acid sequence shown in SEQ ID NO:90 or an amino acid sequence in which one or a few amino acids are substituted, deleted, added or inserted in the amino acid sequence shown in SEQ ID NO:90.
Item 2. The anti-semaphorin 3A antibody or antibody fragment thereof according to item 1, wherein the antibody or antibody fragment thereof is a chimeric antibody, a humanized antibody or an antibody fragment thereof.
Item 3. The anti-semaphorin 3A antibody or antibody fragment thereof according to item 1 or 2, wherein the antibody or antibody fragment thereof is used for acceleration of neuronal regeneration.
Item 4. The anti-semaphorin 3A antibody or antibody fragment thereof according to item 1 or 2, wherein the antibody or antibody fragment thereof is used for prophylaxis and/or treatment of Alzheimer's disease.
Item 5. The anti-semaphorin 3A antibody or antibody fragment thereof according to item 1 or 2, wherein the antibody or antibody fragment thereof is used for prophylaxis and/or treatment of sepsis.
Item 6. The anti-semaphorin 3A antibody or antibody fragment thereof according to item 1 or 2, wherein the antibody or antibody fragment thereof is used for prophylaxis and/or treatment of cancer.
Item 7. Use of the anti-semaphorin 3A antibody or antibody fragment thereof according to item 1 or 2 for the manufacture of a prophylactic and/or therapeutic agent of disseminated intravascular coagulation.
Item 8. The anti-semaphorin 3A antibody or antibody fragment thereof according to item 1 or 2, wherein the antibody or antibody fragment thereof is used for prophylaxis and/or treatment of at least one disease selected from the group consisting of central or peripheral nervous system disease, autoimmune disease, inflammatory disease, infectious disease and allergic disease.
Item 9. A pharmaceutical composition comprising the anti-semaphorin 3A antibody or antibody fragment thereof according to any one of items 1 to 8.
Item 10. The pharmaceutical composition according to item 9, wherein the composition is for acceleration of neuronal regeneration.
Item 11. The pharmaceutical composition according to item 9, wherein the composition is for prophylaxis and/or treatment of Alzheimer's disease.
Item 12. The pharmaceutical composition according to item 9, wherein the composition is for prophylaxis and/or treatment of sepsis.
Item 13. The pharmaceutical composition according to item 9, wherein the composition is for prophylaxis and/or treatment of cancer.
Item 14. The pharmaceutical composition according to item 13, wherein cancer is bowel cancer, colorectal cancer, lung cancer, breast cancer, brain cancer, melanoma, renal cell cancer, leukemia, lymphoma, T-cell lymphoma, stomach cancer, pancreatic cancer, cervical cancer, endometrial cancer, ovarian cancer, esophagus cancer, liver cancer, head and neck squamous cell carcinoma, skin cancer, urinary tract cancer, prostate cancer, choriocarcinoma, pharyngeal cancer or larynx cancer.
Item 15. The pharmaceutical composition according to item 9, wherein the composition is for prophylaxis and/or treatment of disseminated intravascular coagulation.
Item 16. The pharmaceutical composition according to item 15, wherein disseminated intravascular coagulation accompanies at least one selected from the group consisting of sepsis, acute leukemia, solid cancer, premature separation of normally implanted placenta, amniotic fluid embolism, trauma, burn injury, connective tissue disease, shock, aortic aneurysm, acute hepatitis, liver cirrhosis, acute pancreatitis, rhabdomyolysis, thrombosis and severe infectious disease.
Item 17. The pharmaceutical composition according to item 9, wherein the composition is for prophylaxis and/or treatment of at least one disease selected from the group consisting of central or peripheral nervous system disease, autoimmune disease, inflammatory disease, infectious disease and allergic disease.
Item 18. The pharmaceutical composition according to item 17, wherein central or peripheral nervous system disease is neuropathic pain, spinal cord injury or neurodegenerative disease.
Item 19. The pharmaceutical composition according to item 18, wherein neurodegenerative disease is Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, progressive supranuclear palsy, striatonigral degeneration, Shy-Drager syndrome, olivopontocerebellar atrophy or spinocerebellar degeneration.
Item 20. The pharmaceutical composition according to item 17, wherein autoimmune disease is rheumatoid arthritis, type I diabetes mellitus, inflammatory bowel disease, Crohn's disease, systemic lupus erythematosus or multiple sclerosis.
Item 21. The pharmaceutical composition according to item 17, wherein inflammatory disease is sepsis, chronic obstructive pulmonary disease, asthma, arthritis, hepatitis, spondyloarthritis or Sjogren's disease.
Item 22. The pharmaceutical composition according to item 17, wherein infectious disease is bacterial infectious disease, encephalitis, meningitis, endocarditis, hepatitis C, influenza, severe acute respiratory syndrome, pneumonia, sepsis, burn injury- or trauma-induced infectious disease.
Item 23. The pharmaceutical composition according to item 17, wherein allergic disease is allergic rhinitis, allergic conjunctivitis, atopic dermatitis, bronchial asthma or food allergy.
Item 24. A method for neuronal regeneration, comprising administrating the anti-semaphorin 3A antibody or antibody fragment thereof according to item 1 or 2 to a patient in need of neuronal regeneration.
Item 25. A method for treating Alzheimer's disease, comprising administrating the anti-semaphorin 3A antibody or antibody fragment thereof according to item 1 or 2 to a patient with Alzheimer's disease.
Item 26. A method for treating sepsis, comprising administrating the anti-semaphorin 3A antibody or antibody fragment thereof according to item 1 or 2 to a patient with sepsis.
Item 27. A method for treating cancer, comprising administrating the anti-semaphorin 3A antibody or antibody fragment thereof according to item 1 or 2 to a patient with cancer.
Item 28. The treatment method according to item 27, wherein cancer is bowel cancer, colorectal cancer, lung cancer, breast cancer, brain cancer, melanoma, renal cell cancer, leukemia, lymphoma, T-cell lymphoma, stomach cancer, pancreatic cancer, cervical cancer, endometrial cancer, ovarian cancer, esophagus cancer, liver cancer, head and neck squamous cell carcinoma, skin cancer, urinary tract cancer, prostate cancer, choriocarcinoma, pharyngeal cancer or larynx cancer.
Item 29. A method for treating disseminated intravascular coagulation, comprising administrating the anti-semaphorin 3A antibody or antibody fragment thereof according to item 1 or 2 to a patient with disseminated intravascular coagulation.
Item 30. The method for treating disseminated intravascular coagulation according to item 29, wherein the disseminated intravascular coagulation accompanies at least one selected from the group consisting of sepsis, acute leukemia, solid cancer, premature separation of normally implanted placenta, amniotic fluid embolism, trauma, burn injury, connective tissue disease, shock, aortic aneurysm, acute hepatitis, liver cirrhosis, acute pancreatitis, rhabdomyolysis, thrombosis and severe infectious disease.
Item 30. A treatment method comprising administrating the anti-semaphorin 3A antibody or antibody fragment thereof according to item 1 or 2 to a patient with at least one disease selected from the group consisting of central or peripheral nervous system disease, autoimmune disease, inflammatory disease, infectious disease and allergic disease.
Item 31. Use of the anti-semaphorin 3A antibody or antibody fragment thereof according to item 1 or 2 for the manufacture of a medicine for neuronal regeneration.
Item 32. Use of the anti-semaphorin 3A antibody or antibody fragment thereof according to item 1 or 2 for the manufacture of a prophylactic and/or therapeutic agent of Alzheimer's disease.
Item 33. Use of the anti-semaphorin 3A antibody or antibody fragment thereof according to item 1 or 2 for the manufacture of a prophylactic and/or therapeutic agent of sepsis.
Item 34. Use of the anti-semaphorin 3A antibody or antibody fragment thereof according to item 1 or 2 for the manufacture of a prophylactic and/or therapeutic agent of cancer.
Item 35. Use of the anti-semaphorin 3A antibody or antibody fragment thereof according to item 1 or 2 for the manufacture of a prophylactic and/or therapeutic agent of disseminated intravascular coagulation.
Item 36. Use of the anti-semaphorin 3A antibody or antibody fragment thereof according to item 1 or 2 for the manufacture of a prophylactic and/or therapeutic agent of at least one disease selected from the group consisting of central or peripheral nervous system disease, autoimmune disease, inflammatory disease, infectious disease and allergic disease.
Item 37. A method for measuring Sema 3A protein, comprising measuring Sema 3A protein in a sample using immunoassay with the anti-Sema 3A antibody or antibody fragment thereof according to item 1 or 2.
Item 38. A kit for measuring Sema 3A protein containing the anti-Sema 3A antibody or antibody fragment thereof according to item 1 or 2.
Advantages of the Invention
The anti-Sema 3A antibody of the present invention can effectively prevent and/or treat neurodegenerative diseases such as Alzheimer's disease, etc. and significantly ameliorate symptoms associated with the neurodegenerative disease. Without wishing to be bound by limited interpretation for any action mechanism of the anti-Sema 3A antibody of the present invention, there is believed that the anti-Sema 3A antibody of the present invention effectively inhibits a function of Sema 3A protein, thereby to block signaling of Sema 3A, resulting in reduction of accumulation of phosphorylated collapsin response mediator protein (CRMP) in neurofibrillary tangle. The anti-Sema 3A antibody of the present invention is also effective for prophylaxis and/or treatment of central or peripheral nervous system disease other than neurodegenerative disease, autoimmune disease, inflammatory disease, infectious disease and allergic disease, etc. and in particular, can exhibit prominent effects of prophylaxis and/or treatment of inflammatory disease such as sepsis, which is caused by collapsing immunity mechanism due to infection or potent inflammation-inducing stimulus. The anti-Sema 3A antibody of the present invention can effectively suppress migration/invasion activities of cancer cells induced by Sema 3A and remove anticancer drug-unresponsiveness induced by Sema 3A to recover drug sensitivity, and therefore is also effective for prophylaxis and/or treatment of cancer. Furthermore, the anti-Sema 3A antibody of the present invention has the action of suppressing increase of blood level of PAI-1 and therefore, is also effective for prophylaxis and/or treatment of disseminated intravascular coagulation.
The anti-Sema 3A antibody of the present invention can be used in measurement of Sema 3A protein. In particular, the use of the anti-Sema 3A antibody of the present invention allows to measure Sema 3A protein even in the presence of serum in a high accurate manner and therefore measure Sema 3A protein in a biological sample.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows a graph illustrating 13 anti-Sema 3A antibody-producing clones prepared in Example 1.
FIG. 2 shows a graph illustrating results of assessment of binding specificity of anti-Sema 3A avian-mouse chimeric antibody produced in Example 1 to Sema 3A protein.
FIG. 3 shows the results that anti-Sema 3A avian-mouse chimeric antibody, anti-Sema 3A humanized antibody and anti-Sema 3A avian human chimeric antibody is applied to electrophoresis followed by CBB staining.
FIG. 4 shows test results of reactivity of anti-Sema 3A avian-mouse chimeric antibody, anti-Sema 3A humanized antibody and anti-Sema 3A avian-human chimeric antibody to human Sema 3A and mouse Sema 3A antigens.
FIG. 5 shows measured results of affinity of anti-Sema 3A avian-mouse chimeric antibody, anti-Sema 3A humanized antibody and anti-Sema 3A avian-human chimeric antibody to Sema 3A protein by competitive ELISA.
FIG. 6 shows measured results of Sema 3A in serum-free samples by sandwich ELISA with the anti-Sema 3A antibody produced in Examples 2 to 4.
FIG. 7 shows measured results of Sema 3A in serum-containing samples by sandwich ELISA with the anti-Sema 3A antibody produced in Examples 2.
FIG. 8 shows results of collapse assay with chicken-derived Sema 3A protein and anti-Sema 3A avian antibody.
FIG. 9 shows results of collapse assay with chicken-derived Sema 3A protein and anti-Sema 3A avian-mouse chimeric antibody.
FIG. 10 shows results of collapse assay with chicken-derived Sema 3A protein and anti-Sema 3A avian antibody.
FIG. 11 shows results of collapse assay with chicken-derived Sema 3A protein and anti-Sema 3A avian-mouse chimeric antibody.
FIG. 12 shows results of binding assay of anti-Sema 3A avian antibody to human-derived Sema 3A protein.
FIG. 13 shows results of binding assay of anti-Sema 3A avian-mouse chimeric antibody to human-derived Sema 3A protein.
FIG. 14 shows results of binding assay of anti-Sema 3A avian antibody to human-derived Sema 3F protein.
FIGS. 15A and 15B show graphs illustrating results of Example 10. FIG. 15A shows a graph illustrating results of access rate to Object B in acquisition trials of novel object recognition task. FIG. 15B shows a graph illustrating results of access rate to Object C in test trials of novel object recognition task.
FIG. 16 shows a diagram illustrating results of Example 15. The figure is a combined plot of survival curves of three tests of the administration of anti-Sema 3A avian-mouse chimeric antibody by Kaplan-Meier method.
FIG. 17 shows a diagram illustrating results of Example 17. The figure is a plot of survival curves of administered group of anti-Sema 3A humanized antibody of 500 μg one hour after a lipopolysaccharide-inoculated mouse presented inflammatory condition and negative control group by Kaplan-Meier method.
FIG. 18 shows a diagram illustrating results of Example 18. The figure is a diagram illustrating measured results of blood levels of plasminogen activator inhibitor-1 of mice for determining effects of anti-Sema 3A humanized antibody in disseminated intravascular coagulation-model mice.
FIG. 19 shows a graph illustrating results of Example 19. The figure shows analyzed results of effects of Sema 3A protein and/or anti-Sema 3A humanized antibody for cell migration ability in human pancreatic cancer cells (MIAPaCa-2).
FIG. 20 shows a graph illustrating results of Example 20. The figure shows a graph illustrating results of cell invasion assay with human pancreatic cancer cell line (MIAPaCa-2).
FIG. 21 shows a graph illustrating results of Example 20. The figure shows a graph illustrating results of cell invasion assay with human pancreatoblastoma cell line (U87MG).
FIG. 22 shows a graph illustrating results of Example 20. The figure shows a graph illustrating results of cell invasion assay with mouse-derived lung cancer cell line (3LL).
FIG. 23 shows photographic images illustrating results of Example 20. The figure shows microscopic images of invaded cells obtained by staining through cell invasion assay with mouse-derived lung cancer cell line (3LL).
FIG. 24 shows a graph illustrating results of Example 21. The figure shows analyzed results of effect of Sema 3A protein and/or anti-Sema 3A humanized antibody on sensitivity to gemcitabine hydrochloride in human pancreatic cancer cells (MIAPaCa-2).



