WO2019073507A1 - Composition médicinale contenant un antagoniste de plexine a1 pour la prévention et/ou le traitement de la sclérose latérale amyotrophique - Google Patents

Composition médicinale contenant un antagoniste de plexine a1 pour la prévention et/ou le traitement de la sclérose latérale amyotrophique Download PDF

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WO2019073507A1
WO2019073507A1 PCT/JP2017/036585 JP2017036585W WO2019073507A1 WO 2019073507 A1 WO2019073507 A1 WO 2019073507A1 JP 2017036585 W JP2017036585 W JP 2017036585W WO 2019073507 A1 WO2019073507 A1 WO 2019073507A1
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seq
antibody
plexin
human
variable region
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Japanese (ja)
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淳 熊ノ郷
龍禎 奥野
隆祐 大宮
佑理 寺西
慎也 石井
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国立大学法人大阪大学
中外製薬株式会社
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system

Definitions

  • the present invention relates to a pharmaceutical composition for preventing and / or treating myotonic side sclerosis, which comprises a plexin A1 antagonist.
  • Semaphorin was discovered in the early 1990's as a repulsive molecule for nerve growth cones (Non-patent Documents 1 and 2). Semaphorin has more than 20 members identified to date. Semaphorin is characterized by sharing and saving a region consisting of about 500 amino acids called Sema domain outside the cell. Semaphorin is classified into eight subclasses (Sema1-7, V) from the difference in the C-terminal structure following the Sema domain.
  • the receptors responsible for semaphorin activity are mainly plexins (plexin A1 / A2 / A3 / A4, plexin B1 / B2 / B3, plexin C1, plexin D1) and neuropilins (Nrp-1 and Nrp-2). It is done. Semaphorin is also known to bind to integrins, CD72 and Tim-2 (Non-patent Document 3). It is also known that plexin, which is a semaphorin receptor, associates with various co-receptors such as VEGFR-2, c-Met and Trem2 / DAP12. Plexin reflects the diverse functions of semaphorins or constructs complex ligand-receptor relationships.
  • the biological activity of semaphorin is diverse, such as blood vessel and angiogenesis, cancer metastasis / invasion, bone metabolism regulation, retinal homeostasis maintenance, and immune regulation.
  • Involvement of semaphorin in various diseases such as allergic diseases, autoimmune diseases, bone metabolic diseases, neurodegenerative diseases, retinitis pigmentosa, sudden death of the heart, metastasis / invasion of cancer, etc. has been reported sequentially in recent years (Non-Patent Document 3).
  • Non-Patent Document 3 In connection with the biological activity of semaphorin, research aimed at development of a method for diagnosis and treatment of human diseases is currently being vigorously carried out.
  • Plexin A1 is a receptor for class III and class VI semaphorins. Plexin A1 forms a receptor with VEGF receptor and Off-Track during cardiac morphogenesis of chicken, and also forms a receptor for class III semaphorin with Nrp-1 to act as a receptor for neural repulsion factor It has been reported. Furthermore, plexin A1 also acts as a receptor for class VI semaphorins Sema6C and Sema6D, and is also reported to be involved in axonal guidance and cardiac organogenesis.
  • Non-patent Document 4 plexin A1 signal analysis in dendritic cells and osteoclasts confirmed that in these cells plexin A1 forms a heteroreceptor with Trem-2 and DAP-12. Furthermore, stimulation of recombinant soluble Sema6D protein promotes expression of inflammatory cytokines such as IL-12 from dendritic cells and osteoclast differentiation from precursor cells, Sema6D wild type dendritic cells It was shown that while binding to cells, it could hardly bind to dendritic cells of plexin A1-deficient mice.
  • inflammatory cytokines such as IL-12 from dendritic cells and osteoclast differentiation from precursor cells
  • Non-patent Document 5 Plexin A1 inhibition by plexin A1 by shRNA using mouse dendritic cells controls plexin A localization to the immune synapse of dendritic cells and T cells through activation of signal transduction factor Rho (Non-patent document 6).