EMBODIMENTS OF THE INVENTION
1. Anti-Sema 3A Antibody and Antibody Fragment Thereof
The anti-Sema 3A antibody of the present invention is characterized by inclusion of a heavy chain variable region containing CDR1 to CDR3 having specific amino acid sequences and a light chain variable region containing CDR1 to CDR3 having specific amino acid sequences.
The anti-Sema 3A antibody of the present invention can specifically bind to Sema 3A protein and effectively inhibit a function of the protein. Sema 3A protein is an endogenous protein which is encoded by a gene belonging to class III semaphorin subfamily and identified as a factor that collapses nerve growth cone thereby to suppress axon elongation. The amino acid sequence of Sema 3A protein is known (GenBank accession number; NP_006071.1 for human-derived, NP_033178.2 for mouse-derived).
Preferred examples of an aspect of the anti-Sema 3A antibody of the present invention include a heavy chain variable region and a light chain variable region shown in the following items (A) to (E);
(A) a heavy chain variable region containing CDR1 having the amino acid sequence shown in SEQ ID NO:1, CDR2 having the amino acid sequence shown in SEQ ID NO:2 and CDR3 having the amino acid sequence shown in SEQ ID NO:3 and a light chain variable region containing CDR1 having the amino acid sequence shown in SEQ ID NO:4, CDR2 having the amino acid sequence shown in SEQ ID NO:5 and CDR3 having the amino acid sequence shown in SEQ ID NO:6.
(B) a heavy chain variable region containing CDR1 having the amino acid sequence shown in SEQ ID NO:60, CDR2 having the amino acid sequence shown in SEQ ID NO:61 and CDR3 having the amino acid sequence shown in SEQ ID NO:62 and a light chain variable region containing CDR1 having the amino acid sequence shown in SEQ ID NO:64, CDR2 having the amino acid sequence shown in SEQ ID NO:65 and CDR3 having the amino acid sequence shown in SEQ ID NO:66,
(C) a heavy chain variable region containing CDR1 having the amino acid sequence shown in SEQ ID NO:68, CDR2 having the amino acid sequence shown in SEQ ID NO:69 and CDR3 having the amino acid sequence shown in SEQ ID NO:70 and a light chain variable region containing CDR1 having the amino acid sequence shown in SEQ ID NO:72, CDR2 having the amino acid sequence shown in SEQ ID NO:73 and CDR3 having the amino acid sequence shown in SEQ ID NO:74,
(D) a heavy chain variable region containing CDR1 having the amino acid sequence shown in SEQ ID NO:76, CDR2 having the amino acid sequence shown in SEQ ID NO:77 and CDR3 having the amino acid sequence shown in SEQ ID NO:78 and a light chain variable region containing CDR1 having the amino acid sequence shown in SEQ ID NO:80, CDR2 having the amino acid sequence shown in SEQ ID NO:81 and CDR3 having the amino acid sequence shown in SEQ ID NO:82,
(E) a heavy chain variable region containing CDR1 having the amino acid sequence shown in SEQ ID NO:84, CDR2 having the amino acid sequence shown in SEQ ID NO:85 and CDR3 having the amino acid sequence shown in SEQ ID NO:86 and a light chain variable region containing CDR1 having the amino acid sequence shown in SEQ ID NO:88, CDR2 having the amino acid sequence shown in SEQ ID NO:89 and CDR3 having the amino acid sequence shown in SEQ ID NO:90.
In the anti-Sema 3A antibody of the present invention, amino acid sequences of CDRs 1 to 3 in a heavy chain and a light chain (SEQ ID NOs:1-6, 60-62, 64-66, 68-70, 72-74, 76-78, 80-82, 84-86 and 88-90) may be those in which one or a few amino acids are substituted, deleted, added and/or inserted. Anti-Sema 3A antibody containing CDR sequences in which one or a few amino acids are substituted, deleted, added and/or inserted has desirably a binding activity to Sema 3A protein equal to or more than that of anti-Sema 3A antibody containing unmodified CDR sequences. The number of amino acids substituted, deleted, added and/or inserted preferably includes but are not particularly limited to 1 to 3, more preferably 1 to 2, particularly preferably 1 per CDR. In amino acid sequences of CDRs 1 to 3 in a heavy chain and a light chain of the anti-Sema 3A antibody of the present invention, amino acids may be substituted, deleted, added and/or inserted in one amino acid sequence and may be substituted, deleted, added and/or inserted in two or more amino acid sequences.
In substitution of an amino acid in the amino acid sequence of CDR, a substitution by the corresponding amino acid (i.e. conservative amino acid substitution) is preferred since the binding activity of antibody is expected not to change through the substitution. In particular, the following category is established based on properties of amino acid side chains:
Basic amino acid: lysine, arginine and histidine,
Acidic amino acid: glutamic acid and aspartic acid,
Neutral amino acid: glycine, alanine, serine, threonine, methionine, cysteine, phenylalanine, tryptophan, tyrosine, leucine, isoleucine, valine, glutamine, asparagine and proline.
Furthermore, the neutral amino acid described above can be categorized into one having a polar side chain (asparagine, glutamine, serine, threonine, tyrosine and cysteine), one having a non-polar side chain (glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine and tryptophan), one having an amide-containing side chain (asparagine and glutamine), one having a sulfur-containing side chain (methionine and cysteine), one having an aromatic side chain (phenylalanine, tryptophan and tyrosine), one having a hydroxyl-containing side chain (serine, threonine and tyrosine) and one having an aliphatic side chain (alanine, leucine, isoleucine and valine), etc.
A method of substitution of one or several amino acid residues to other target desired amino acids include, for example, site-directed mutagenesis (Hashimoto-Gotoh T. et al., Gene, Vol. 152, p. 271-275 (1995); Zoller M J. 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 T A., Proc Natl Acad Sci USA., Vol. 82, p. 488-492 (1985) and others), and the use of such site-directed mutagenesis allows to perform amino acid substitution in the amino acid sequence of CDR. Other method of substitution to other amino acid include a library technique described in WO2005/080432.
In the anti-Sema 3A antibody of the present invention, amino acid sequences in the framework region of variable region and in the constant region are not particularly limited unless the amino acid sequences substantially affect the binding activity to Sema 3A protein.
Amino acid sequences of CDRs 1 to 3 (SEQ ID NOs:1-6, 60-62, 64-66, 68-70, 72-74, 76-78, 80-82, 84-86 and 88-90) are derived from an avian antibody, and the anti-Sema 3A antibody of the present invention may be an avian antibody, preferably a chimeric antibody and a humanized antibody.
A chimeric antibody is an antibody in which regions having different origins are conjugated with each other. In the case where a chimeric antibody is used as the anti-Sema 3A antibody of the present invention, as pharmaceutical compositions and therapeutic agents of various diseases, used is desirably an avian-human chimeric antibody which is composed of an avian antibody-derived variable region and a human antibody-derived constant region or an avian-mouse chimeric antibody which is composed of an avian antibody-derived variable region and a mouse antibody-derived constant region, more preferably an avian-human chimeric antibody. In the case where a chimeric antibody is used in immunoassay of and a kit for measurement of Sema 3A protein, an avian-human chimeric antibody and an avian-mouse chimeric antibody can be used and added to which is also an avian-rabbit chimeric antibody which is composed of an avian antibody-derived variable region and a rabbit antibody-derived constant region and an avian-goat chimeric antibody which is composed of an avian antibody-derived variable region and a goat antibody-derived constant region. Amino acid sequences shown in SEQ ID NOs: 7, 8, 59, 67, 75 and 83 are exemplified as examples of an amino acid sequence of the heavy chain variable region used for an avian-human chimeric antibody, an avian-mouse chimeric antibody, an avian-rabbit chimeric antibody or an avian-rabbit chimeric antibody. Amino acid sequences shown in SEQ ID NOs: 9, 10, 63, 71, 79 and 87 are also exemplified as examples of an amino acid sequence of the light chain variable region used for an avian-human chimeric antibody, an avian-mouse chimeric antibody, an avian-rabbit chimeric antibody or an avian-rabbit chimeric antibody. When these amino acid sequences of variable regions (SEQ ID NOs: 7 to 10, 59, 63, 67, 71, 75, 79, 83 and 87) each have a binding activity to Sema 3A protein equal to or greater than that of an unmodified amino acid sequence, one or several amino acids may be substituted, deleted, added and/or inserted. The number of amino acids substituted, deleted, added and/or inserted is not particularly limited, and for example, 1 to 21, preferably 1 to 14, more preferably 1 to 3 in the heavy chain variable region (SEQ ID NOs: 7, 8, 59, 67, 75, 83) and/or the light chain variable region (SEQ ID NOs: 9, 10, 63, 71, 79, 87). Note that in the heavy chain variable region (SEQ ID NOs: 7, 8, 59, 67, 75, 83) and/or the light chain variable region (SEQ ID NOs: 9, 10, 63, 71, 79, 87), amino acids are desirably substituted, deleted, added and/or inserted in other region except amino acid sequences of CDRs 1 to 3. In amino acid sequences of the heavy chain and light chain variable regions, the substitution of amino acids is desirably conservative amino acid substitution as described above.
A humanized antibody is one in which a non-human derived CDR sequence is grafted on a framework region of a human antibody and one which contains a non-human antibody-derived CDR sequence, a human antibody-derived framework region and a human antibody-derived constant region. A humanized antibody has a decreased antigenicity in the human body and therefore, is suitable for pharmaceutical applications of the anti-Sema 3A antibody of the present invention. The amino acid sequence shown in SEQ ID NO: 11 is exemplified as an example of amino acid sequences of the heavy chain variable region used for a humanized antibody. Also, the amino acid sequences shown in SEQ ID NOs: 12 and 13 is exemplified as examples of amino acid sequences of the light chain variable region used for a humanized antibody. When these amino acid sequences of variable regions (SEQ ID NOs: 11 to 13) each has a binding activity to Sema 3A protein equal to that of unmodified amino acid sequences, one or several amino acids may be substituted, deleted, added and/or inserted. The number of amino acids substituted, deleted, added and/or inserted is not particularly limited, and is, for example, 1 to 21, preferably 1 to 14, more preferably 1 to 3 in the heavy chain variable region (SEQ ID NO: 11) and/or the light chain variable region (SEQ ID NOs: 12 and 13). Note that in the heavy chain variable region (SEQ ID NO: 11) and/or the light chain variable region (SEQ ID NOs: 12 and 13), amino acids are desirably substituted, deleted, added and/or inserted in other region except amino acid sequences of CDRs 1 to 3. In amino acid sequences in a heavy chain and light chain variable regions, the substitution of amino acids is desirably a conservative amino acid substitution as described above.
A chimeric antibody can be produced, for example, by substituting an avian antibody-constant region containing the heavy chain variable region and light chain variable region having each of amino acid sequences of CDRs 1 to 3 with a human antibody-constant region (e.g. Morrison et al., Proc. Natl. Acad. Sci., Vol. 81, p. 6851-6855 (1984); Neuberger et al., Nature, Vol. 312, p. 604-608 (1984); Takeda et al., Nature, Vol. 314, p. 452-454 (1985) and others). Nucleotide sequences of DNA encoding SEQ ID NOs: 7, 8, 59, 67, 75 and 83, amino acid sequences in the heavy chain variable region used for an avian-human chimeric antibody are shown in SEQ ID NOs: 14, 15, 103, 111, 119 and 127, respectively. Nucleotide sequences of DNA encoding SEQ ID NOs: 9, 10, 63, 71, 79 and 87, amino acid sequences in the light chain variable region used for an avian-human chimeric antibody are shown in SEQ ID NOs: 16, 17, 104, 112, 120 and 128, respectively. Known amino acid sequences can be used in the human antibody-constant region. In particular, an avian-human chimeric antibody can be produced by the following process.
Firstly, DNA encoding an avian heavy chain variable region containing a CDR having a given amino acid sequence is produced through chemical synthesis, biochemical cleavage, reconjugation and the like. The obtained heavy chain variable region-encoding DNA is ligated with human heavy chain constant region-encoding DNA, which then is incorporated to a vector for expression to produce a heavy chain expression vector. A light chain expression vector is produced in the same manner as described above. The obtained heavy chain expression vector and light chain expression vector are used to cotransform a host cell such as HEK293 cell line, CHO cells, SP2/0 cells, etc. The transformant is cultivated, followed by separation of target chimeric antibody from the transformant culture solution. Alternatively, amino acids of a framework region in the antibody variable region may be substituted so that a CDR of an avian-human chimeric antibody forms a suitable antigen-biding site (Sato, K. et al., Cancer Research, Vol. 53, p. 851-856 (1993)).
A humanized antibody can be produced, for example, by grafting CDRs 1 to 3 containing the amino acid sequences on a framework region of a human antibody (e.g. 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)). Nucleotide sequences of DNA encoding heavy chain CDR 1 (SEQ ID NOs: 1, 60, 68, 76 and 84) are shown in SEQ ID NOs: 18, 97, 105, 113 and 121, respectively. Nucleotide sequences of DNA encoding heavy chain CDR 2 (SEQ ID NOs: 2, 61, 69, 77 and 85) are shown in SEQ ID NOs: 19, 98, 106, 114 and 122, respectively. Nucleotide sequences of DNA encoding heavy chain CDR 3 (SEQ ID NOs: 3, 62, 70, 78 and 86) are shown in SEQ ID NOs: 20, 99, 107, 115 and 123, respectively. Nucleotide sequences of DNA encoding light chain CDR 1 (SEQ ID NOs: 4, 64, 72, 80 and 88) are shown in SEQ ID NOs: 21, 100, 108, 116 and 124, respectively. Nucleotide sequences of DNA encoding light chain CDR 2 (SEQ ID NOs: 5, 65, 73, 81 and 89) are shown in SEQ ID NOs: 22, 101, 109, 117 and 125, respectively. Nucleotide sequences of DNA encoding light chain CDR 3 (SEQ ID NOs: 6, 66, 74, 82 and 90) are shown in SEQ ID NOs: 23, 102, 110, 118 and 126, respectively. In particular, a humanized antibody can be produced by the following process.
A humanized antibody can be produced by treating CDRs 1 to 3 having given amino acid sequences and heavy chain variable region-encoding DNA ligated with four framework regions derived from human antibodies in a given order through chemical synthesis, biochemical cleavage, reconjugation, etc. Here, amino acids of the framework regions may be mutated by substitution, deletion and/or addition, etc. so that the CDRs of humanized antibody form suitable antigen-biding sites (Sato, K. et al., Cancer Research, Vol. 53, p. 851-856 (1993)). The obtained heavy chain variable region-encoding DNA is ligated with a human heavy chain constant region-encoding DNA, which then is incorporated to a vector for expression to produce a heavy chain expression vector. A light chain expression vector is produced in the same manner as described above. The obtained heavy chain expression vector and light chain expression vector are used to cotransform host cells such as FreeStyle 293 cell line (Life Technologies), CHO cells, SP2/0 cells, etc. The transformant is cultivated, followed by separation of target humanized antibody from the transformant culture solution. A nucleotide sequence of DNA encoding the amino acid sequence, SEQ ID NO: 11 in the heavy chain variable region used for a humanized antibody is shown in SEQ ID NO: 24. Nucleotide sequences of DNA encoding the amino acid sequences, SEQ ID NOs: 12 and 13 in the light chain variable region used for a humanized antibody is shown in SEQ ID NOs: 25 and 26. Known amino acid sequences can be used in a human antibody-constant region.
Isotypes of the anti-Sema 3A antibody of the present invention are not particularly limited and examples thereof include IgG (IgG1, IgG2, IgG3 and IgG4), IgA (IgA1 and IgA2), IgM, IgD and IgE, for example. Among them, IgG is preferred.
In the present invention, an antibody fragment can be also used as far as the antigen binding region of anti-Sema 3A antibody is contained therein. Examples of antibody fragments include Fab, Fab′, F(ab′)2, scFv, dsFv, etc. These antibody fragments can be produced according to the conventional methods.
The anti-Sema 3A antibody or antibody fragment thereof of the present invention may be an antibody or its antibody fragment conjugated with various compounds such as polyethylene glycol, a radioactive substance, toxin, etc. The anti-Sema 3A antibody or antibody fragment thereof of the present invention may be treated by modifying a bound sugar chain or fusing with other proteins, as necessary.
2. Pharmaceutical Composition
The present invention provides a pharmaceutical composition containing anti-Sema 3A antibody or its antibody fragment. The pharmaceutical composition of the present invention can effectively inhibit a function of Sema 3A protein and achieve various drug efficacy by containing anti-Sema 3A antibody or its antibody fragment.
In the pharmaceutical composition of the present invention, anti-Sema 3A antibody or its antibody fragment, which is contained as an active component, may be in any form of the aspects (A) to (E), and preferred is the anti-Sema 3A antibody or antibody fragment thereof of the aspect (A) in order to effectively inhibit a function of Sema 3A protein and achieve prominent drug efficacy.
The pharmaceutical composition of the present invention may simply contain an effective amount of the anti-Sema 3A antibody or its antibody fragment and otherwise may contain a pharmaceutically acceptable carrier or additive. Examples of such a carrier or an addictive include a surfactant, an excipient, a colorant, a flavorincagent, a preservative, a stabilizer, a buffer, a pH buffer, a disintegrant, a solubilizer, a solubilizing aid, a tonicity agent, a binder, a disintegrant, a lubricant, a diluent, a corrigent, etc, for example. The carrier or addictive is not limited to them and other carriers or additives commonly used in pharmaceutical compositions can be also used, as applicable.
An administration form of the pharmaceutical composition of the present invention may be in either oral or parenteral form, and particular examples thereof include oral administration; and parenteral administration such as intravenous, intramuscular, intraperitoneal, subcutaneous, transnasal, pulmonary, dermal, transmucosal, intraocular administrations, etc.
A formulation of the pharmaceutical composition of the present invention can be appropriately set depending on the administration form to be adopted. For example, in the case of oral administration, the composition may be prepared in the formulation of a powder, a granule, a capsule, a syrup, a suspension, etc., and in the case of parenteral administration, the composition may be prepared in the formulation of a liquid, a suspension, an emulsion, a spray, a suppository, an eye drop, etc.
The pharmaceutical composition of the present invention can effectively inhibit a function of Sema 3A protein by the action of anti-Sema 3A antibody or its antibody fragment and therefore is useful for prophylaxis and/or treatment of diseases involving Sema 3A protein. Examples of diseases involving Sema 3A protein include, for example, central or peripheral nervous system disease, autoimmune disease, inflammatory disease, infectious disease, allergic disease and cancer, etc. Particular examples of central or peripheral nervous system disease include neuropathic pain, spinal cord injury, and neurodegenerative disease (e.g. Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, progressive supranuclear palsy, striatonigral degeneration, Shy-Drager syndrome, olivopontocerebellar atrophy and spinocerebellar degeneration, etc.). Particular examples of autoimmune disease include rheumatoid arthritis, type I diabetes mellitus, inflammatory bowel disease, Crohn's disease, systemic lupus erythematosus, multiple sclerosis, etc. Particular examples of inflammatory disease include sepsis, chronic obstructive pulmonary disease, asthma, arthritis, hepatitis, spondyloarthritis, Sjogren's syndrome, etc. Particular examples of infectious disease include bacterial infectious disease, encephalitis/meningitis, endocarditis, hepatitis C, influenza/severe acute respiratory syndrome (SARS), pneumonia, sepsis, burn injury, trauma infectious disease, etc. Particular examples of allergic disease include allergic rhinitis, allergic conjunctivitis, atopic dermatitis, bronchial asthma, food allergy, etc. Particular examples of cancer include bowel cancer, colorectal cancer, lung cancer, breast cancer, brain cancer, melanoma, renal cell cancer, leukemia, lymphoma, T-cell lymphoma, stomach cancer, pancreatic cancer, cervical cancer, endometrial cancer, ovarian cancer, esophagus cancer, liver cancer, head and neck squamous cell carcinoma, skin cancer, urinary tract cancer, prostate cancer, choriocarcinoma, pharyngeal cancer, larynx cancer, etc.
Anti-Sema 3A antibody or its antibody fragment can effectively suppress neuronal regeneration-inhibitory ability of Sema 3A protein to accelerate regeneration and elongation of nerves and therefore the pharmaceutical composition of the present invention is useful as a pharmaceutical composition for regenerating and elongating nerves (i.e. pharmaceutical composition for neuronal regeneration and elongation). The pharmaceutical composition of the present invention also allows to regenerate and elongate nerves themselves by anti-Sema 3A antibody or its antibody fragment and therefore, is specifically useful as a pharmaceutical composition for prophylaxis and/or treatment of neurodegenerative disease.
Furthermore, anti-Sema 3A antibody or its antibody fragment exhibits prominent effects of prophylaxis and/or treatment of inflammatory disease containing sepsis, other disease associated with cytokine storm, such as graft versus host disease (GVHD), acute respiratory distress syndrome (ARDS), avian influenza, smallpox, systemic inflammatory response syndrome (SIRS), drug-induced cytokine storm, etc. which are caused by collapsing immunity mechanism due to infection or potent inflammation-inducing stimulus and therefore the antibody or its antibody fragment is highly useful as a pharmaceutical composition for prophylaxis and/or treatment of these diseases.
Anti-Sema 3A antibody or its antibody fragment can effectively suppress migration/invasion activities of cancer cells induced by Sema 3A and exhibits prominent effects of prophylaxis and/or treatment of diseases associated with exacerbation/progression of cancer and therefore, is highly useful as a pharmaceutical composition for prophylaxis and/or treatment of cancerous diseases. When the pharmaceutical composition of the present invention is used for applications of prophylaxis and/or treatment of cancer, other anticancer drug may be used in combination. Anti-Sema 3A antibody or its antibody fragment can specifically remove anticancer drug-unresponsiveness of cancer cells induced by Sema 3A to recover drug sensitivity, and therefore exhibit prominent effects of prophylaxis and/or treatment of cancer in combination with other anticancer drugs. In the case of combined use of anti-Sema 3A antibody or its antibody fragment with other anticancer drug, anti-Sema 3A antibody or its antibody fragment and other anticancer drug may be prepared in single pharmaceutical composition for formulation and anti-Sema 3A antibody or its antibody fragment and other anticancer drug may be prepared in different pharmaceutical compositions for formulation.
The pharmaceutical composition of the present invention can suppress increase of blood level of PAI-1 by the action of anti-Sema 3A antibody or its antibody fragment and therefore, is also effective for prophylaxis and/or treatment of disseminated intravascular coagulation. Examples of underlying diseases associated with disseminated intravascular coagulation include sepsis, acute leukemia, solid cancer, premature separation of normally implanted placenta, amniotic fluid embolism, trauma, burn injury, connective tissue disease, shock, aortic aneurysm, acute hepatitis, liver cirrhosis, acute pancreatitis, thrombosis, severe infectious disease, etc.
Dosage and frequency of the pharmaceutical composition of the present invention varies depending on administration method, age and body weight of the patient, types of disease and degrees of symptom severity, etc. and therefore, cannot be uniformly defined. However, neuronal regeneration commonly requires from a few days to a few months or more and therefore, the composition is preferably administered at the necessary frequency to suppress semaphorin activity during neuronal regeneration process. For example, the composition can be administered at the corresponding amount to 0.1 mg to 1000 mg, preferably 1 mg to 500 mg by weight of anti-Sema 3A antibody or its antibody fragment per a dose at once per about 1 to 30 days. Note that the composition can be also administered in the form of a sustained release agent or portionwise over a long period through an osmotic pump in order to reduce frequency of administration. In all of these administration methods, preferably used is an administration route and an administration method where the composition has a concentration which allows to sufficiently inhibit Sema 3A protein activity at the action site.
3. Measurement Method and Measurement Kit of Sema 3A Protein
The present invention provides a measurement method of Sema 3A protein using anti-Sema 3A antibody or its antibody fragment. The measurement method of the present invention immunologically measures Sema 3A in a sample through antigen-antibody reaction of anti-Sema 3A antibody or its antibody fragment with Sema 3A in the sample.
In the measurement method of the present invention, anti-Sema 3A antibody or its antibody fragment reactive with Sema 3A in a sample may be any of the aspects (A) to (E) and the anti-Sema 3A antibody or antibody fragment thereof of the aspect (A) is preferably mentioned in order to measure Sema 3A protein in a higher accurate manner.
A sample is not particularly limited as far as the measurement of Sema 3A is required, and examples thereof include, for example, biological samples such as blood, serum, plasma, urine, spinal fluid, joint fluid, lymph fluid, amnion fluid, breast fluid, various tissue fluids, hippocampus extract and various tissue extracts, etc.
For the measurement method of the present invention, any immunoassay containing sandwich method, competitive method, coagulation method, etc., may be adopted. In the case where sandwich method, which uses a capture antibody trapping an antigen and a primary antibody binding to the antigen conjugated with the capture antibody, is adopted as the measurement method of the present invention, the capture antibody can be any one selected from the anti-Sema 3A antibody or antibody fragment thereof in the aspects (A) to (E), and the different one in the aspects (A) to (E) from the selected capture antibody can be used as the primary antibody. In the case where Sandwich method is adopted, there are particularly preferred that the anti-Sema 3A antibody or antibody fragment thereof in the aspect (A) is used as the capture antibody and the anti-Sema 3A antibody or antibody fragment thereof in the aspect (B) or (C) is used as a primary antibody since Sema 3A protein can be measured particularly in the presence of serum in a higher accurate manner.
Immunoassay includes enzyme-linked immunosorbent assay (ELISA), fluorescent immunoassay, radioimmunoassay depending on marker types, and any of these methods can be used for the measurement method of the present invention. Enzyme-linked immunosorbent assay is preferably mentioned in the light of simpleness and promptness of measurement.
Immunoassay using antigen-antibody reaction is known per se and the measurement method of the present invention can be carried out by any known approach depending on measuring mechanisms and marker types of immunoassay.
The present invention also provides a measurement kit used in the measurement method. The measurement kit of the present invention includes anti-Sema 3A antibody or its antibody fragment.
The measurement kit of the present invention may contain other reagents and instruments together with anti-Sema 3A antibody or its antibody fragment depending on measuring mechanisms and marker types for immunoassay. For example, in the case where enzyme-linked immunosorbent assay is selected, the kit may contain a measuring plate, a chromogenic substrate solution, a quenching solution, a washing solution, a standard sample, etc. together with anti-Sema 3A antibody or its antibody fragment. In the case where sandwich method is adopted, anti-Sema 3A antibody or its antibody fragment used as a capture antibody can be provided in the state immobilized in a solid phase.
EXAMPLES
The present invention is described with reference to Examples in detail below, however, it should be construed that the present invention is not limited to these examples.
Example 1: Preparation of Anti-Sema 3A Antibody (Avian Antibody and Avian-Mouse Chimeric Antibody)
1) Cell Culture
Cell culture of DT40 cells derived from chicken B cells was carried out according to the following process. A CO2 thermostat is used as an incubator, in which culturing was carried out in the presence of 5% CO2 at 39.5° C. IMDM medium (Life Technologies) is used, to which were added 10 vol % FBS, 1 vol % chicken serum, 100 units/mL of penicillin, 100 μg/mL of streptomycin and 55 μM 2-mercaptoethanol for use herein. Trichostatin A (Wako Pure Chemical Industries, Ltd.) was dissolved in DMSO to 5 mg/mL to prepare a stock solution and the stock solution was appropriately diluted in the medium to the final concentration of 1.25 ng/mL or 2.5 ng/mL before use.
2) Isolation of Anti-Sema 3A Antibody-Producing Cells
Mouse Sema 3A protein was produced from mouse Sema 3A protein expressing cell possessed by Molecular Pharmacology and Neurobiology, Yokohama City University Graduate School of Medicine, which protein was used as an antigen to isolate antibody-producing cells using Autonomously Diversifying Library (ADLib) system (Chiome Bioscience, Inc.). In particular, the following experimental process was carried out.
2-1) Preparation of Magnetic Beads with Immobilized Antigen
Immobilization of mouse Sema 3A protein to magnetic beads for His-tag was carried out according to the following process. Dynabeads TALON (Life Technologies) was used as magnetic beads and Dynal MPC (Life Technologies) was used as a magnetic stand.
Beads (22.5 μL) was washed with 45 μL of PBS buffer three times, which then reacted with 6.1 μg of mouse Sema 3A protein in PBS buffer at 4° C. for 10 minutes while stirring in rotating manner. Subsequently, the beads conjugated with mouse Sema 3A protein was washed four times with 45 μL of buffer C (PBS buffer containing 0.1% BSA) and then suspended in 45 μL of buffer C.
2-2) Selection of Antibody-Producing Clone Through Magnetic Beads with Immobilized Antigen
Wild-type DT40 cells were cultivated for 5 weeks or more in IMDM medium containing 1.25 ng/mL or 2.5 ng/mL of trichostatin A and 8×108 of the cultivated cells were divided into 8 portions by 1×108 cells each, each of which was washed one time with 10 mL of washing solution (PBS buffer containing 1 wt % BSA), followed by washing one time in 1 mL of the same washing solution and then mixed with 5×106 of the magnetic beads with immobilized antigen prepared in 2-1) in 1 mL of washing solution. Then the mixture was incubated at 4° C. for 30 minutes while gently rotating and subsequently washed three times with 1.7 mL of washing solution for 3 minutes using KingFisher mL (Thermo Fisher Scientific K.K.). Finally, the cells associated with magnetic beads with immobilized antigen were suspended in 500 μL of medium, which was added to 20 mL of medium and then dispensed into a 96 well plate by 200 μL each and incubated at 39.5° C. In the following process, IMDM medium (Life Technologies) containing 10 vol % FBS, 100 units/mL of penicillin, 100 μg/mL of streptomycin and 55 μM 2-mercaptoethanol was used as a cultivation medium.
2-3) Screening of Anti-Sema 3A Antibody-Producing Clone
A direct solid phase ELISA was carried out according to the following process. In Day 6 after the step 2-2), 2.5 μg/mL of mouse Sema 3A protein was dispensed into a 384 well Maxisorp (Nunc) by 20 μL each and left to stand overnight. Ovalbumin (OA) and rabbit IgG (rIgG) were immobilized in the plate as negative controls in similar manner in order to examine the specificity of antibodies. On the next day, the content in the plate was removed and 45 μL of blocking solution (PBS buffer containing 1% BSA) was added thereto, followed by incubation at room temperature for one hour. The plate was washed five times with 120 μL/well of ELISA washing buffer (PBS containing 0.05 wt % Tween 20), to which were added culture supernatants derived from the colony obtained by selection in the step 2-2) by 25 μL/well and then incubated at room temperature for one hour. The plate was washed five times with 120 μL/well of ELISA washing solution and then to which was added 25 μL of diluent in which a secondary antibody was diluted 2000 times with a blocking buffer and incubated at room temperature for 45 minutes. Anti-chicken IgM-HRP (Bethyl Laboratories, Inc.) was used as a secondary antibody. The each well was washed five times with 120 μL of ELISA washing buffer, to which was added 25 μL of TMB+(Dako) and incubated for 30 minutes. Subsequently, the reaction was quenched with 25 μL of 1N sulfuric acid to determine the absorbance at 450 nm.
Consequently, thirteen cell strains exhibiting positive for anti-Sema 3A antibody production through direct solid phase ELISA were obtained through the selection by direct association with magnetic beads with immobilized antigen, as shown in FIG. 1.
Among the positive strains obtained, cloning of cell strains No. 4 and No. 6 for antibody producing cells was carried out by limiting dilution, according to the following process.
Each of both strains was prepared to 1×103 cells/mL, 150 μL of which was added to 60 mL of medium and the suspension was seeded in three 96 well plates (Thermo Fisher Scientific K.K.) in an amount of 200 μL/well, followed by stationary culturing for 7 days. Twenty clones from the strains, which was observed to form cell colonies, were subjected to screening of mouse Sema 3A antibody producing clones according to the method described in the step 2-3). Clones exhibiting positive for mouse Sema 3A antibody production were selected by 2 clones each from the strains and then subjected to high-density cell culture.
In high-density cell culture, clones were subjected to extended culture and amplified to 4×107 cells. Clones were observed to be amplified to enough cell counts and the clones were cultivated with CELLine CL-1000 (BD biosciences) in AIM-V medium containing 20 vol % chicken serum component (Life Technoligies). A chicken serum component is a chicken serum in which antibodies were removed. The preparation process comprised removing immunoglobulin as a precipitate with 50% saturated ammonium sulfate from chicken serum (Life Technologies), dialyzing the supernatant with PBS buffer, and concentrating the supernatant using a Centri Prep (Amicon) to adjust the increased volume caused by dialyzing. After culturing for about 96 hours, culturing was continued until a survival rate of cells was 50% or less while measuring the survival rate. As the survival rate decreased to 50% or less, the culture supernatant was harvested.
3) Preparation of Culture Supernatant for ELISA
Culture supernatant for analyzing a titer by ELISA was prepared as the following process. in order to remove serum-derived IgM, etc. A chicken serum component was added to AIM-V serum-free medium (Life Technoligies) at a concentration of 3%. Cells were added to the prepared medium at a concentration of 1×106 cells/mL and cultivated for 2 days to obtain culture supernatant.
4) Selection of Anti-Sema 3A Antibody-Producing Clones
The culture supernatant for ELISA obtained in the step 3) was subjected to ELISA to measure a concentration of anti-Sema 3A antibody and thereby to obtain clone No. 4-2 strain having high anti-Sema 3A antibody production ability.
5) Preparation of Culture Supernatant Containing Anti-Sema 3A Avian Antibody (IgM)
Anti-Sema 3A avian antibody-producing clone No. 4-2 strain obtained in the step 4) was cultivated with IMDM medium in a CO2 incubator in the presence of 5% CO2 at 39.5° C. and the culture supernatant was harvested.
6) Preparation of Anti-Sema 3A Avian-Mouse Chimeric Antibody (IgG)
cDNA was synthesized from total RNA extracted from the anti-Sema 3A antibody producing clone No. 4-2 strain obtained in the step 4) by reverse transcription with a reverse transcriptase (SuperScript III, Life Technoligies) and an oligo (dT) primer. The obtained cDNA was used as a template and light chain variable region genes were amplified by PCR using a sense primer containing the avian antibody 2 light chain variable region sequence (primer 1: GAAGATCTAAGCTTGCCATGGCCTGGGCTCCTCTCCTCCT (SEQ ID NO: 27)) and an antisense primer containing the avian antibody λ light chain variable region sequence and the mouse antibody light chain constant region sequence (primer 2: TGGCGAAGACTTCGGCTGGCCTAGGAC (SEQ ID NO: 28)). At the same time, heavy chain variable region genes were amplified by PCR using a sense primer containing the avian antibody heavy chain variable region sequence (primer 3: GAAGATCTAAGCTTACCATGAGCCCACTCG (SEQ ID NO: 29)) and an antisense primer containing the avian antibody heavy chain variable region and mouse IgG2a antibody constant region (primer 4: CGATGGGGCTGTTGTTTTGGCGGAGGAGACGATGACTTC (SEQ ID NO: 30)). On the other hand, the DNA sequence of mouse antibody 2 light chain constant region was used as a template and mouse light chain constant region genes were amplified by PCR using a sense primer containing a sense primer sequence of the avian antibody λ light chain variable region and the mouse antibody λ light chain constant region sequence (primer 5: AAGTCTTCGCCATCAGTCACCCTGTTTCCA (SEQ ID NO: 31)) and an antisense primer (primer 6: TATGCGGCCGCTTACTAGGAACAGTCA (SEQ ID NO: 32)). Additionally, the cDNA sequence of mouse IgG2a antibody heavy chain constant region was used as a template and mouse heavy chain constant region genes were amplified by PCR using a sense primer containing a sense primer sequence of the avian antibody heavy chain variable region and the mouse IgG2a antibody heavy chain constant region sequence (primer 7: GCCAAAACAACAGCCCCATCGGTCTATCCACTGGCCCCT (SEQ ID NO: 33)) and an antisense primer (primer 8: AGATAGCGGCCGCTTATCATTTACCCGG (SEQ ID NO: 34)).
The amplified light chain variable region fragment and light chain constant region fragment were used as a template and avian-mouse chimeric antibody light chain genes were amplified by PCR using primer 1 and primer 6. Furthermore, the amplified heavy chain variable region fragment and heavy chain constant region fragment were used as a template and avian-mouse chimeric antibody heavy chain genes were amplified by PCR using primer 3 and primer 8.
The avian-mouse chimeric antibody light chain genes and avian-mouse chimeric antibody heavy chain genes were digested with restriction enzymes Hind III and Not I, respectively, each of which then was cloned into Hind III-Not I site of a plasmid pCEP4 for expressing mammal cells (Life Technoligies). The sequence of the cloned antibody gene was analyzed by sequencing using a DNA sequencer. Amino acid sequences of the avian-mouse chimeric antibody light chain and the avian-mouse chimeric antibody heavy chain were translated based on the analyzed nucleotide sequences. The final translated amino acid sequence of the avian-mouse chimeric antibody heavy chain is shown in SEQ ID NO: 35 and the final translated amino acid sequence of the avian-mouse chimeric antibody light chain is shown in SEQ ID NO: 36.
The constructed plasmid in the above step was transfected into FreeStyle 293 cell line (Life Technoligies) using polyethyleneimine method and subjected to shaking culture at 37° C., 8% CO2, and 135 rpm for 7 days to collect culture supernatant.
The concentration of anti-Sema 3A avian-mouse chimeric antibody (IgG) contained in the culture supernatant was quantified by ELISA. Anti-Sema 3A avian-mouse chimeric antibody was purified from the culture supernatant using a column prepared by loading Protein G Sepharose 4 Fast Flow (GE Healthcare) to Poly-Prep Chromatography Columns (Bio-Rad Laboratories, Inc). A solvent of the eluted antibody was exchanged with PBS buffer using a PD-10 column (GE Healthcare).
The antigen specificity of the purified antibody was measured by ELISA with PBS buffer. 2.5 μg/mL of antigen was dispensed to a 96 well Maxi Sorp Plate (Nunc) and reacted overnight at 4° C. to coat the plate with an antigen (mouse Sema 3A protein). Ovalbumin, rabbit IgG and skim milk were immobilized in the plate as negative controls in similar manner in order to examine specificity of antibodies. The next day, the plate was washed three times with 200 μL/well of washing solution (PBS containing 0.05% Tween 20), to which was added 200 μL/well of blocking solution (PBS containing 0.5% skim milk) and the mixture was reacted for 30 minutes. The plate was washed three times with 200 μL/well of washing solution, to which was added 100 μL of culture supernatant containing antibody and the mixture was reacted for one hour. The plate was washed five times with 200 μL/well of washing solution, to which was added 100 μL/well of diluent in which anti-mouse IgG2A goat antibody labeled with horseradish peroxidase (HRP) (Bethyl Laboratories, Inc.) was diluted 2000 times with PBS buffer and the mixture was reacted for one hour. The plate was washed five times with 200 μL/well of washing solution, to which was added 100 μL/well of TMB+(Dako) and the mixture was chromogenically reacted for five minutes. Subsequently, to the plate was added 100 μL of 1M sulfuric acid to quench the reaction. The absorbance was measured at 450 nm using μQuantBiomolecular Spectrometer (Bio Tek Instruments, Inc.) Results are shown in FIG. 2.
7) Determination of CDR Sequences in Variable Region of Anti-Sema 3A Avian-Mouse chimeric antibody
CDR sequence of the anti-Sema 3A avian-mouse chimeric antibody obtained in the above step was determined. The CDR sequence was determined according to the method of Kabat et al. (Sequences of Proteins of Immunological Interest, NIH publication, 91-3242). Consequently, the following sequences were specified as the amino acid sequences of CDRs 1 to 3 of a heavy chain and a light chain.