  • plexin A1 has been reported to be involved in dendritic cell migration to lymph nodes and in antigen-specific T cell responses. Furthermore, expression of Sema3A but not Sema6C or Sema6D is required for dendritic cell migration as it passes through lymphatic endothelial cells, where Sema3A stimulates myosin-II activity to induce actomyosin contraction has been reported (Patent Document 1).
  • Non-Patent Document 8 cites the above-mentioned Non-Patent Document 7 and introduces the structure of a ternary complex of “Sema3A-plexin A1-Nrp-1”. However, Non-Patent Document 7 does not disclose the structure of the “Sema3A-plexin A1-Nrp-1” ternary complex.
  • Sema3A transmits a signal through Nrp-1 / plexin A1, but does not bind to plexin A1, but only to Nrp-1.
  • Sema3A neutralizing antibody, Nrp-1 neutralizing antibody, soluble Nrp-1 disclosed as substances that inhibit the binding of Nrp-1 / plexin A1 heteroreceptor to Sema 3A and therapeutic agents for cellular immune diseases It has been suggested that it could be (Patent Document 1).
  • Patent Document 9 it has been reported that enhanced expression of Sema3A is found in the motor cortex and spinal cord of the brain of ALS patients.
  • Non-patent Document 10 results of the effect of an anti-Nrp-1 antibody in ALS model mice (Non-patent Document 10) and the Rotarod test in ALS model mice knocked out of Nrp-1 have been reported (Non-patent document 11).
  • a plexin A1 antagonist for example, an anti-plexin A1 antibody
  • ALS amyotrophic lateral sclerosis
  • the present invention has been made in view of the above situation, and provides a pharmaceutical composition for preventing and / or treating amyotrophic lateral sclerosis (ALS) containing a plexin A1 antagonist, and a kit comprising the pharmaceutical composition.
  • An object of the present invention is to provide an anti-plexin A1 antibody, a pharmaceutical composition used for the prevention and / or treatment of a disease associated with the biological activity of 3A.
  • ALS amyotrophic lateral sclerosis
  • plexin A1 antagonists exhibit efficacy in the ALS model.
  • the present inventors succeeded in obtaining an anti-plexin A1 antibody that inhibits the biological activity of semaphorin 3A.
  • the present invention is based on such findings, and relates to [1] to [11] below.
  • a pharmaceutical composition for the prevention and / or treatment of myotrophic lateral sclerosis which comprises a plexin A1 antagonist.
  • the antagonist is selected from the group consisting of anti-plexin A1 antibody or antibody fragment thereof, antisense to plexin A1, double stranded RNA (dsRNA), siRNA, miRNA, short hairpin RNA, RNA aptamer and ribozyme
  • the pharmaceutical composition according to any one of [1] to [3].
  • [5] The pharmaceutical composition of any one of [1] to [4], wherein the antagonist is an anti-plexin A1 antibody or an antibody fragment thereof.
  • [6] The pharmaceutical composition of [4] or [5], wherein the antibody or the antibody fragment thereof is an anti-human mouse cross plexin A1 antibody.
  • a kit comprising the pharmaceutical composition of any one of [1] to [7].
  • a pharmaceutical composition for suppressing muscle weakness or muscle atrophy comprising a plexin A1 antagonist.
  • the present invention also relates to the following [12] to [18].