TABLE 1





Anti-Sema 3A avian-mouse chimeric antibody


(clone No. 4-2-derived)
















Heavy chain CDR 1
SYPMG (SEQ ID NO: 1)



(corresponding to positions



31 to 35 of SEQ ID NO: 35)


 


Heavy chain CDR 2
GIDDDGDSDTRYAPAVKG (SEQ ID NO: 2)



(corresponding to positions



50 to 67 of SEQ ID NO: 35)


 


Heavy chain CDR 3
HTGIGANSAGSIDA (SEQ ID NO: 3)



(corresponding to positions



100 to 113 of SEQ ID NO: 35)


 


Light chain CDR 1
SGGGSYTGSYYYG (SEQ ID NO: 4)



(corresponding to positions



21 to 33 of SEQ ID NO: 36)


 


Light chain CDR 2
YNNKRPS (SEQ ID NO: 5)



(corresponding to positions



50 to 56 of SEQ ID NO: 36)


 


Light chain CDR 3
GSADNSGDA (SEQ ID NO: 6)



(corresponding to positions



89 to 97 of SEQ ID NO: 36)









Example 2: Preparation of Anti-Sema 3A Antibody (Humanized Antibody and Avian-Human Chimeric Antibody)
1) Construction of Humanized Antibody (Humanized-1 and Humanized-2) Genes
Two humanized antibodies (Humanized-1 and Humanized-2) were designed by altering to amino acids in the framework region of a human antibody from ones in the framework region of the variable region of anti-Sema 3A antibody produced by the clone No: 4-2 strain obtained in the step 4) of Example 1.
The designed heavy chain (the sequences of Humanized-1 and 2 are same) and Humanized-1 light chain amino acid sequence-encoding genes were separately synthesized with consideration of codon optimization to express in human cells. Alternatively, Humanized-1 light chain gene sequence was used as a template and the amplified product by PCR using a sense primer (primer Aγ: GAAGATCTAAGCTTCCACCATGGCAT (SEQ ID NO: 37)) and an antisense primer (primer Bγ: TTGTAATAGATCACTGTCACGGGA (SEQ ID NO: 38)) and the amplified product by PCR using a sense primer (primer Cγ: TCCCGTGACAGTGATCTATTACAA (SEQ ID NO: 39)) and an antisense primer (primer Dγ: AGATAGCGGCCGCTTAGGAACATTC (SEQ ID NO: 40)) were subjected to assembly PCR using a sense primer Aγ and an antisense primer Dγ in order to synthesize Humanized-2 light chain.
2) Construction of Avian-Human Chimeric Antibody Genes
Avian-human chimeric antibody genes were constructed as follows. cDNA of the obtained clone No. 4-2 strain in the step 4) of Example 1 was used as a template and light chain variable region genes were amplified by PCR using a sense primer containing the avian antibody λ light chain variable region sequence (primer Eγ: GAAGATCTAAGCTTCCACCATGGCCTGGGCTCCTCT (SEQ ID NO: 41)) and an antisense primer containing the avian antibody λ light chain variable region sequence and human antibody λ light chain constant region sequence (primer Fγ: CTTTGGGCTGGCCTAGGACGGTCAGGGTTGT (SEQ ID NO: 42)). Alternatively, heavy chain variable region genes were amplified by PCR using a sense primer containing the avian antibody heavy chain variable region sequence (primer Gγ: GAAGATCTAAGCTTCCACCATGAGCCCACTCG (SEQ ID NO: 43)) and an antisense primer containing the avian antibody heavy chain variable region and human antibody IgG1 constant region (primer Hγ: GCCCCTTTGTACTAGCGGAGGAGACGATGACTTC (SEQ ID NO: 44)). On the other hand, the DNA sequence of synthesized Humaized-1 human antibody λ light chain constant region was used as a template and human λ light chain constant region genes were amplified by PCR using a sense primer containing the human antibody λ light chain constant region sequence (primer Iγ: GGCCAGCCCAAAGCCAACCCTACCGTG (SEQ ID NO: 45)) and an antisense primer (primer Jγ: AGATAGCGGCCGCTTATTAGGAACATTCGGTT (SEQ ID NO: 46)). Furthermore, the synthesized DNA sequence of Humanized-1 human IgG1 antibody heavy chain constant region was used as a template and human IgG1 heavy chain constant region genes were amplified by PCR using a sense primer containing the human IgG1 antibody heavy chain constant region sequence (primer Kγ: GCTAGTACAAAGGGGCCCTCAGTGTTCCCACTG (SEQ ID NO: 47)) and an antisense primer (primer Lγ: AGATAGCGGCCGCTTATTATTTTCCAGGTGACAG (SEQ ID NO: 48)).
The amplified light chain variable region fragment and light chain constant region fragment were used as a template and avian-human chimeric antibody light chain genes were amplified by PCR using the primer Eγ and primer Jγ. Furthermore, the amplified heavy chain variable region fragment and heavy chain constant region fragment were used as a template and avian-human chimeric antibody heavy chain genes were amplified by PCR using the primer Gγ and primer Lγ.
3) Construction of Expression Vector and Expression, Purification and Concentration Measurement of Protein
The synthesized Humanized-1 light chain gene and heavy chain gene, Humanized-2 light chain gene and heavy chain gene, and avian-human chimeric antibody light chain gene and heavy chain gene were digested with restriction enzymes Hind III and Not I, each of which was cloned into Hind III-Not I site of a plasmid pCEP4 for expressing mammal cells (Life Technoligies). The presence of the cloned antibody gene sequences was analyzed by sequencing using a DNA sequencer. Amino acid sequences of a humanized antibody (Humanized-1 and Humanized-2) and an avian-human chimeric antibody heavy chain and light chain were translated based on the sequenced nucleotide sequences.
The final determined amino acid sequence of humanized antibodies (Humanized-1 and Humanized-2) heavy chain is shown in SEQ ID NO: 49 and the amino acid sequence-encoding nucleotide sequence is shown in SEQ ID NO: 50. The amino acid sequence of the heavy chain (SEQ ID NO: 49) contains the amino acid sequence of the variable region shown in SEQ ID NO: 11. Furthermore, the final determined amino acid sequence of humanized antibody (Humanized-1) light chain is shown in SEQ ID NO: 51 and the amino acid sequence-encoding nucleotide sequence is shown in SEQ ID NO: 52. The amino acid sequence of the light chain (SEQ ID NO: 51) contains the amino acid sequence of the variable region shown in SEQ ID NO: 12. Additionally, the final determined amino acid sequence of humanized antibody (Humanized-2) light chain is shown in SEQ ID NO: 53 and the amino acid sequence-encoding nucleotide sequence is shown in SEQ ID NO: 54. The amino acid sequence of the light chain (SEQ ID NO: 53) contains the amino acid sequence of the variable region shown in SEQ ID NO: 13.
The final determined amino acid sequence of avian-human chimeric antibody (clone No. 4-2-derived) heavy chain is shown in SEQ ID NO: 55 and the amino acid sequence-encoding nucleotide sequence is shown in SEQ ID NO: 56. The amino acid sequence of the heavy chain (SEQ ID NO: 55) contains the amino acid sequence of the variable region shown in SEQ ID NO: 8. Furthermore, the final determined amino acid sequence of avian-human chimeric antibody (clone No. 4-2-derived) light chain is shown in SEQ ID NO: 57 and the amino acid sequence-encoding nucleotide sequence is shown in SEQ ID NO: 58. The amino acid sequence of the light chain (SEQ ID NO: 57) contains the amino acid sequence of the variable region shown in SEQ ID NO: 10.
The constructed expression plasmid in the above step was transfected into FreeStyle 293 cell line (Life Technoligies) by polyethyleneimine and subjected to shaking culture at 37° C., 8% CO2, and 135 rpm for 7 days to collect culture supernatant.
Anti-Sema 3A humanized antibodies (Humanized-1 and Humanized-2) and avian-human chimeric antibody were purified from the culture supernatant using a column prepared by loading Protein A Sepharose 4 Fast Flow (GE Healthcare) to Poly-Prep Chromatography Columns (Bio-Rad Laboratories, Inc). A solvent of the eluted antibody was exchanged with PBS buffer using a PD-10 column (GE Healthcare).
The molecular absorbance coefficient was calculated from amino acid composition of the purified antibody and the concentration was determined by ultraviolet absorbance method. 500 ng of the purified antibody in reduced or non-reduced state was subjected to electrophoresis and the gel was stained with Coomassie Brilliant Blue (CBB). Results are shown in FIG. 3. FIG. 3 also shows results of electrophoresis with CBB stain of the obtained avian-mouse antibody in Example 1 and CL18M(+) avian-mouse chimeric antibody (control).
Example 3: Preparation of Anti-Sema 3A Antibody (Avian-Mouse Chimeric Antibody)
Eight cell strains exhibiting positive for anti-Sema 3A antibody production were obtained in the same antibody screening process as the steps 1) and 2) of Example 1 except that human Sema 3A protein was used as an antigen and DT40 cells were used in which the constant region of chicken IgM was replaced with that of mouse IgG. Used were No. 165 and No. 582 strains among clones exhibiting positive for anti-Sema 3A antibody production for preparation and purification of anti-Sema 3A antibody to obtain anti-Sema 3A avian-mouse chimeric antibody (IgG) (clone No. 165 strain and clone No. 582 strain-derived). The variable regions of the heavy chain and light chain of anti-Sema 3A avian-mouse chimeric antibody and CDR sequencings were carried out, respectively. The CDR sequence was determined according to the method of Kabat et al. (Sequences of Proteins of Immunological Interest, NIH publication, 91-3242). Results are shown in Tables 2 and 3.







TABLE 2





Anti-Sema 3A avian-mouse chimeric antibody


(clone No. 165 strain-derived)
















Heavy chain
AVTLDESGGGLQTPGGGLSLVCKASGFTFS


variable region
SYEMQWVRQAPGKGLEWVAGIYTGSTWYGA



AVKGRATISRDNGQSTVRLQLNNLRAEDTA



IYYCAKSGIGVNSAAFIDAWGHGTEVIVSS



(SEQ ID NO: 59)


 


Heavy chain CDR 1
SYEMQ (SEQ ID NO: 60)



(corresponding to positions



31 to 35 of SEQ ID NO: 59)


 


Heavy chain CDR 2
GIYTGSTWYGAAVKG (SEQ ID NO: 61)



(corresponding to positions



50 to 64 of SEQ ID NO: 59)


 


Heavy chain CDR 3
SGIGVNSAAFIDA (SEQ ID NO: 62)



(corresponding to positions 



97 to 109 of SEQ ID NO: 59)


 


Light chain
ALTQPASVSANPGETVKITCSGGGSYGGSY


variable region
YYGWYQQKAPGSAPVTVIYNNNKRPSNIPS



RFSGSLSGSTNTLTITGVRADDEAVYFCGS



ADNSGTAFGAGTTLTVL (SEQ ID NO: 63)


 


Light chain CDR 1
SGGGSYGGSYYYG (SEQ ID NO: 64)



(corresponding to positions



21 to 33 of SEQ ID NO: 63)


 


Light chain CDR 2
NNNKRPS (SEQ ID NO: 65)



(corresponding to positions



50 to 56 of SEQ ID NO: 63)


 


Light chain CDR 3
GSADNSGTA (SEQ ID NO: 66)



(corresponding to positions



89 to 97 of SEQ ID NO: 63)
















TABLE 3





Anti-Sema 3A avian-mouse chimeric antibody


(clone No. 582 strain-derived)
















Heavy chain
AVTLDESGGGLQTPGGALSLVCKASGFTMS


variable region
SYEMQWVRQAPGKGLEWVAAIYTRSTWYGA



AVKGRATISRDNGQSTVRLQLNNLRAEDTG



TYYCAKSGIGLNSAAFIDAWGHGTEVIVSS



(SEQ ID NO: 67)


 


Heavy chain CDR 1
SYEMQ (SEQ ID NO: 68)



(corresponding to positions



31 to 35 of SEQ ID NO: 67)


 


Heavy chain CDR 2
AIYTRSTWYGAAVKG (SEQ ID NO: 69)



(corresponding to positions



50 to 64 of SEQ ID NO: 67)


 


Heavy chain CDR 3
SGIGLNSAAFIDA (SEQ ID NO: 70)



(corresponding to positions



97 to 109 of SEQ ID NO: 67)


 


Light chain
ALTQPASVSANPGETVKITCSGGGSYGGSY


variable region
YYGWYQQKAPGSAPVTVIYTNNKRPSNIPS



RFSGSLSGSTNTLTITGVRADDEAVYFCGS



ADNSGTAFGAGTTLTVL (SEQ ID NO: 71)


 


Light chain CDR 1
SGGGSYGGSYYYG (SEQ ID NO: 72)



(corresponding to positions



21 to 33 of SEQ ID NO: 71)


 


Light chain CDR 2
TNNKRPS (SEQ ID NO: 73)



(corresponding to positions



50 to 56 of SEQ ID NO: 71)


 


Light chain CDR 3
GSADNSGTA (SEQ ID NO: 74)



(corresponding to positions



89 to 97 of SEQ ID NO: 71)









Example 4: Preparation of Anti-Sema 3A Antibody (Avian Antibody and Avian-Human Chimeric Antibody)
1) Preparation of Avian Antibody (IgM)
Mouse Sema 3A protein was used as an antigen and antibody screening was carried out in the same process as the steps 1) and 2) in Example 1 to obtain clones exhibiting positive for anti-Sema 3A antibody production. Among the clones, 240-40 strain and 255-72 strain were used for preparation and purification of anti Sema 3A antibody to obtain anti-Sema 3A avian antibody (clone No. 240-40 strain and clone No. 255-72 strain-derived). The variable regions of the heavy chain and light chain of anti-Sema 3A avian antibody and CDR sequencings were carried out, respectively. The CDR sequence was determined according to the method of Kabat et al. (Sequences of Proteins of Immunological Interest, NIH publication, 91-3242). Results are shown in Tables 4 and 5.







TABLE 4





Anti-Sema 3A avian antibody


(clone No. 240-40 strain-derived)
















Heavy chain
AVTLDESGGGLQTPGGALSLVCKASGFTFSS


variable region
YDMNWVRQAPGKGLEWVAGIYSGSSTYYGAA



VKGRATISRDNGQSTLRLQLNNLRAEDTGIY



YCAKSAIPVNSAGSIDAWGHGTEVIVSS



(SEQ ID NO: 75)


 


Heavy chain CDR 1
SYDMN (SEQ ID NO: 76)



(corresponding to positions



31 to 35 of SEQ ID NO: 75)


 


Heavy chain CDR 2
GIYSGSSTYYGAAVKG (SEQ ID NO: 77)



(corresponding to positions



50 to 65 of SEQ ID NO: 75)


 


Heavy chain CDR 3
SAIPVNSAGSIDA (SEQ ID NO: 78)



(corresponding to positions



98 to 110 of SEQ ID NO: 75)


 


Light chain
ALTQPASVSANPGETVKITCSGGGSYGGSYY


variable region
YSWHQQKSPGSALVTVIYYNNKRPSDIPSRF



SGSLSGSTNTLTITGVRADDEAVYFCGSADT



SGTAFGAGTTLTVL (SEQ ID NO: 79)


 


Light chain CDR 1
SGGGSYGGSYYYS (SEQ ID NO: 80)



(corresponding to positions



21 to 33 of SEQ ID NO: 79)


 


Light chain CDR 2
YNNKRPS (SEQ ID NO: 81)



(corresponding to positions



50 to 56 of SEQ ID NO: 79)


 


Light chain CDR 3
GSADTSGTA (SEQ ID NO: 82)



(corresponding to positions



89 to 97 of SEQ ID NO: 79)
















TABLE 5





Anti-Sema 3A avian antibody


(clone No. 255-72 strain-derived)
















Heavy chain
AVTLDESGGGLQTPGGALSLVCKASGFTFSS


variable region
YEMQWVRQAPGKGLEWVAGIYSGSTWYGAAV



KGRATISRDNGQSTVRLQLNNLRAEDTGTYY



CAKSGIGFNSAGSIDAWGHGTEVIVSS



(SEQ ID NO: 83)


 


Heavy chain CDR 1
SYEMQ (SEQ ID NO: 84)



(corresponding to positions



31 to 35 of SEQ ID NO: 83)


 


Heavy chain CDR 2
GIYSGSTWYGAAVKG (SEQ ID NO: 85)



(corresponding to positions



50 to 64 of SEQ ID NO: 83)


 


Heavy chain CDR 3
SGIGFNSAGSIDA (SEQ ID NO: 86)



(corresponding to positions



97 to 109 of SEQ ID NO: 83)


 


Light chain
ALTQPASVSANPGETVKITCSGGGSYGGSYY


variable region
YSWHQQKSPGSALVTVIYYNNKRPSDIPSRF



SGSLSGSTNTLTITGVRADDEAVYFCGSADN



SGSAFGAGTTLTVL (SEQ ID NO: 87)


 


Light chain CDR 1
SGGGSYGGSYYYS (SEQ ID NO: 88)



(corresponding to positions



21 to 33 of SEQ ID NO: 87)


 


Light chain CDR 2
YNNKRPS (SEQ ID NO: 89)



(corresponding to positions



50 to 56 of SEQ ID NO: 87)


 


Light chain CDR 3
GSADNSGSA (SEQ ID NO: 90)



(corresponding to positions



89 to 97 of SEQ ID NO: 87)









2) Preparation of Avian-Human Chimeric Antibody (IgG1)

cDNA was synthesized from total RNA extracted from anti-Sema 3A antibody-producing clone No. 240-40 strain and 255-72 strain by reverse transcription with a reverse transcriptase (SuperScript III, Life Technologies). The obtained cDNA was used as a template and light chain variable region genes were amplified by PCR using a sense primer containing an avian antibody λ light chain variable region sequence (primer Mγ: AGCTTGCTAGCGGCCACCATGGCCTGGGCTCCTCTC (SEQ ID NO: 91)) and an antisense primer (primer Nγ: TCTGGCGGCCGCTAGACTCACCTAGGACGGTCAGGGTTGTC (SEQ ID NO: 92)). Alternatively, heavy chain variable region genes were amplified by PCR using a sense primer containing an avian antibody heavy chain variable region sequence (primer Oγ: AGCTTGCTAGCGGCCACCATGAGCCCACTCGTCTCC (SEQ ID NO: 93)) and an antisense primer (primer Pγ: TCTGGCGGCCGCTAGACTCACCGGAGGAGACGATGACTTC (SEQ ID NO: 94)).
DNA sequences shown in SEQ ID NO: 95 in the heavy chain constant region and SEQ ID NO:96 in the light chain constant region were produced by total synthesis of their genes and both sequences were digested with Not I and Eag I, which then were linked to Not I site of a vector pCEP4 (Life Technologies).
The amplified variable region sequence was cloned to the vector containing the constant region constructed in the above manner. In particular, both of a heavy chain constant region vector and a light chain constant region vector were digested with Not I and heavy chain variable region cDNA and light chain variable region cDNA were linked with each vector using In-fusion HD Cloning Kit (TAKARA BIO INC.)
The constructed plasmid in the above step was transfected into FreeStyle 293 cell line (Life Technoligies) by polyethyleneimine and subjected to shaking culture at 37° C., 8% CO2, 135 rpm to recover culture supernatant, which was purified by Protein A to obtain anti-Sema 3A avian-human chimeric antibody (IgG1) (clone No. 240-40 strain and clone No. 255-72 strain-derived).
Example 5: Measurement of Antigenic Specificity
The obtained avian-mouse chimeric antibody in Example 1 and the obtained humanized antibodies (Humanized-1 and Humanized-2) and avian-human chimeric antibody in Example 2 were measured for antigenic specificity by antigen solid phase ELISA with PBS buffer. The obtained avian-mouse chimeric antibody in Example 1, which was labeled with biotin, was also measured for antigenic specificity. CL18M (+) avian-mouse chimeric antibody, which served as a negative control, was also measured for antigenic specificity. Particular measurement conditions were as follows.
Firstly, 2.5 μg/mL of antigen (mouse and human Sema 3A protein) was dispensed into a 384 Well MaxiSorp Plate (Nunc) and reacted overnight at 4° C. in order to coat the plate with the antigen. Otherwise, ovalbumin (OVA) and bovine serum albumin (BSA) were immobilized in the plate as negative controls in similar manner in order to examine specificity of antibodies. After reaction, the plate was washed three times with 50 μL/well of washing solution (PBS containing 0.05% Tween 20), to which was added 25 μL/well of blocking solution (PBS buffer containing 1% bovine serum albumin), and the mixture was reacted for 30 minutes. The resultant product was washed three times with 50 μL/well of washing solution, to which were added 20 μL/well of respective antibodies and the mixture was reacted for one hour. The resultant product was washed five times with 50 μL/well of washing solution, to which was added 20 μL of diluent in which anti-human λ light chain goat antibody labeled with horseradish peroxidase (HRP) (SouthernBiotech) was diluted 4000 times with PBS buffer, in which anti-mouse IgG2A goat antibody labeled with horseradish peroxidase (HRP) (Bethyl Laboratories, Inc.) was diluted 2000 times with PBS buffer or in which streptavidin labeled with horseradish peroxidase (HRP) (Thermo Fisher Scientific K.K.) was diluted 10000 times with PBS buffer and the mixture was reacted for one hour. The plate was washed five times with 50 μL/well of washing solution, to which was added 20 μL/well of TMB+(Dako) and the mixture was chromogenically reacted for 20 minutes. Subsequently, to the each well was added 20 μL of 1M sulfuric acid to quench the reaction. The absorbance was measured at 450 nm using Infinite M1000 (Tecan Trading AG).
Results are shown in FIG. 4. As apparently shown in FIG. 4, the antibodies obtained in Examples 1 and 2 are observed to exhibit reactivity with human Sema 3A and mouse Sema 3A antigen, but not to exhibit reactivity with ovalbumin and BSA used as negative antigens. Results show that antibodies obtained in Examples 1 and 2 exhibit cross reactivity specific to Sema 3A.
Example 6: Measurement of Affinity to Antigen-1
The obtained clone No. 4-2-derived avian-mouse chimeric antibody in Example 1, the obtained humanized antibodies in Example 2 (Humanized-1 and Humanized-2) and avian-human chimeric antibody were subjected to competitive ELISA to evaluate affinity to the antigen. CL18M (+) avian-mouse chimeric antibody, which served as a negative control, was also measured for affinity to the antigen. Particular measurement conditions were as follows.
Firstly, 2.5 μg/mL of antigen (mouse and human Sema 3A protein) was dispensed into a 384 Well MaxiSorp Plate (Nunc) and reacted overnight at 4° C. in order to coat the plate with the antigen. The next day, the plate was washed three times with 50 μL/well of washing solution (PBS containing 0.05% Tween 20), to which was added 25 μL/well of blocking solution (PBS containing 1% bovine serum albumin) and the mixture was reacted for 30 minutes. Each of humanized antibodies (Humanized-1 and Humanized-2), avian-human chimeric antibody and avian-mouse chimeric antibody was subjected to serial doubling dilution, to which was mixed anti-Sema 3A avian-mouse chimeric antibody labeled with biotin to the final concentration of 60 ng/mL, and the mixture was reacted with the antigen solid phase plate which was washed three times with 50 μL/well of washing solution for one hour. The plate was washed five times with 50 μL/well of washing solution, to which was added 20 μL/well of diluent in which streptavidin labeled with horseradish peroxidase (HRP) (Thermo Fisher Scientific K.K.) was diluted 10000 times with PBS buffer and the mixture was reacted for 30 minutes. The plate was washed five times with 50 μL/well of washing solution, to which was added 20 μL/well of TMB+(Dako) and the mixture was chromogenically reacted for 20 minutes. Subsequently, to the each well was added 20 μL, of 1M sulfuric acid to quench the reaction. The absorbance was measured at 450 nm using Infinite M1000 (Tecan Trading AG).
Results are shown in FIG. 5. Table 6 also shows results of calculated 50% inhibitory concentration IC50 (μg/mL) of anti-Sema 3A avian-mouse chimeric antibody on antigen-antibody reaction for antibodies. Consequently, humanized antibodies (Humanized-1 and Humanized-2), avian-human chimeric antibody and avian-mouse chimeric antibody were almost comparable for affinity and therefore, humanized antibodies are not observed to have significant changes for affinity.









TABLE 6









IC50 (μg/mL)










Human Sema
Mouse Sema


Clone No. 4-2-derived antibodies
3A antigen
3A antigen





Humanized antibody (Humanized-1)
0.071
0.120


Humanized antibody (Humanized-2)
0.101
0.157


Avian-human chimeric antibody
0.054
0.112


Avian-mouse chimeric antibody
0.074
0.147


CL18M (+) avian-mouse antibody
N.D.
N.D.


(negative control)









Example 7: Measurement of Affinity to Antigen-2
The obtained clone No. 4-2-derived avian-mouse chimeric antibody in Example 1, the obtained humanized antibodies (Humanized-1 and Humanized-2) in Example 2 and avian-human chimeric antibody were evaluated for affinity to the antigen using BIACORE (GE Healthcare). Particular conditions were as follows.
A sensor chip CM5 (GE Healthcare) was activated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) and then reacted with Protein A (NACALAI TESQUE, INC.) diluted to 50 μg/mL with Acetate 5.0 (GE Healthcare), that is, subjected to amine coupling reaction. Antibodies were reacted at concentrations of capture amount of about 200 RU, with which were reacted antigens (mouse and human Sema 3A protein) at a concentration of 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM or 3.125 nM. The association time was 3 minutes and the dissociation time was 4 minutes, from which reaction, the obtained sensorgram was analyzed by Langmuir binding model (BlAevaluation Version 4.1) (GE Healthcare) to calculate KD (dissociation constant).
Consequently, KD values for human Sema 3A were 3-4 nM for humanized antibodies (Humanized-1 and Humanized-2), 14-15 nM for avian-human chimeric antibody and avian-mouse chimeric antibody. KD values for mouse Sema 3A were also 4-5 nM for humanized antibodies (Humanized-1 and Humanized-2), 17-18 nM for avian-human chimeric antibody and avian-mouse chimeric antibody. Both humanized antibodies of Humanized-1 and Humanized-2 had low KD values for Sema 3A compared to ones of the chimeric antibodies, suggesting affinity improvement by humanization.
Example 8: Measurement of Human Sema 3A by Sandwich ELISA
With use of the obtained anti-Sema 3A antibodies in Examples 2-4, human Sema 3A was measured by sandwich ELISA. Particular conditions were as follows.
Firstly, capture antibodies shown in Table 7 were diluted 1:2000, which was dispensed to a 384 Well Maxi Sorp Plate (Nunc) to react overnight at 4° C. in order to coat the plate with the capture antibodies. After reaction, each well was washed three times with a washing solution (PBS containing 0.05% Tween 20, pH 7.2), to which was added 45 μL of blocking solution (PBS containing 1% bovine serum albumin, pH 7.2) and the mixture was reacted for two hours at room temperature. Subsequently, the plate was washed three times with a washing solution (PBS containing 0.05% Tween 20, pH 7.2), to which was added 20 μL of antibodies and the mixture was reacted for one hour. Then, test samples were added in an amount of 25 μL to each well and the mixture was reacted at room temperature for one hour. Note that for the test sample, used was a diluent in which recombinant human Sema 3A-Fc chimeric protein (R&D systems, #1250-S3-02J) was serially diluted with PBS or a diluent in which recombinant human Sema 3A-Fc chimeric protein (R&D systems, #1250-S3-02J) was serially diluted with human serum type AB (Sigma-Aldrich Japan, #H4522) diluted five times with PBS. Subsequently, the wells were washed three times with a washing solution (PBS containing 0.05% Tween 20, pH 7.2), to each of which was added 25 μL of a primary antibody shown in Table 7 (diluted with PBS containing 1% bovine serum albumin (pH 7.2), at a concentration of 0.1 μg/mL) and the mixture was reacted at room temperature for one hour. The resultant product was washed five times with a washing solution (PBS containing 0.05% Tween 20, pH 7.2) and then reaction and detection of a secondary antibody were carried out by the following methods.
(Cases of Conditions 1, 2 and 5-8)
Anti-mouse IgG antibody labeled with horseradish peroxidase (HRP) (GE #NA931; diluted 1:1000 with PBS containing 1% bovine serum albumin (pH 7.2)) was added in an amount of 25 μL to each well and the mixture was reacted at room temperature for one hour, followed by washing five times with a washing solution (PBS containing 0.05% Tween 20, pH 7.2). Subsequently, to the each well was added 20 μL of TMB (Dako, #S1599) and the mixture was chromogenically reacted for 20 minutes, to which was added 20 μL of 1N sulfuric acid to quench the reaction. The absorbance was measured at 450 nm using Infinite M1000 (Tecan Trading AG).
(Cases of Conditions 3 and 4)
Mouse anti-avian IgM antibody labeled with biotin (BeckmanCoulter Inc. #733087; diluted 1:20000 with PBS containing 1% bovine serum albumin (pH 7.2)) was added in an amount of 25 μL to each well and the mixture was reacted at room temperature for one hour, followed by washing five times with a washing solution (PBS containing 0.05% Tween 20, pH 7.2). Subsequently, streptavidin labeled with horseradish peroxidase (HRP) (Thermo Fisher Scientific K.K. 21130; diluted 10000 times with PBS containing 1% bovine serum albumin (pH 7.2)) was added in an amount of 25 μL to each well and the mixture was reacted at room temperature for one hour, followed by washing five times with a washing solution (PBS containing 0.05% Tween 20, pH 7.2). Subsequently, 20 μL of TMB (Dako, #S1599) was added to the each well and the mixture was chromogenically reacted for 20 minutes, to which 20 μL of 1N sulfuric acid was added to quench the reaction. The absorbance was measured at 450 nm using Infinite M1000 (Tecan Trading AG).
(Case of Condition 9)
Anti-goat IgG antibody labeled with horseradish peroxidase (HRP) (Bethyl Laboratories, Inc. #A50; diluted 1:10000 with PBS containing 1% bovine serum albumin (pH 7.2)) was added in an amount of 25 μL to each well and the mixture was reacted at room temperature for one hour, followed by washing five times with a washing solution (PBS containing 0.05% Tween 20, pH 7.2). Subsequently, to the 20 μL of TMB (Dako, #S1599) was added to the each well and the mixture was chromogenically reacted for 20 minutes, to which 20 μL, of 1N sulfuric acid was added to quench the reaction. The absorbance was measured at 450 nm using Infinite M1000 (Tecan Trading AG).
The obtained results are shown in Table 7, and FIGS. 6 and 7. Note that FIG. 6 shows a relationship of antigen (Sema 3A) and OD 450 (absorbance at 450 nm) obtained by measuring test samples not containing serum in Conditions 1 to 4 and 8. FIG. 7 also shows a relationship of antigen (Sema 3A) and OD 450 (absorbance at 450 nm) obtained by measuring test samples containing serum in Conditions 1 and 2. These results apparently shows that use of anti-Sema 3A antibodies obtained in Examples 1 to 4 allows to measure Sema 3A by ELISA. In the case where the obtained humanized antibody (Humanized-2) in Example 2 was used as a capture antibody and the obtained avian-mouse chimeric antibodies (clone No. 165 strain and clone No. 582 strain-derived) in Example 3 were used as the primary antibodies (Conditions 1 and 2), Sema 3A in serum also can be measured in a high accurate manner, which was found that Sema 3A in an analyte derived from biological entity can be measured. On the other hand, in the case where anti-Sema 3A antibody available commercially was used (Condition 9), Sema 3A could not be detected.









TABLE 7









Test results













Test samples




Test samples
(cases of 




(cases of serum-free)
serum-containing)















Absorbance 

Absorbance 





at 450 nm 

at 450 nm 



Test conditions

of Sema 

of Sema 














Capture antibodies
Primary antibodies
Results
3A of 1 μM
Results
3A of 1 μM
















Condition 1
Humanized antibody 
Avian-mouse chimeric 
Detectable
0.706
Detectable
0.807



obtained in Example 2 
antibody obtained in 







(Humanized-2)
Example 3 (clone No.








166 strain-derived)






Condition 2
Humanized antibody 
Avian-mouse chimeric 
Detectable
1.799
Detectable
0.811



obtained in Example 2 
antibody obtained in 







(Humanized-2)
Example 3 (clone No. 








582 strain-derived)
















Condition 3
Humanized antibody 
Avian antibody obtained 
Detectable
0.191
Not performed



obtained in Example 2 
in Example 4 (clone No. 






(Humanized-2)
240-40 strain-derived)





Condition 4 
Humanized antibody 
Avian antibody obtained 
Detectable
0.174
Not performed



obtained in Example 2 
in Example 4 (clone No. 






(Humanized-2)
255-72 strain-derived)





Condition 5
Avian antibody 
Avian-mouse chimeric 
Detectable
3.403
Not performed



obtained in Example 4 
antibody obtained in 






(clone No. 240-40 
Example 1 (clone No.






strain-derived)
4-2 strain-derived)





Condition 6
Avian antibody 
Avian-mouse chimeric 
Detectable
2.916
Not performed



obtained in Example 4 
antibody obtained in 






(clone No. 255-72 
Example 1 (clone No.






strain-derived)
4-2 strain-derived)





Condition 7 
Avian-human chimeric 
Avian-mouse chimeric 
Detectable
3.288
Not performed



antibody obtained in 
antibody obtained in 






Example 4 (clone No.
Example 1 (clone No.






240-40 strain-derived)
4-2 strain-derived)





Condition 8 
Avian-human chimeric 
Avian-mouse chimeric 
Detectable
1.889
Not performed



antibody obtained in 
antibody obtained in 






Example 4 (clone No.
Example 1 (clone No.






255-72 strain-derived)
4-2 strain-derived)





Condition 9
Humanized antibody 
Goat anti-Sema 3A 
Undetectable
—
Not performed



obtained in Example 2 
polyclonal IgG






(Humanized-2)
(marketed product)









Example 9: Collapse Assay-1
1) Test Method
Dorsal root ganglion (DRG) of Day 7 chicken embryo was isolated from a chicken. The isolated DRG was subjected to stationary culture in a 24 well dish coated with PLL and laminin containing 250 μL of NGF-containing medium at 37° C. for 16 to 20 hours, from which was observed elongation of NGF-sensitive DRG neurons. Aside from this, a mixed solution of chicken-derived Sema 3A protein (3 nM) and the obtained anti-Sema 3A avian antibody in Example 1 (culture supernatant) (at an antibody concentration of 10 μg/mL) or anti-Sema 3A avian-mouse chimeric antibody (10 μg/mL) was pre-incubated on ice for 30 minutes. The mixed solution was added to the 24 well dish so as to provide 0, 0.1 or 0.3 nM of the final concentration of the chicken-derived Sema 3A protein and the mixed solution was subjected to stationary culture at 37° C. for 30 minutes. Subsequently, the neurons were immobilized with a 3.7% formalin solution, and the growth cone, which was formed at the end of axons of the culture neurons, was visualized with Alexa488-conjugated Phalloidin. The rate (%) of neurons with collapsed growth cones to total NGF-sensitive DRG neurons was fluorescence-microscopically determined. Anti-rabbit IgG antibody or mouse IgG was also used as a control instead of anti-Sema 3A avian antibody or anti-Sema 3A avian-mouse chimeric antibody and collapse assay was carried out in the same manner as described above.
2) Results
FIG. 8 shows results of collapse assay with anti-Sema 3A avian antibody and FIG. 9 shows results of collapse assay with anti-Sema 3A avian-mouse chimeric antibody. As apparently shown in FIGS. 8 and 9, anti-Sema 3A avian antibody and anti-Sema 3A avian-mouse chimeric antibody are observed to have the action of suppressing growth cone collapse by Sema 3A protein. That is, there is revealed that anti-Sema 3A antibody containing CDRs 1 to 3 having specific amino acid sequences can effectively suppress the ability of collapse induction of growth cone by Sema 3A protein.
Example 10: Collapse Assay-2
1) Test Method
Collapse assay was carried out in the same process as Example 9 except that human-derived Sema 3A protein was used instead of chicken-derived Sema 3A protein.
2) Results
FIG. 10 shows results of collapse assay with anti-Sema 3A avian antibody. As shown in FIG. 10, anti-Sema 3A avian antibody could effectively suppress a function of human-derived Sema 3A protein thereby to suppress growth cone collapse. That is, there is revealed that anti-Sema 3A antibody containing CDRs 1 to 3 having specific amino acid sequences can also effectively suppress the ability of collapse induction of growth cone by Sema 3A protein in human.
Example 11: Collapse Assay-3
1) Test Method
Dorsal root ganglion (DRG) of Day 7 chicken embryo was isolated from a chicken. The isolated DRG was subjected to stationary culture in a 24 well dish coated with PLL and laminin containing 250 μL of NGF-containing medium at 37° C. for 16 to 20 hours, from which was observed elongation of NGF-sensitive DRG neurons. Aside from this, a mixed solution of human-derived Sema 3A protein (3 nM) and the obtained anti-Sema 3A humanized antibody in Example 2 (Humanized-2) (culture supernatant) (at an antibody concentration of 100 μg/mL) was pre-incubated on ice for 30 minutes. The mixed solution was added to the 24 well dish so as to provide 0, 0.05, 0.1 or 0.3 nM of the final concentration of the human-derived Sema 3A protein and the mixed solution was subjected to stationary culture at 37° C. for 30 minutes. Subsequently, the neurons were immobilized with a 3.7% formalin solution, and the growth cone, which was formed at the end of axons of the culture neurons, was visualized with Alexa488-conjugated Phalloidin. The rate (%) of neurons with collapsed growth cones to total NGF-sensitive DRG neurons was fluorescence-microscopically determined. Human IgG-lambda was used as a control instead of anti-Sema 3A humanized antibody and collapse assay was carried out in the same manner as described above.
2) Results
FIG. 11 shows results of collapse assay with anti-Sema 3A humanized antibody. As apparently shown in FIG. 11, anti-Sema 3A humanized antibody is observed to have the action of suppressing growth cone collapse by Sema 3A protein. That is, there is revealed that anti-Sema 3A antibody containing CDRs 1 to 3 having specific amino acid sequences can effectively suppress the ability of collapse induction of growth cone by Sema 3A protein also in the case of being humanized.
Example 12: Binding Assay to Human Sema 3A
1) Test Method
Prepared was wild type COS-7 cells or COS-7 cells transformed so as to express EGFP (Enhanced Green Fluorescent Protein)-fused NRP1 (Neuropilin-1). Aside from this, DMEM medium containing alkaline phosphatase-conjugated human-derived Sema 3A protein (0.1 nM) and the obtained avian anti-Sema 3A antibody in Example 1 (culture supernatant) (at an antibody concentration of 10 μg/mL) or anti-Sema 3A avian-mouse chimeric antibody (10 μg/mL) was pre-incubated on ice for 30 minutes, 500 μL of which was added to wild-type COS-7 cells or NRP1-expressing COS-7 cells which was previously blocked with HBH buffer (Hanks' balanced salt solution containing 20 mM sodium HEPES, pH 7.00, 0.05 vol % BSA and 10 vol % FBS) and the mixture was left to stand on ice for one hour. Subsequently, the cells were washed four times with HBH buffer and then immobilized with a 4% formaldehyde. The immobilized cells was washed once with HH buffer (Hanks' balanced salt solution containing 20 mM sodium HEPES, pH 7.00), to which then was added 500 μL of alkaline phosphatase substrate (NBT/BCIP: nitro-blue tetrazolium chloride/5-bromo-4-chloro-3′-indolyphosphate p-toluidine salt) and the mixture was left to stand at room temperature overnight. Subsequently, cells were observed for a degree of chromogenic reaction using microscopy. Control assay was carried out in the same process as described above except that alkaline phosphatase binding human-derived Sema 3A protein was not added, or rabbit IgG was used instead of anti-Sema 3A avian antibody or anti-Sema 3A avian-mouse chimeric antibody. Note that NRP1-expressing COS-7 cells used in the test is confirmed to have no variation of NRP1 expression level based on the fluorescence intensity of EGFP from the cells.
2) Results
FIG. 12 shows results of binding assay with anti-Sema 3A avian antibody and FIG. 13 shows results of binding assay with anti-Sema 3A avian-mouse chimeric antibody. As apparently shown in FIGS. 12 and 13, in the case of addition of the mixture of avian anti-Sema 3A antibody or anti-Sema 3A avian-mouse chimeric antibody and human Sema 3A protein, binding of human-derived Sema 3A protein to NRP 1-expressing COS-7 cells was inhibited. That is, results reveal that anti-Sema 3A antibody containing CDRs 1 to 3 having specific amino acid sequences binds to human-derived Sema 3A protein and can inhibit binding of human-derived Sema 3A protein and NRP 1.
Example 13: Binding Assay to Human Sema 3F
1) Test Method
Binding assay was carried out in the same process as Example 12 except for use of COS-7 cells transformed so as to express EGFP-fused NRP 2 (Neuropilin-2) (NRP 2-expressing COS-7 cells) instead of NRP 1-expressing COS-7 cells and of alkaline phosphatase-conjugated human-derived Sema 3F protein instead of alkaline phosphatase-conjugated human-derived Sema 3A protein.
2) Results
FIG. 14 shows results of binding assay with anti-Sema 3A avian antibody. As apparently shown in FIG. 14, even in the case of addition of a mixture of anti-Sema 3A avian antibody and human-derived Sema 3F protein, binding of human-derived Sema 3F protein to NRP 2-expressing COS-7 cells was not inhibited. That is, results reveal that anti-Sema 3A antibody containing CDRs 1 to 3 having specific amino acid sequences dose not bind to Sema 3F protein which is a subfamily molecule having high homology with Sema 3A protein, suggesting possibility to specifically bind to Sema 3A protein.
Example 14: Analysis of Influence of Anti-Sema 3A Antibody on Cognitive Function
1) Test Materials
1-1) Aβ (25-35) and Aβ (35-25)
Aβ (25-35) (Amyloid β-protein (25-35)) (Bachem, #H-1192) and Aβ (35-25) (Amyloid β-protein (35-25)) (Bachem, #H-2964) were prepared with distilled water to 1 mg/mL and then incubated at 37° C. for 4 days to prepare an Aβ (25-35) solution and an Aβ (35-25) solution. Through this process, Aβ (25-35) aggregates and achieves cell cytotoxicity. On the other hand, Aβ (35-25) is an inactivated Aβ which has the reversed amino acid sequence of activated Aβ 25-35 and does not exhibit cell cytotoxicity even after the process. Aβ (35-25) was used as a negative control.
1-2) Anti-Sema 3A Antibody
The produced anti-Sema 3A avian-mouse chimeric antibody in Example 1 was prepared with physiological saline to 1 mg/mL to prepare an anti-Sema 3A antibody solution.
1-4) Normal IgG
For a normal IgG solution, 1 mg/mL of normal mouse IgG (obtained from Calbiochem) was used.
1-5) Mouse
Mice (C57BL/6J) aged 3 weeks old purchased from CHARLES RIVER LABORATORIES JAPAN, INC. were used.
2) Test Method
Novel Object Recognition Task (NORT) was carried out according to the following method.
Firstly, mice aged 3 weeks old were prepared and acclimated to an experimenter by handling such that mice were put on experimenter's hand for 10 minutes a day for 2 weeks. Secondly, mice were divided into 5 groups shown in Table 8 and intracerebroventricularly administered with Aβ and an antibody under conditions shown in Table 8. These solutions were administered at 1 mm right inferior of bregma. Administration was carried out using a microsyringe equipped with a teflon tube (Eicom Corporation) connected at the needle tip of the syringe and a 27 G injection needle (TERUMO CORPORATION) bent into L shape at 3.3 mm from the tip of the needle and inserted into the tip of the tube.