  • An anti-plexin A1 antibody or an antibody fragment thereof according to any one of the following (1) to (4): (1) H chain variable region comprising CDR1 set forth in SEQ ID NO: 1, CDR2 set forth in SEQ ID NO: 2, and CDR3 set forth in SEQ ID NO: 3, and CDR1 set forth in SEQ ID NO: 4, SEQ ID NO: 4 An antibody or an antibody fragment thereof comprising a light chain variable region comprising CDR2 of 5: and CDR3 of SEQ ID NO: 6; (2) H chain variable region comprising CDR1 of SEQ ID NO: 7, CDR2 of SEQ ID NO: 8, and CDR3 of SEQ ID NO: 9, and CDR1 of SEQ ID NO: 10, SEQ ID NO: An antibody or an antibody fragment thereof comprising a light chain variable region comprising CDR2 of SEQ ID NO: 11 and CDR3 of SEQ ID NO: 12; (3) H chain variable region comprising CDR1 of SEQ ID NO: 13, CDR2
  • a method for the prophylaxis and / or treatment of ALS which comprises the step of administering a plexin A1 antagonist to a patient.
  • a pharmaceutical composition for preventing and / or treating amyotrophic lateral sclerosis (ALS) containing a plexin A1 antagonist a kit comprising the pharmaceutical composition, muscle weakness or muscle atrophy comprising the plexin A1 antagonist
  • a pharmaceutical composition for suppressing degeneration or loss of the motor nerve containing the plexin A1 antagonist a pharmaceutical composition for suppressing degeneration or loss of the motor nerve containing the plexin A1 antagonist, prevention of a disease associated with biological activity of semaphorin 3A containing the plexin A1 antagonist and /
  • a pharmaceutical composition for use in therapy an anti-plexin A1 antibody.
  • a pharmaceutical composition for preventing and / or treating amyotrophic lateral sclerosis (ALS) containing an anti-plexin A1 antibody, and an anti-plexin A1 antibody that inhibits the biological activity of semaphorin 3A were provided.
  • FIG. 5 shows antagonist activity of anti-human mouse cross Plexin A1 antibody against human semaphorin 3A. It is a figure which shows the antagonist activity with respect to mouse semaphorin 3A of an anti-human mouse cross Plexin A1 antibody.
  • FIG. 6 is a graph showing relative survival time when a control antibody or anti-PlexinA1 antagonist antibody hPASK # 188 is administered to G93A mutant SOD1 transgenic mice, and a relative value of a change in time until the mouse falls in the rotarod test.
  • Anti-PlexinA1 antagonist antibody hPASK # 188 has a survival time (P ⁇ 0.005, log rank test), time to drop of mouse in Rotarod test (P ⁇ 0.05 at 18 and 19 weeks in comparison with control antibody) , Wilcoxon rank sum test, one-tailed test) was significantly extended.
  • the present invention relates to a pharmaceutical composition for the prevention and / or treatment of muscle atrophy lateral sclerosis (ALS) containing a plexin A1 antagonist.
  • plexin A1 for example, human or mouse plexin A1 can be used, but it is not particularly limited thereto.
  • the amino acid sequence and nucleotide sequence of mouse plexin A1 are described, for example, in Kameyama T et al. "Biochemical and biophysical research communications.” Biochem Biophys Res Commun. 1996, 226 (2), 524-9, Accession No. D 86948 of Genbank, NCBI. It is published in Reference Sequence NP_032907.1.
  • plexin A1 amino acid sequence and nucleotide sequence of human plexin A1 are disclosed in, for example, Tamagnone L et al. Cell. 1999, 99 (1), 71-80, Genbank Accession No. X87832, NCBI Reference Sequence NP_115618, NCBI Reference Sequence NM_032242 ing.
  • Plexin A1 can be easily cloned based on these sequence information.
  • the amino acid sequence or base sequence of plexin A1 can be appropriately modified and used within the intended range of use.
  • the amino acid sequence of plexin A1 is well conserved between human and mouse.
  • the plexin A1 antagonist means, for example, a compound that specifically binds to plexin A1 protein and prevents or reduces its signal transduction activity, for example, an antibody (the antibody is an anti-plexin A1 antibody, an antibody against anti-plexin A1) And aptamers.
  • Another plexin A1 antagonist refers to a compound that prevents or reduces expression of plexin A1 protein, and includes, for example, antisense nucleic acid, ribozyme nucleic acid, and nucleic acid having RNAi activity.