TABLE 8





Group
Administration conditions







Intact group (N = 24)
Non-treated (no Aβ and antibody-treated)


Aβ (35-25)-treated group
ICV administration of Aβ (35-25) solution


(N = 34)
of 3 μL


Aβ (25-35)-treated group
ICV administration of Aβ (25-35) solution


(N = 43)
of 3 μL


Aβ (25-35) + normal
ICV administration of Aβ (25-35) solution


IgG-treated group (N = 22)
of 3 μL and normal IgG solution of 3 μL



at the same time


Aβ (25-35) + anti-Sema
ICV administration of Aβ (25-35) solution


3A antibody-treated
of 3 μL and anti-Sema 3A antibody solution


group (N = 22)
of 3 μL at the same time









3 days after intracerebroventricular administration, mice were transferred into a test cage with 35 cm×35 cm×35 cm and acclimated in the test cage for 10 minutes. The next day (Day 4 after administration), Objects A and B were placed on given positions in the test cage and an amount of time to access to each of the Objects for 10 minutes was measured (acquisition trial). At this time, an amount of time to access within 1 cm or less of distance between the Object and nose of the mouse was measured as an access time. In the next day (Day 5 after administration), an amount of access time to each of the Object A and a new Object C instead of the Object B for 10 minutes was measured (test trial).
Note that for the Objects A, B and C, used were those with different shapes and colors. The Objects A, B and C particularly have the following shapes and color.
Object A: A shape composed of a base portion (green) stretched horizontally and an extension portion (green) stretched vertically from the center of the base portion and provided with T shaped form in the front view. The base and extension portions each are in cuboid form.
Object B: the same shape as the Object A except that a base portion color is yellow and an extension portion is in columnar form and its color is red.
Object C: the same shape as the Object A except that a base portion color is yellow and an extension portion is cut out to circular arc form in the lateral front view and is yellow.
Objects A, B and C were placed such that their base portions were in contact with the bottom of the test cage and their extension portions were stretched upwardly.
3) Statistical Analysis
In the acquisition trial, the rate of access time to the Object B to total access time to the Objects A and B (access rate to Object B) was calculated. When the acquisition trial was carried out, the Objects A and B were unknown objects and therefore, an amount of access time to each of the both was expected to be comparable levels. When an amount of access time to each of the both was disproportionally high on either one, cognition function, etc. was possibly impaired due to a drug administration and therefore, an estimation was considered to be impossible in the experiment. In the test trial, the rate of access time to the Object C to total access time to the Objects A and C (access rate to Object C) was calculated. When the test trial was carried out, the object C was only an unknown object and therefore, disproportional amount of access time to the Object C was expected when memory of the Object A was acquired. As described above, the calculated access rates to the Object B and to the Object C each was subjected to statistical analysis using one-way analysis of variance. Comparison between groups was performed by Tukey-Kramer HSD test.
4) Results
The obtained results are shown in FIGS. 15A (acquisition trial) and 15B (test trial).
As apparently shown in FIG. 15A, the access rates to Object B in all groups was substantially about 50% in the acquisition trial and no significant differences were observed between all groups. Thus, impairment of cognitive function was not observed, suggesting that estimation of memory acquisition in the experiment was possible. On the other hand, as shown in FIG. 15B, Intact group and Aβ (35-25)-treated group (negative control) each had about 75% of access rate to the Object C in test trial. However, Aβ (25-35)-treated group had only about 50% of access rate to the Object C, resulting in significant decrease of preference to the Object C compared to Intact group (p<0.0001, vs. Intact group). Results reveal that memory of the Object A was not acquired due to impairment of memory caused by Aβ (25-35) administration.
Furthermore, as apparently shown in FIG. 15B, Aβ (25-35)+anti-Sema 3A antibody-treated group had about 75% of access rate to the object C, resulting in significant recovery compared to Aβ (25-35)-treated group and Aβ (25-35)+normal IgG-treated group (p<0.0001, vs. Aβ (25-35)-treated group or Aβ (25-35)+normal IgG-treated group).
These results demonstrate that anti-Sema 3A antibody containing CDRs having specific amino acid sequences exhibits effects to inhibit memory impairment induction induced with Aβ. Increased expression level of Sema 3A protein has been already observed in the postmortem brain in a patient with Alzheimer's disease, and Collapsin Response Mediator Protein (CRMP), which is phosphate-modified in Sema 3A signaling pathway, is known to be highly phosphate-modified CRMP in neurofibrillary tangle, a lesion of Alzheimer's disease. Accordingly, such drug efficacy is considered to be due to blocking of Sema 3A-CRMP signal via anti-Sema 3A antibody.
Example 15: Analysis of Effects of Anti-Sema 3A Avian-Mouse Chimeric Antibody on Immune/Inflammatory Diseases
Effects of the obtained avian-mouse chimeric antibody in Example 2 on lethal inflammatory condition was analyzed in lipopolysaccharide-inoculated model, a simulated septic condition model.
1) Test Materials
1-1) Lipopolysaccharide
Lipopolysaccharide (hereinafter referred to as LPS, Sigma-Aldrich Japan, Lot No: 032M4082V) was prepared with physiological saline to 6 mg/mL.
1-2) Avian-Mouse Anti-Sema 3A Antibody
The produced anti-Sema 3A avian-mouse chimeric antibody in Example 2 was diluted with physiological saline to 5 mg/mL, which was used as anti-Sema 3A antibody solution.
1-3) Chimeric Antibody for Negative Control
Non-specific avian-mouse chimeric antibody (IgG) (chimeric antibody for negative control), which was produced using an antibody library constructed by chicken B cell-derived DT40 cells, was diluted with physiological saline to 5 mg/mL, which was used as a chimeric antibody solution for negative control.
1-4) Mouse
Male mice aged 6 weeks old (C57BL/6J) purchased from CHARLES RIVER LABORATORIES JAPAN, INC. were used.
2) Test Method
After purchased, C57BL/6J mice were conditioned in a mice rearing room and divided into 5 mice a group and among them, a test group was set as shown in Table 9. Antibodies were administered via tail vein and after 30 minutes, LPS was intraperitoneally inoculated. The day in which these treatments were carried out was set as Day 0 and each mouse was observed to be alive or dead until Day 4, which was used as an indicator of efficacy determination. The same test was carried out total three times to confirm reproducibility.
After three tests were confirmed to be non-dissociative, results obtained from these tests were combined, based on which, determined were improvement effects of survival rate and its efficacy at Day 4. Kaplan-Meiyer method was used to prepare the survival curve, based on which, effects of survival period-extension were determined.








TABLE 9





Group
Administration conditions







Negative control
Chimeric antibody solution for negative control was


group (N = 5)
administered in an amount of 0.1 mL (500 μg of dose



of chimeric antibody for negative control) via tail vein



and after 30 minutes, LPS was intraperitoneally



inoculated to 60 mg/kg of dose.


Anti-Sema 3A
Anti-Sema 3A antibody solution was administered in


avian-
an amount of 0.1 mL (500 μg of dose of anti-Sema 3A


mouse chimeric
avian-mouse chimeric antibody) via tail vein and after


antibody-treated
30 minutes, LPS was intraperitoneally inoculated to


group (N = 5)
60 mg/kg of dose.









3) Statistical Analysis

In the case of being dead earlier than Day 4 after administration, the survival period-extension was not confirmed (no effective) and in the case of being alive until Day 4, the survival period-extension was confirmed (effective). From these binarized values, consistency among the three tests was determined by Breslow-Day test and then, the difference of efficacy rate obtained by combining the three tests was determined by Cochran-Mantel-Haenzel test. Log-rank test was performed to determine effects of the survival period-extension.
4) Results
The obtained results are shown in Table 10. As apparently shown in Table 10, in all three tests, the survival rate of anti-Sema 3A avian-mouse chimeric antibody-treated group was superior than that of non-specific sequence chimeric antibody-treated group, a negative control. Bresslow-Day test, which was performed for confirmation of non-dissociative among these tests, does not indicate significance and therefore, the three tests are confirmed to be non-dissociative (p=0.6202). On that basis, results of the three tests were combined, based on which, Cochran-Mantel-Haenzel test was performed and in addition to P=0.0364, statistical significance difference is observed at 5% level of significance on the survival rate between the negative control group and the anti-Sema 3A avian-mouse chimeric antibody group. These results reveal that anti-Sema 3A avian-mouse chimeric antibody exhibits effects to improve the survival rate of model animals with lethal inflammatory condition and the effects indicate reproducibility.













TABLE 10








Number of


Survival



Group
mouse
Dead
Alive
rate





















Test I
Negative control
N = 5
3
2
40%



Anti-Sema 3A chimeric
N = 5
2
3
60%



antibody


Test II
Negative control
N = 5
4
1
20%



Anti-Sema 3A chimeric
N = 5
1
4
80%



antibody


Test III
Negative control
N = 5
4
1
20%



Anti-Sema 3A chimeric
N = 5
2
3
60%



antibody


Combined
Negative control
N = 15
11
4
26.7% 


three tests
Anti-Sema 3A chimeric
N = 15
5
10
66.7% 



antibody









The survival curve of the combined three tests was prepared by Kaplan-Meier method and log-rank test was performed to determine the significance. Results are shown in FIG. 16. As shown in FIG. 16, analysis of the survival period-extension also indicates significance, i.e. p=0.0115, and reveals that anti-Sema 3A avian-mouse chimeric antibody exhibits effects of survival period-extension.
Example 16: Analysis of Effect of Anti-Sema 3A Humanized Antibody on Immune/Inflammatory Diseases
Effects of the obtained anti-Sema 3A humanized antibody in Example 2 on LPS-induced inflammatory disease model was also analyzed in the same manner as Example 12.
1) Test Materials
1-1) LPS
Preparation of LPS was carried out in the same manner as Example 15.
1-2) Anti-Sema 3A Humanized Antibody
The produced anti-Sema 3A humanized antibody (Humanized-2) in Example 2 was diluted with physiological saline to obtain a solution of anti-Sema 3A humanized antibody. In anti-Sema 3A humanized antibody 500 μg-treated group, anti-Sema 3A humanized antibody (Humanized-2) was prepared to 5 mg/mL and in anti-Sema 3A humanized antibody 100 μg-treated group, anti-Sema 3A humanized antibody (Humanized-2) was prepared to 1 mg/mL, for use herein.
1-3) Human Antibody for Negative Control
Non-specific human polyclonal antibody (human IgG POLYCLONAL Isotype Control; BioXCell, #BE0092) was diluted with physiological saline to 5 mg/mL, which was used as a solution of human antibody for negative control.
1-4) Mouse
Male mice aged 6 weeks old (C57BL/6J) purchased from CHARLES RIVER LABORATORIES JAPAN, INC. were used.
2) Test Method
After purchased, C57BL/6J mice were conditioned in a mouse rearing room and divided into 10 mice a group and among them, test groups were set as shown in Table 11. Antibodies were administered via tail vein and after 30 minutes, LPS was intraperitoneally inoculated. The day in which these treatments were carried out was set as Day 0 and each mouse was observed to be alive or dead until Day 4, which was used as an indicator of efficacy determination.








TABLE 11





Group
Administration conditions







Negative control
A solution of human antibody for negative control was


group (N = 10)
administered in an amount of 0.1 mL (500 μg of dose



of human antibody for negative control) via tail vein



and after 30 minutes, LPS was intraperitoneally



inoculated to 60 mg/kg of dose.


Anti-Sema 3A
A solution of anti-Sema 3A humanized antibody was


humanized
administered in an amount of 0.1 mL (100 μg of dose


antibody
of anti-Sema 3A humanized antibody) via tail vein


100 μg-treated
and after 30 minutes, LPS was intraperitoneally


group (N = 10)
inoculated to 60 mg/kg of dose.


Anti-Sema 3A
A solution of anti-Sema 3A humanized antibody was


humanized
administered in an amount of 0.1 mL (500 μg of dose


antibody
of anti-Sema 3A humanized antibody) via tail vein


500 μg-treated
and after 30 minutes, LPS was intraperitoneally


group (N = 10)
inoculated to 60 mg/kg of dose.









3) Statistical Analysis

The binarized survival rates of no effective and effective values in the same manner as Example 12 were subjected to one-sided Cochran-Armitage test to determine dose reactivity relative to improvement effects of the survival rate, wherein the following three dose reactivity types were supposed: linear increasing type, low dose-saturation type and high dose-rising type, and contrasts of these types were predetermined. Log-rank test was performed to determine effects of the survival period-extension.
4) Results
Survival rates of each group are shown in Table 12. In anti-Sema 3A humanized antibody 100 μg-treated group, the survival rate was twice (60%) as the negative control group (30%) and in anti-Sema 3A humanized antibody 500 μg-treated group, the survival rate was observed to be higher (80%) than that of the 100 μg-treated group. In each of both anti-Sema 3A humanized antibody-treated groups, survival rates were improved and dose-dependent trend was found.











TABLE 12






Number


Survival


Group
of mouse
Dead
Alive
rate







Negative control group
N = 10
7
3
30%


Anti-Sema 3A humanized antibody
N = 10
4
6
60%


100 μg-treated group


Anti-Sema 3A humanized antibody
N = 10
2
8
80%


500 μg-treated group









These results apparently show that anti-Sema 3A humanized antibody also suppresses LPS-induced lethal inflammatory condition and exhibits effects to improve the survival rate similarly to anti-Sema 3A avian-mouse chimeric antibody shown in Example 15.
Example 17: Analysis of Effects of Anti-Sema 3A Humanized Antibody on Lethal Inflammatory Diseases after the Onset
From the obtained results in Example 16, effects of anti-Sema 3A humanized antibody was determined in the case where LPS was inoculated before the onset and its treatment was made after the onset.
1) Test Material
1-1) LPS
Preparation of LPS was carried out in the same manner as Example 15.
1-2) Anti-Sema 3A Humanized Antibody
The produced anti-Sema 3A humanized antibody (Humanized-2) in Example 2 was diluted with physiological saline to obtain an anti-Sema 3A humanized antibody solution. In each of anti-Sema 3A humanized antibody 500 μg, 250 μg and 125 μg-treated groups, anti-Sema 3A humanized antibody (Humanized-2) was prepared to 5, 2.5 and 1.25 mg/mL, respectively, for use herein.
1-3) Human Antibody for Negative Control
A solution of human antibody for negative control was prepared in the same manner as Example 16.
1-4) Mouse
Male mice aged 6 weeks old (C57BL/6J) purchased from CHARLES RIVER LABORATORIES JAPAN, INC. were used.
2) Test Method
After purchased, C57BL/6J mice were conditioned in a mouse rearing room and divided into 10 mice a group and among them, test groups were set as shown in Table 13. In the test, differently from Examples 15 and 16, actual medical care-based use was supposed and therefore, efficacy determination was made in administrating after inflammatory condition had already induced. Firstly, LPS was intraperitoneally inoculated to induce inflammatory reaction. Antibodies were administered via tail vein one hour after intraperitoneal inoculation of LPS. The day in which these treatments were made was set as Day 0 and each mouse was observed to be alive or dead until Day 4, which was used as an indicator of efficacy determination.








TABLE 13





Group
Administration conditions







Negative
LPS was intraperitoneally inoculated to 60 mg/kg of


control
dose and after one hour, a solution of human antibody


group (N = 10)
for negative control was administered in an amount of



0.1 mL (500 μg of dose of a solution of human antibody



for negative control) via tail vein.


Anti-Sema 3A
LPS was intraperitoneally inoculated to 60 mg/kg of


humanized
dose and after one hour, an anti-Sema 3A humanized


antibody
antibody solution was administered in an amount of


125 μg-treated
0.1 mL (125 μg of dose of anti-Sema 3A humanized


group (N = 10)
antibody) via tail vein.


Anti-Sema 3A
LPS was intraperitoneally inoculated to 60 mg/kg of


humanized
dose and after one hour, an anti-Sema 3A humanized


antibody
antibody solution was administered in an amount of


250 μg-treated
0.1 mL (250 μg of dose of anti-Sema 3A humanized


group (N = 10)
antibody) via tail vein.


Anti-Sema 3A
LPS was intraperitoneally inoculated to 60 mg/kg of


humanized
dose and after one hour, an anti-Sema 3A humanized


antibody
antibody solution was administered in an amount of


500 μg-treated
0.1 mL (500 μg of dose of anti-Sema 3A humanized


group (N = 10)
antibody) via tail vein.









3) Statistical Analysis

In dose reactivity types which were indicated by 4 groups containing negative control group, the following 7 types were supposed: (1) observed dose-linear regression, (2) linear increasing, (3) middle dose-rising, (4) high dose-rising, (5) middle dose-saturation, (6) low dose-saturation and (7) middle dose-rising and saturation types. Cochran-Armitage test was applied to all of the 7 reaction patterns and p-values, which were multiple-adjusted by sorting and re-extraction method, were calculated and among the calculated p-values, a contrast reactivity type with the smallest p-value was adopted as an optimal dose reactivity type. Log-rank test was performed to determine effects of the survival period-extension.
4) Results
Survival rates of each group of Day 4 after administration are shown in Table 14. In the negative control group, the survival rate was 10%, however, in respective groups treated with 125, 250 and 500 μg of anti-Sema 3A humanized antibody one hour after LPS inoculation, survival rates were 30, 40 and 100%, respectively. Accordingly, trend was observed in which a survival rate increases as a dose increases. In particular, in anti-Sema 3A humanized antibody 500 μg-treated group, surprising effects were observed in which all 10 mice were alive.











TABLE 14






Number of


Survival


Group
mouse
Dead
Alive
rate



















Negative control group
N = 10
9
1
10%


Anti-Sema 3A humanized antibody
N = 10
7
3
30%


125 μg-treated group


Anti-Sema 3A humanized antibody
N = 10
6
4
40%


250 μg-treated group


Anti-Sema 3A humanized antibody
N = 10
0
10
100% 


500 μg-treated group









The 7 dose reactivity types, which were supposed to be indicated by the binarized survival rates, were predetermined and subjected to Cochran-Armitage test multiple-adjusted by sorting and re-extraction method. Results of the calculated p-values are shown in Table 15. Note that unnecessarily-multitudes of digit numbers are provided in order to express the rank order of p-values.










TABLE 15








Multiple-adjusted



Dose-reaction patterns
p-values









Observed dose-linear regression type
0.00001790



Linear increasing type
0.00004540



Middle dose-rising type
0.00005730



High dose-rising type
0.00008040



Middle dose-saturation type
0.00133730



Middle dose-rising and saturation type
0.00252090



Low dose-saturation type
0.01135610










As analytical results, results were obtained that all of the supposed dose-reaction types indicated significance. Dose-reaction type with the smallest p-value was the observed dose-linear increasing type (p=0.00001790), one with the second smaller p-value was the linear increasing type (p=0.0000454), followed by middle dose-rising type (p=0.00005730) and high dose-rising type (p=0.00008040) in this order. These results reveal that obvious dose-dependency was scientifically indicated for effects of survival rate improvement in the test as well as anti-Sema 3A humanized antibody had efficacy to control lethal inflammatory condition and to be able to improve the survival rate.
FIG. 17 shows the survival curve of anti-Sema 3A humanized antibody 500 μg-treated group, all cases of which were alive and which exhibited obvious effects of survival rate improvement. In log-rank test, results were obtained that significance level with p<0.0001 was satisfied. There is obviously found that anti-Sema 3A humanized antibody controls lethal inflammatory condition even after the onset of the condition and exhibits effects on the extension of the survival period.
Example 18: Effects of Anti-Sema 3A Humanized Antibody on Disseminated Intravascular Coagulation Model Mouse
Disseminated intravascular coagulation (hereinafter, referred to as DIC) associated with sepsis, solid tumor, etc. causes increase of blood level of plasminogen activator inhibitor-1 (hereinafter, referred to as PAI-1) thereby to suppress the action of fibrinolytic system, leading to development of multiple organ failure with serious conditions. There is believed that suppression of increase of blood level of PAI-1 can lead to inhibition of the progression of DIC and therefore, effects of anti-Sema 3A humanized antibody on blood level of PAI-1 were examined.
1) Test Material
1-1) LPS
LPS (Sigma-Aldrich Japan, Lot No: 102M4017V) was prepared with physiological saline to a concentration of 1.5 mg/mL.
1-2) Anti-Sema 3A Humanized Antibody
The produced anti-Sema 3A humanized antibody (Humanized-2) in Example 2 was prepared with physiological saline to a concentration of 5 mg/mL.
1-3) Mouse
Male mice aged 6 weeks old (C57BL/6J) purchased from CHARLES RIVER LABORATORIES JAPAN, INC. were used.
2) Test Method
2-1) Grouping
After purchased, C57BL/6J mice were conditioned in a mouse rearing room and divided into 5 mice a group and among them, test groups were set as shown in Table 16.
2-2) LPS Inoculation and Antibody Administration
LPS was intraperitoneally inoculated so as to be inoculated in an amount of 15 mg/kg. 0.1 mL of 5 mg/mL solution of the antibody solution was administered via tail vein so as to be inoculated in an amount of 500 μg/mouse. Note that Condition-1 group in which LPS was not inoculated was intraperitoneally inoculated with physiological saline.
2-3) Measurement of Mouse PAI-1
Plasma was collected at 1.5, 3 and 9 hours after LPS inoculation to measure blood levels of PAI-1 in the treated animal using mouse PAI-1 ELISA kit (Innovative Research, Inc).











TABLE 16







LPS
Antibody-treated
Blood collecting



(mg/kg)
(500 μg)
time



















Condition 1
—
—
Test starting time


Condition 2
15
—
1.5 hr after LPS





inoculation


Condition 3
15
30 min before LPS
1.5 hr after LPS




inoculation
inoculation


Condition 4
15
1 hr after LPS
1.5 hr after LPS




inoculation
inoculation


Condition 5
15
—
3 hr after LPS





inoculation


Condition 6
15
30 min before LPS
3 hr after LPS




inoculation
inoculation


Condition 7
15
1 hr after LPS
3 hr after LPS




inoculation
inoculation


Condition 8
15
—
9 hr after LPS





inoculation


Condition 9
15
30 min before LPS
9 hr after LPS




inoculation
inoculation


Condition 10
15
1 hr after LPS
9 hr after LPS




inoculation
inoculation









3) Statistical Analysis

For antibody-untreated and antibody-treated groups, two-tailed Student's t test was performed to calculate p-value and the p-value was evaluated to indicate statistical significance in the case of the significance level of 5% or lower.
4) Results
FIG. 18 shows the measured results of blood levels of PAI-1. In LPS-untreated group (Condition 1), the blood level of PAI-1 was very low, but the blood level of PAI-1 after LPS inoculation indicated to gradually increase after 1.5 hours and significantly increased 3 and 9 hours after LPS inoculation compared to the LPS-untreated animal (Conditions 2, 5 and 8).
In antibody-treated after 3 hours of LPS inoculation group, both of antibody-treated before LPS inoculation group (Condition 6, p=0.0027) and antibody-treated after LPS inoculation group (Condition 7, p=0.0051) suppressed increase of blood levels of PAI-1 compared to the antibody-untreated group. This effect of suppressing increase of PAI-1 by anti-Sema 3A humanized antibody persisted even 9 hours after LPS inoculation and resulted in suppressing increase of blood levels of PAI-1 in both of antibody-treated before LPS inoculation group (Condition 9, p<0.0001) and antibody-treated after LPS inoculation group (Condition 10, p=0.0029) similarly to that of antibody-treated 3 hours after LPS inoculation group.
These results reveal that the produced anti-Sema 3A humanized antibody (humanized-2) in Example 2 has the action of suppressing the increase of blood level of PAI-1, which is one of aggravating factors of DIC.
Example 19: Effects of Anti-Sema 3A Humanized Antibody on Migration Ability of Cancer Cells
Disseminated and distal metastases caused after surgical treatment or remission induction by chemical therapy are known to greatly decrease the survival rate of a cancer patient. Activation of migration ability of cancer cells allows to release cancer cells from primary tumor, resulting in disseminated and distal metastases and therefore, effects of anti-Sema 3A humanized antibody on malignant alteration of cancer cells induced by Sema 3A was examined.
1) Test Method
The produced anti-Sema 3A humanized antibody (Humanized-2) in Example 2 was used to evaluate effects on migration ability of cancer cells, which is induced by Sema 3A. Herein, cells of pancreatic cancer in which patients with high Sema 3A expression level have poor outcome were used to examine the effects. Particular experimental process was carried out as follows.
Fibronectin was diluted with PBS buffer to 0.1 mg/mL to prepare a fibronectin diluent and 10 μL of the fibronectin diluent was applied on a lower surface of a filter in a 24 well-type chamber (chemotaxicell chamber manufactured by KURABO INDUSTRIES LTD. 8 μm pore) and the filter was left to stand and dried at room temperature for one hour, a migration chamber provided with which was prepared.
Subsequently, the prepared migration chamber was set in a 24 well plate, to the outer layer of which was added 600 μL of DMEM medium containing 0.1% fetal bovine serum. To the inner layer of the chamber were added 2×105 cells/mL of human pancreatic cancer cell line (MIAPaCa-2) and 200 μL of serum-free DMEM medium containing additive components shown in Table 17, which was cultivated in the presence of 5% CO2 at 37° C. for 4 hours. Subsequently, the chamber was removed and cancer cells in the inner layer of the chamber were drawn and removed. Furthermore, the residual cells in the chamber were removed using a cotton swab wetted with PBS buffer. Then, the chamber was immersed with a cell staining solution (Diff-Quick, SYSMEX INTERNATIONAL REAGENTS CO., LTD) for 10 minutes or more, followed by washing twice with ultrapure water and drying. After drying, cell counts migrated to the lower surface of the chamber filter were measured by a microscope.









TABLE 17









Final concentration of additive



components in inner layer of chamber











Human
Anti-Sema 3A humanized
Negative



Sema
antibody (Humanized-2)
control human



3A/Fc
obtained in Example
antibody



(ng/mL)
2 (μg/mL)
(μg/mL)














Condition 1
—
—
—


Condition 2
100
—
—


Condition 3
100
0.1
—


Condition 4
100
1
—


Condition 5
100
10
—


Condition 6
100
—
0.1


Condition 7
100
—
1


Condition 8
100
—
10









2) Statistical Analysis

With consideration for multiplicity, two-sided Dunnett's test was applied to Conditions 3 to 5 and Conditions 6 to 8 relative to Condition 2 to calculate p-values and when the p-values were significance level of 5% or lower, they were evaluated to indicate statistical significance.
3) Test Results
The obtained results are shown in FIG. 19. A migration activity of pancreatic cancer cell MIAPaCa-2 stimulated by human Sema 3A was enhanced two times or more (Condition 2). However, a migration activity of pancreatic cancer cells induced by Sema 3A was suppressed in the presence of anti-Sema 3A humanized antibody (Conditions 3 to 5). In anti-Sema 3A antibody 1 μg/ml-treated group (P=0.0031) and 10 μg/ml-treated group (P<0.001), migration of cancer cells were suppressed to the same level as Sema 3A-untreated group (Condition 1). On the other hand, in negative control group, cancer cell migration induced by Sema 3A was not suppressed (Conditions 6 to 8). That is, results reveal that anti-Sema 3A antibody containing CDR having a specific amino acid sequence specifically suppresses pancreatic cancer cell migration induced by Sema 3A.
Example 20: Effects of Anti-Sema 3A Humanized Antibody on Invasion Ability of Cancer Cells
Cancer cells having activated migration ability lyse and invade the surrounding extracellular substrates and then destroys basal membrane to transfer to blood and lymph vessels, eventually transferring to distant organs. Suppression of invasion and metastasis of cancer cells leads to suppression of metastatic recurrence and therefore is considered to be useful for survival rate improvement of cancer patients. In addition to suppressing effects on enhancement of Sema 3A-induced migration ability examined in Example 19, effects of anti-Sema 3A humanized antibody on invasion ability of cancer cells was examined.
1) Test Method
Extracellular substrates removed growth factors (Becton, Dickinson and Company, Matrigel (trademark) Growth Factor Redused) was loaded in the inner layer of invasion chamber (Becton, Dickinson and Company, BioCoat (trademark), 8 μm pore, #354483), which was used to evaluate effects of anti-Sema 3A humanized antibody on invasion ability of cancer cells induced by Sema 3A. Particular experimental process was carried out as follows.
For cancer cells, human pancreatic cancer cell line (MIAPaCa-2), human glioblastoma line (U87MG) and mouse-derived lung cancer cell line (3LL) were used. Firstly, the extracellular substrates loaded in an invasion chamber were treated by swelling and then, the invasion chamber was set in a 24 well plate, to the outer layer of which was added 0.75 mL of DMEM medium containing 0.1% fetal bovine serum (1% fetal bovine serum for 3LL cells) and to the inner layer of which was added 2×105 cells/mL of cancer cells and 125 μL of serum-free DMEM medium containing additive components at concentrations shown in Table 18 and the chamber was cultivated in the presence of 5% CO2 at 37° C. for 24 hours. Subsequently, the invasion chamber was removed, followed by removing residual cells in the inner layer of the invasion chamber in the same manner as Example 19 and then, the invaded cell counts, which transferred into the lower surface of the filter, were measured by a microscope.









TABLE 18









Components added to inner layer of invasion chamber











Human
Anti-Sema 3A humanized
Negative



Sema
antibody (Humanized-2)
control human



3A
obtained in Example
antibody



(g/mL)
2 (μg/mL)
(μg/mL)














Condition 1
—
—
—


Condition 2
—
0.1
—


Condition 3
—
1.0
—


Condition 4
—
10.0 
—


Condition 5
100
—
—


Condition 6
100
0.1
—


Condition 7
100
1.0
—


Condition 8
100
10.0 
—


Condition 9
100
—
0.1


Condition 10
100
—
1.0


Condition 11
100
—
10.0









2) Statistical Analysis

For inhibitory action of Sema 3A-induced invasion activity, with consideration for multiplicity, two-sided Dunnett's test was applied to Conditions 6 to 8 and Conditions 9 to 10 relative to Condition 5 to calculate p-values and when the p-values were significance level of 5% or lower, they were evaluated to indicate statistical significance. A list of p-values is shown in Table 19.
3) Test Results
Measured results of invaded cell counts are shown in FIG. 20 for MIAPaCa-2 cells, in FIG. 21 for U87MG cells and in FIG. 22 for 3LL cells under the above-mentioned Conditions. In the case of addition of Sema 3A (Condition 5), all cancer cell lines exhibited obviously-enhanced invasion ability compared to Sema 3A-unstimulated group (Condition 1). In the case of addition of human Sema 3A together with anti-Sema 3A humanized antibody, all cancer cell lines exhibited suppressed invasion to the same extent as Sema 3A-unstimulated group (Conditions 6 to 8). Under non Sema 3A-stimulated condition, surprising invasion-suppressing effects were not observed except 3LL (Conditions 1 to 4). On the other hand, in antibody-containing negative control group, enhancement of cancer cell invasion ability induced by Sema 3A was little suppressed (Conditions 9 to 11). FIG. 23 shows microscopic images of invaded 3LL cells. There are visually observed that in Condition 5 in which Sema 3A was added, cancer cells significantly invaded compared to Condition 1 in which Sema 3A was not added, and in Conditions 7 and 8 in which anti-Sema 3A humanized antibody was treated in the presence of Sema 3A, cancer cell invasion activity induced by Sema 3A was apparently suppressed. As apparently shown in these results, there is revealed that anti-Sema 3A antibody containing a CDR having a specific amino acid sequence has the action of suppressing cancer cell invasion activity induced by Sema 3A to the same extent as Sema 3A-unstimulated group.










TABLE 19









Results of Dunnett's test












MIAPaCa-2
U87MG
3LL
















Condition 6
p = 0.4339
p < 0.001
p = 0.3278



Condition 7
p = 0.0012
p < 0.001
p = 0.0091



Condition 8
p < 0.001
p < 0.001
p = 0.0022



Condition 9
p = 0.952
p = 0.8182
p = 0.302



Condition 10
p = 0.920
p = 0.8676
p = 0.142



Condition 11
p = 0.987
p = 0.0046
p = 0.903










Example 21: Effects of Anti-Sema 3A Humanized Antibody on Anticancer Agent-Unresponsiveness Induced by Sema 3A
Pancreatic cancer is known to have very low 5 year-survival rate among cancer. The etiology includes: in pancreatic cancer tissue which is in a hypovascular state, the cancer cells can proliferate and develop even in such a nutrient starvation state and often have tolerance for an anticancer agent such as gemcitabine hydrochloride (hereinafter, referred to as GEM). That is, in pancreatic cancer treatment, overcoming unresponsiveness to an anticancer agent is an important subject and therefore, drug-tolerance induced by Sema 3A and removal action of the tolerance by anti-Sema 3A humanized antibody in the nutrient starvation state were evaluated.
1) Test Method
In a nutrient starvation state characteristic of pancreatic cancer, effects of humanized anti-Sema 3A antibody (the produced humanized-2 in Example 2) on GEM-unresponsiveness induced by Sema 3A was evaluated. Particular experimental process was carried out as follows.
A cell suspension in which 4×104 cells/mL of human pancreatic cells (MIAPaCa-2) was suspended in DMEM medium containing 10% fetal bovine serum was seeded into each well of a 96 well plate in an amount of 100 μL for each one. The plate was cultivated in the presence of 5% CO2 at 37° C. for 24 hours and then the medium was replaced with 100 μL of DMEM medium containing 0.1% fetal bovine serum, followed by cultivation for another 24 hours. Subsequently, to each well was added 100 μL of DMEM medium containing 0.1% fetal bovine serum with given amounts of components shown in Table 20, followed by cultivation for 2 days. Then, the culture supernatant was removed from each well in an amount of 100 μL and to each well was added CellTiter reagent (CellTiter AQueous One Solution Proliferation Assay; Promega) of 20 μL, followed by cultivation in the presence of 5% CO2 at 37° C. for one hour and then the absorbance was measured at 490 nm.









TABLE 20









Final concentration of components in each well














Anti-Sema 3A






humanized antibody
Negative



Human

(Humanized-2)
control



Sema
Gemcitabine
obtained in
human



3A/Fc
hydrochloride
Example 2
antibody



(ng/mL)
(nM)
(μg/mL)
(μg/mL)















Condition 1
—
—
—
—


Condition 2
—
10
—
—


Condition 3
100
10
—
—


Condition 4
100
10
0.1
—


Condition 5
100
10
1
—


Condition 6
100
10
10
—


Condition 7
100
10
—
0.1


Condition 8
100
10
—
1


Condition 9
100
10
—
10









2) Statistical Analysis

With consideration for multiplicity, two-sided Dunnett's test was applied to Conditions 4 to 6 and Conditions 7 to 9 relative to Condition 3 to calculate p-values and when the p-values were significance level of 5% or lower, they were evaluated to indicate statistical significance. The p-values are shown in Table 21.
3) Test Results
FIG. 24 shows results of assessment of pancreatic cell proliferation. GEM suppressed proliferation of pancreatic cells (Condition 2 relative to Condition 1), however, human Sema 3A reduced sensitivity to GEM and induced drug-unresponsiveness (Condition 3 relative to Condition 2). The GEM tolerance induced by Sema 3A was removed with anti-Sema 3 A humanized antibody of 1 or 10 μg/ml. and GEM sensitivity was recovered to the same extent as a condition in the absence of Sema 3A (Conditions 4 to 6 relative to Condition 3). On the other hand, human antibody-containing negative control group was not able to recover GEM sensitivity to the same extent as a condition in the absence of Sema 3A (Conditions 7 to 9). Results reveal that the produced humanized anti-Sema 3A antibody in Example 2 has ability to remove anticancer agent tolerance induced by Sema 3A in such a nutrient starvation as pancreatic cancer tissue.