  • the plexin A1 antagonists include anti-plexin A1 antibodies.
  • plexin A1 antagonists include plexin A1 antagonists that inhibit the biological activity of semaphorin 3A, and plexin A1 antagonists that inhibit the regression of cell forms.
  • plexin A1 antagonists include anti-plexin A1 antibodies that inhibit the biological activity of semaphorin 3A, and anti-plexin A1 antibodies that inhibit the regression of cell forms. More specifically, examples of the anti-plexin A1 antibody include the antibodies described later.
  • Semaphorin 3A may be, for example, human or mouse semaphorin 3A, but is not particularly limited thereto.
  • the amino acid sequence of human semaphorin 3A is published, for example, in NCBI Reference Sequence NP — 006071.1.
  • the amino acid sequence of mouse semaphorin 3A is disclosed, for example, in NCBI Reference Sequence NM_009152.
  • Semaphorin 3A can be easily cloned based on these sequence information.
  • the amino acid sequence of semaphorin 3A can be appropriately modified and used within the scope of the purpose of use. In the present specification, semaphorin 3A is also described as Sema 3A.
  • the biological activity of semaphorin 3A refers to an activity that semaphorin 3A can exert in vitro or in vivo, and means, for example, the following activities.
  • Activity to retract cell morphology eg dendritic cell morphology
  • activity to promote migration of dendritic cells to regional lymph nodes US Patent Publication 2012/0322085
  • inhibit osteoclast differentiation include activity, activity to promote osteoblast differentiation (Hayashi M et al. Nature 2012, 485, 69-74), and nerve extension inhibitory activity (US Pat. No. 7,642,362).
  • the antagonist of the present invention is only required to inhibit at least one of the biological activities of semaphorin 3A, preferably to inhibit the regression of cell form, and more preferably the regression of cell form of dendritic cell Inhibit.
  • Dendritic cells may be derived, for example, from bone marrow or monocytes.
  • Diseases associated with biological activity of semaphorin 3A include, for example, amyotrophic lateral sclerosis (ALS).
  • ALS amyotrophic lateral sclerosis
  • SOD1 gene has been reported as the causative gene for familial amyotrophic lateral sclerosis (familial ALS), and G37R, H46R, G85R, G93A, etc. are known as gene mutations.
  • Transgenic mice having these mutations develop symptoms similar to ALS, and are widely used for research and development of therapeutic agents for ALS as ALS model animals.
  • SOD1 transgenic mice carrying the G93A mutation can be used to confirm the effect of ALS therapeutics, but SOD1 transgenic mice carrying other mutations can also be used.
  • inhibitors of the biological activity of semaphorin 3A means that the biological activity of semaphorin 3A is reduced by 5% or more in the presence of the antagonist of the present invention as compared to the absence, preferably 10% or more, 20 % Or more, 30% or more, 50% or more, 75% or more, 80% or more, 90% or more, or 95% or more.
  • Sema3A induces dendritic cell migration by retracting nerve growth cones of neurons and suppressing axonal outgrowth, or inducing regression of dendritic cell morphology in the process of dendritic cells passing through micro lymphatics. It controls a variety of biological reactions including neural network formation and immune reaction by showing the reductive activity to various cells, such as controlling
  • Methods known to those skilled in the art can be appropriately used as a method for measuring the regression of the cell form, for example, a method of directly analyzing the cell form, changes in cell form and adhesion, xCELLigence (registered trademark) And so on), but it is not limited thereto.
  • Cells such as various tumor cells, endothelial cells including HUVEC, dorsal root ganglion (DRG) neurons and dendritic cells are seeded on a 96 well cell culture plate and cultured for several hours to 1 day to allow the cells to adhere.
  • Sema3A is added, and culture is further performed for 30 minutes to several hours at 37 ° C. in a 5% CO 2 incubator. At this time, a well to which Sema3A is not added is set as a comparison control.