TABLE 21







Antibody
Results of Dunnett's test










concentration
Anti-Sema 3A
Negative control


(μg/mL)
humanized antibody
human antibody












0.1
p = 0.0150
p = 0.0414


1
p < 0.001
p = 0.0179


10
p < 0.001
p = 0.0147









SEQUENCE LISTING FREE TEXT
SEQ ID NO: 1 is the amino acid sequence of heavy chain CDR1 of avian-mouse chimeric antibody (clone No. 4-2 strain-derived).
SEQ ID NO: 2 is the amino acid sequence of heavy chain CDR2 of avian-mouse chimeric antibody (clone No. 4-2 strain-derived).
SEQ ID NO: 3 is the amino acid sequence of heavy chain CDR3 of avian-mouse chimeric antibody (clone No. 4-2 strain-derived).
SEQ ID NO: 4 is the amino acid sequence of light chain CDR1 of avian-mouse chimeric antibody (clone No. 4-2 strain-derived).
SEQ ID NO: 5 is the amino acid sequence of light chain CDR2 of avian-mouse chimeric antibody (clone No. 4-2 strain-derived).
SEQ ID NO: 6 is the amino acid sequence of light chain CDR3 of avian-mouse chimeric antibody (clone No. 4-2 strain-derived).
SEQ ID NO: 7 is the amino acid sequence of heavy chain variable region of avian-human chimeric antibody (clone No. 4-2 strain-derived).
SEQ ID NO: 8 is the amino acid sequence of heavy chain variable region of avian-human chimeric antibody (clone No. 4-2 strain-derived).
SEQ ID NO: 9 is the amino acid sequence of light chain variable region of avian-human chimeric antibody (clone No. 4-2 strain-derived).
SEQ ID NO: 10 is the amino acid sequence of light chain variable region of avian-human chimeric antibody (clone No. 4-2 strain-derived).
SEQ ID NO: 11 is the amino acid sequence of heavy chain variable region of humanized antibody (clone No. 4-2 strain-derived).
SEQ ID NO: 12 is the amino acid sequence of light chain variable region of humanized antibody (Humanized-1) (clone No. 4-2 strain-derived).
SEQ ID NO: 13 is the amino acid sequence of light chain variable region of humanized antibody (Humanized-2) (clone No. 4-2 strain-derived).
SEQ ID NO: 14 is the nucleotide sequence encoding the amino acid sequence (SEQ ID NO: 7) of heavy chain variable region of avian-human chimeric antibody (clone No. 4-2 strain-derived).
SEQ ID NO: 15 is the nucleotide sequence encoding the amino acid sequence (SEQ ID NO: 8) of heavy chain variable region of avian-human chimeric antibody (clone No. 4-2 strain-derived).
SEQ ID NO: 16 is the nucleotide sequence encoding the amino acid sequence (SEQ ID NO: 9) of light chain variable region of avian-human chimeric antibody (clone No. 4-2 strain-derived).
SEQ ID NO: 17 is the nucleotide sequence encoding the amino acid sequence (SEQ ID NO: 10) of light chain variable region of avian-human chimeric antibody (clone No. 4-2 strain-derived).
SEQ ID NO: 18 is the nucleotide sequence encoding the amino acid sequence of heavy chain CDR1 of avian-mouse chimeric antibody (clone No. 4-2 strain-derived).
SEQ ID NO: 19 is the nucleotide sequence encoding the amino acid sequence of heavy chain CDR2 of avian-mouse chimeric antibody (clone No. 4-2 strain-derived).
SEQ ID NO: 20 is the nucleotide sequence encoding the amino acid sequence of heavy chain CDR3 of avian-mouse chimeric antibody (clone No. 4-2 strain-derived).
SEQ ID NO: 21 is the nucleotide sequence encoding the amino acid sequence of light chain CDR1 of avian-mouse chimeric antibody (clone No. 4-2 strain-derived).
SEQ ID NO: 22 is the nucleotide sequence encoding the amino acid sequence of light chain CDR2 of avian-mouse chimeric antibody (clone No. 4-2 strain-derived).
SEQ ID NO: 23 is the nucleotide sequence encoding the amino acid sequence of light chain CDR3 of avian-mouse chimeric antibody (clone No. 4-2 strain-derived).
SEQ ID NO: 24 is the nucleotide sequence encoding the amino acid sequence (SEQ ID NO: 11) of heavy chain variable region of humanized antibody (clone No. 4-2 strain-derived).
SEQ ID NO: 25 is the nucleotide sequence encoding the amino acid sequence (SEQ ID NO: 12) of light chain variable region of humanized antibody (Humanized-1) (clone No. 4-2 strain-derived).
SEQ ID NO: 26 is the nucleotide sequence encoding the amino acid sequence (SEQ ID NO: 13) of light chain variable region of humanized antibody (Humanized-2) (clone No. 4-2 strain-derived).
SEQ ID NO: 27 is the nucleotide sequence of primer-1.
SEQ ID NO: 28 is the nucleotide sequence of primer-2.
SEQ ID NO: 29 is the nucleotide sequence of primer-3.
SEQ ID NO: 30 is the nucleotide sequence of primer-4.
SEQ ID NO: 31 is the nucleotide sequence of primer-5.
SEQ ID NO: 32 is the nucleotide sequence of primer-6.
SEQ ID NO: 33 is the nucleotide sequence of primer-7.
SEQ ID NO: 34 is the nucleotide sequence of primer-8.
SEQ ID NO: 35 is the amino acid sequence of heavy chain of avian-mouse chimeric antibody (clone No. 4-2 strain-derived).
SEQ ID NO: 36 is the amino acid sequence of light chain of avian-mouse chimeric antibody (clone No. 4-2 strain-derived).
SEQ ID NO: 37 is the nucleotide sequence of primer-Aγ.
SEQ ID NO: 38 is the nucleotide sequence of primer-Bγ.
SEQ ID NO: 39 is the nucleotide sequence of primer-Cγ.
SEQ ID NO: 40 is the nucleotide sequence of primer-Dγ.
SEQ ID NO: 41 is the nucleotide sequence of primer-Eγ.
SEQ ID NO: 42 is the nucleotide sequence of primer-Fγ.
SEQ ID NO: 43 is the nucleotide sequence of primer-Gγ.
SEQ ID NO: 44 is the nucleotide sequence of primer-Hγ.
SEQ ID NO: 45 is the nucleotide sequence of primer-Iγ.
SEQ ID NO: 46 is the nucleotide sequence of primer-Jγ.
SEQ ID NO: 47 is the nucleotide sequence of primer-Kγ.
SEQ ID NO: 48 is the nucleotide sequence of primer-Lγ.
SEQ ID NO: 49 is the amino acid sequence of heavy chain of humanized antibodies (Humanized-1 and Humanized-2) (clone No. 4-2 strain-derived).
SEQ ID NO: 50 is the nucleotide sequence encoding the amino acid sequence of heavy chain of humanized antibodies (Humanized-1 and Humanized-2) (clone No. 4-2 strain-derived).
SEQ ID NO: 51 is the amino acid sequence of light chain of humanized antibody (Humanized-1) (clone No. 4-2 strain-derived).
SEQ ID NO: 52 is the nucleotide sequence encoding the amino acid sequence of light chain of humanized antibody (Humanized-1) (clone No. 4-2 strain-derived).
SEQ ID NO: 53 is the amino acid sequence of light chain of humanized antibody (Humanized-2) (clone No. 4-2 strain-derived).
SEQ ID NO: 54 is the nucleotide sequence encoding the amino acid sequence of light chain of humanized antibody (Humanized-2) (clone No. 4-2 strain-derived).
SEQ ID NO: 55 is the amino acid sequence of heavy chain of avian-human chimeric antibody (clone No. 4-2 strain-derived).
SEQ ID NO: 56 is the nucleotide sequence encoding the amino acid sequence of heavy chain of avian-human chimeric antibody (clone No. 4-2 strain-derived).
SEQ ID NO: 57 is the amino acid sequence of light chain of avian-human chimeric antibody (clone No. 4-2 strain-derived).
SEQ ID NO: 58 is the nucleotide sequence encoding the amino acid sequence of light chain of avian-human chimeric antibody (clone No. 4-2 strain-derived).
SEQ ID NO: 59 is the amino acid sequence of heavy chain variable region of avian-mouse chimeric antibody (clone No. 165 strain-derived).
SEQ ID NO: 60 is the amino acid sequence of heavy chain CDR1 of avian-mouse chimeric antibody (clone No. 165 strain-derived).
SEQ ID NO: 61 is the amino acid sequence of heavy chain CDR2 of avian-mouse chimeric antibody (clone No. 165 strain-derived).
SEQ ID NO: 62 is the amino acid sequence of heavy chain CDR3 of avian-mouse chimeric antibody (clone No. 165 strain-derived).
SEQ ID NO: 63 is the amino acid sequence of light chain variable region of avian-mouse chimeric antibody (clone No. 165 strain-derived).
SEQ ID NO: 64 is the amino acid sequence of light chain CDR1 of avian-mouse chimeric antibody (clone No. 165 strain-derived).
SEQ ID NO: 65 is the amino acid sequence of light chain CDR2 of avian-mouse chimeric antibody (clone No. 165 strain-derived).
SEQ ID NO: 66 is the amino acid sequence of light chain CDR3 of avian-mouse chimeric antibody (clone No. 165 strain-derived).
SEQ ID NO: 67 is the amino acid sequence of heavy chain variable region of avian-mouse chimeric antibody (clone No. 582 strain-derived).
SEQ ID NO: 68 is the amino acid sequence of heavy chain CDR1 of avian-mouse chimeric antibody (clone No. 165 strain-derived).
SEQ ID NO: 69 is the amino acid sequence of heavy chain CDR2 of avian-mouse chimeric antibody (clone No. 165 strain-derived).
SEQ ID NO: 70 is the amino acid sequence of heavy chain CDR3 of avian-mouse chimeric antibody (clone No. 165 strain-derived).
SEQ ID NO: 71 is the amino acid sequence of light chain variable region of avian-mouse chimeric antibody (clone No. 582 strain-derived).
SEQ ID NO: 72 is the amino acid sequence of light chain CDR1 of avian-mouse chimeric antibody (clone No. 582 strain-derived).
SEQ ID NO: 73 is the amino acid sequence of light chain CDR2 of avian-mouse chimeric antibody (clone No. 582 strain-derived).
SEQ ID NO: 74 is the amino acid sequence of light chain CDR3 of avian-mouse chimeric antibody (clone No. 582 strain-derived).
SEQ ID NO: 75 is the amino acid sequence of heavy chain variable region of avian antibody (clone No. 240-40 strain-derived).
SEQ ID NO: 76 is the amino acid sequence of heavy chain CDR1 of avian antibody (clone No. 240-40 strain-derived).
SEQ ID NO: 77 is the amino acid sequence of heavy chain CDR2 of avian antibody (clone No. 240-40 strain-derived).
SEQ ID NO: 78 is the amino acid sequence of heavy chain CDR3 of avian antibody (clone No. 240-40 strain-derived).
SEQ ID NO: 79 is the amino acid sequence of light chain variable region of avian antibody (clone No. 240-40 strain-derived).
SEQ ID NO: 80 is the amino acid sequence of light chain CDR1 of avian antibody (clone No. 240-40 strain-derived).
SEQ ID NO: 81 is the amino acid sequence of light chain CDR2 of avian antibody (clone No. 240-40 strain-derived).
SEQ ID NO: 82 is the amino acid sequence of light chain CDR3 of avian antibody (clone No. 240-40 strain-derived).
SEQ ID NO: 83 is the amino acid sequence of heavy chain variable region of avian antibody (clone No. 255-72 strain-derived).
SEQ ID NO: 84 is the amino acid sequence of heavy chain CDR1 of avian antibody (clone No. 255-72 strain-derived).
SEQ ID NO: 85 is the amino acid sequence of heavy chain CDR2 of avian antibody (clone No. 255-72 strain-derived).
SEQ ID NO: 86 is the amino acid sequence of heavy chain CDR3 of avian antibody (clone No. 255-72 strain-derived).
SEQ ID NO: 87 is the amino acid sequence of light chain variable region of avian antibody (clone No. 255-72 strain-derived).
SEQ ID NO: 88 is the amino acid sequence of light chain CDR1 of avian antibody (clone No. 255-72 strain-derived).
SEQ ID NO: 89 is the amino acid sequence of light chain CDR2 of avian antibody (clone No. 255-72 strain-derived).
SEQ ID NO: 90 is the amino acid sequence of light chain CDR3 of avian antibody (clone No. 255-72 strain-derived).
SEQ ID NO: 91 is the nucleotide sequence of primer-Mγ.
SEQ ID NO: 92 is the nucleotide sequence of primer-Nγ.
SEQ ID NO: 93 is the nucleotide sequence of primer-Oγ.
SEQ ID NO: 94 is the nucleotide sequence of primer-Pγ.
SEQ ID NO: 95 is the nucleotide sequence encoding the amino acid sequence of heavy chain constant region of humanized antibody.
SEQ ID NO: 96 is the nucleotide sequence encoding the amino acid sequence of light chain constant region of humanized antibody.
SEQ ID NO: 97 is the nucleotide sequence encoding the amino acid sequence (SEQ ID NO: 60) of heavy chain CDR1 of avian-mouse chimeric antibody (clone No. 165 strain-derived).
SEQ ID NO: 98 is the nucleotide sequence encoding the amino acid sequence (SEQ ID NO: 61) of heavy chain CDR2 of avian-mouse chimeric antibody (clone No. 165 strain-derived).
SEQ ID NO: 99 is the nucleotide sequence encoding the amino acid sequence (SEQ ID NO: 62) of heavy chain CDR3 of avian-mouse chimeric antibody (clone No. 165 strain-derived).
SEQ ID NO: 100 is the nucleotide sequence encoding the amino acid sequence (SEQ ID NO: 64) of light chain CDR1 of avian-mouse chimeric antibody (clone No. 165 strain-derived).
SEQ ID NO: 101 is the nucleotide sequence encoding the amino acid sequence (SEQ ID NO: 65) of light chain CDR2 of avian-mouse chimeric antibody (clone No. 165 strain-derived).
SEQ ID NO: 102 is the nucleotide sequence encoding the amino acid sequence (SEQ ID NO: 66) of light chain CDR3 of avian-mouse chimeric antibody (clone No. 165 strain-derived).
SEQ ID NO: 103 is the nucleotide sequence encoding the amino acid sequence (SEQ ID NO: 59) of heavy chain variable region of avian-mouse chimeric antibody (clone No. 165 strain-derived).
SEQ ID NO: 104 is the nucleotide sequence encoding the amino acid sequence (SEQ ID NO: 63) of light chain variable region of avian-mouse chimeric antibody (clone No. 165 strain-derived).
SEQ ID NO: 105 is the nucleotide sequence encoding the amino acid sequence (SEQ ID NO: 68) of heavy chain CDR1 of avian-mouse chimeric antibody (clone No. 582 strain-derived).
SEQ ID NO: 106 is the nucleotide sequence encoding the amino acid sequence (SEQ ID NO: 69) of heavy chain CDR2 of avian-mouse chimeric antibody (clone No. 582 strain-derived).
SEQ ID NO: 107 is the nucleotide sequence encoding the amino acid sequence (SEQ ID NO: 70) of heavy chain CDR3 of avian-mouse chimeric antibody (clone No. 582 strain-derived).
SEQ ID NO: 108 is the nucleotide sequence encoding the amino acid sequence (SEQ ID NO: 72) of light chain CDR1 of avian-mouse chimeric antibody (clone No. 582 strain-derived).
SEQ ID NO: 109 is the nucleotide sequence encoding the amino acid sequence (SEQ ID NO: 73) of light chain CDR2 of avian-mouse chimeric antibody (clone No. 582 strain-derived).
SEQ ID NO: 110 is the nucleotide sequence encoding the amino acid sequence (SEQ ID NO: 74) of light chain CDR3 of avian-mouse chimeric antibody (clone No. 582 strain-derived).
SEQ ID NO: 111 is the nucleotide sequence encoding the amino acid sequence (SEQ ID NO: 67) of heavy chain variable region of avian-mouse chimeric antibody (clone No. 582 strain-derived).
SEQ ID NO: 112 is the nucleotide sequence encoding the amino acid sequence (SEQ ID NO: 71) of light chain variable region of avian-mouse chimeric antibody (clone No. 582 strain-derived).
SEQ ID NO: 113 is the nucleotide sequence encoding the amino acid sequence (SEQ ID NO: 76) of heavy chain CDR1 of avian antibody (clone No. 240-40 strain-derived).
SEQ ID NO: 114 is the nucleotide sequence encoding the amino acid sequence (SEQ ID NO: 77) of heavy chain CDR2 of avian antibody (clone No. 240-40 strain-derived).
SEQ ID NO: 115 is the nucleotide sequence encoding the amino acid sequence (SEQ ID NO: 78) of heavy chain CDR3 of avian antibody (clone No. 240-40 strain-derived).
SEQ ID NO: 116 is the nucleotide sequence encoding the amino acid sequence (SEQ ID NO: 80) of light chain CDR1 of avian antibody (clone No. 240-40 strain-derived).
SEQ ID NO: 117 is the nucleotide sequence encoding the amino acid sequence (SEQ ID NO: 81) of light chain CDR2 of avian antibody (clone No. 240-40 strain-derived).
SEQ ID NO: 118 is the nucleotide sequence encoding the amino acid sequence (SEQ ID NO: 82) of light chain CDR3 of avian antibody (clone No. 240-40 strain-derived).
SEQ ID NO: 119 is the nucleotide sequence encoding the amino acid sequence (SEQ ID NO: 75) of heavy chain variable region of avian antibody (clone No. 240-40 strain-derived).
SEQ ID NO: 120 is the nucleotide sequence encoding the amino acid sequence (SEQ ID NO: 79) of light chain variable region of avian antibody (clone No. 240-40 strain-derived).
SEQ ID NO: 121 is the nucleotide sequence encoding the amino acid sequence (SEQ ID NO: 84) of heavy chain CDR1 of avian antibody (clone No. 255-72 strain-derived).
SEQ ID NO: 122 is the nucleotide sequence encoding the amino acid sequence (SEQ ID NO: 85) of heavy chain CDR2 of avian antibody (clone No. 255-72 strain-derived).
SEQ ID NO: D123 is the nucleotide sequence encoding the amino acid sequence (SEQ ID NO: 86) of heavy chain CDR3 of avian antibody (clone No. 255-72 strain-derived).
SEQ ID NO: 124 is the nucleotide sequence encoding the amino acid sequence (SEQ ID NO: 88) of light chain CDR1 of avian antibody (clone No. 255-72 strain-derived).
SEQ ID NO: 125 is the nucleotide sequence encoding the amino acid sequence (SEQ ID NO: 89) of light chain CDR2 of avian antibody (clone No. 255-72 strain-derived).
SEQ ID NO: 126 is the nucleotide sequence encoding the amino acid sequence (SEQ ID NO: 90) of light chain CDR3 of avian antibody (clone No. 255-72 strain-derived).
SEQ ID NO: 127 is the nucleotide sequence encoding the amino acid sequence (SEQ ID NO: 83) of heavy chain variable region of avian antibody (clone No. 255-72 strain-derived).
SEQ ID NO: 128 is the nucleotide sequence encoding the amino acid sequence (SEQ ID NO: 87) of light chain variable region of avian antibody (clone No. 255-72 strain-derived).

## Claims

1. An anti-semaphorin 3A antibody comprising:
a heavy chain CDR1 having an amino acid sequence shown in SEQ ID NO:1, CDR2 having an amino acid sequence shown in SEQ ID NO:2, and CDR3 having an amino acid sequence shown in SEQ ID NO:3; and
a light chain CDR1 having an amino acid sequence shown in SEQ ID NO:4, CDR2 having an amino acid sequence shown in SEQ ID NO:5, and CDR3 having an amino acid sequence shown in SEQ ID NO:6.

2. The anti-semaphorin 3A antibody or antibody fragment thereof according to claim 1, wherein the antibody or antibody fragment thereof is a chimeric antibody, a humanized antibody or an antibody fragment thereof.

3. A pharmaceutical composition comprising the anti-semaphorin 3A antibody or antibody fragment thereof according to claim 1.

4. A method for measuring Sema 3A protein, comprising measuring Sema 3A protein in a sample using immunoassay with the anti-Sema 3A antibody or antibody fragment thereof according to claim 1.

5. A kit for measuring Sema 3A protein, comprising the anti-Sema 3A antibody or antibody fragment thereof according to claim 1.