  • cell morphology is photographed using a cell image analyzer for high content screening such as microscopic observation or ArrayScan (registered trademark), and changes in the cell morphology are quantified by image analysis software (Cellomics-vHCSTM: Scan etc.).
  • a cell image analyzer for high content screening such as microscopic observation or ArrayScan (registered trademark)
  • Change in the cell morphology are quantified by image analysis software (Cellomics-vHCSTM: Scan etc.).
  • an index used in quantifying cell shape change for example, a method using an index "% High Object Convex Hull Area ratio" can be mentioned. This is a calculation of the ratio of the area of the cell itself to the area of the convex hull to calculate the percentage of cells above a certain threshold, and this number is compared between when Sema3A is not added and when it is added.
  • Regression of the cell form means that the above value decreased by 5% or more when Sema3A is added compared to when Sema3A is not added, preferably 10% or more, 20% or more, 30% or more, 50% or more, 75 % Or more, 80% or more, 90% or more, or 95% or more.
  • Average neurite outgrowth / neuron by using neurite outgrowth application of image analysis software (Cellomics-vHCSTM: Scan etc.) as an example of an index used in quantifying another cell shape change
  • the index quantified as / well can be used.
  • Regression of the cell form means that the above value decreased by 5% or more when Sema3A is added compared to when Sema3A is not added, preferably 10% or more, 20% or more, 30% or more, 50% or more, 75 % Or more, 80% or more, 90% or more, or 95% or more.
  • a method of calculating the area of cells from the captured image and determining that the cells smaller than a certain threshold value are determined to be regressed can also be used.
  • Cells such as various tumor cells, endothelial cells including HUVEC, dorsal root ganglion (DRG) neurons and dendritic cells are seeded on tissue culture E-plates with electrodes accumulated on the bottom of the wells, and can be performed for several hours to 1 day Adhere cells by culturing.
  • Sema3A is added, and the cells are cultured in an incubator at 37 ° C., 5% CO 2 for about one more hour. At this time, a well to which Sema3A is not added is set as a comparison control.
  • Changes in cell morphology and adhesion are detected as electrical impedance using xCELLigence®.
  • This electrical impedance is detected using RTCA software (registered trademark), which is analysis software for xCELLigence (registered trademark), and changes relative to the current cell condition as a unitless parameter called cell index (CI). It is created from The cell shape regression-inducing activity of Sema3A is measured by comparing Sema3A with and without this CI value.
  • Regression of the cell form means that the CI value decreased by 5% or more when Sema3A was added compared to when Sema3A was not added, and preferably 10% or more, 20% or more, 30% or more, 50% or more, 75 % Or more, 80% or more, 90% or more, or 95% or more.
  • Sema3A acts on the heteroreceptor complex of Plexin-A1 and Neuropilin-1 on dendritic cells and shows activity to induce actomyosin contraction, and induces changes in cell morphology to pass through the microlymphatic cell gap I have control.
  • a method of measuring the activity methods known to those skilled in the art can be used as appropriate, for example, a method of measuring using a cell migration assay using a Boyden chamber in vitro, It is not limited to these.
  • transwells (Corning) are placed in a 24-well plate containing 0.6 mL of 0.1% BSA in RPMI 1640 containing chemokines such as CCL21 and CXCL12. Dendritic cells are added to the upper chamber of the transwell and incubated at 37 ° C. for 1-3 hours. The cells in the lower chamber are then counted.
  • lymphatic endothelial cells and vascular endothelial cells are overlaid on the upper chamber. Briefly, cells of SVEC 4-10 or HMVEC-dLy are seeded at the top or bottom of a 2 ⁇ g / mL fibronectin coated transwell insert.
  • transendothelial cell migration assay is performed according to the same method as the cell migration assay described above. In performing these assays, add Sema 3A with dendritic cells to the upper chamber of the transwell and compare the migration results of dendritic cells to the lower chamber with wells without Sema 3A. The promoting activity of cell migration is measured.
  • the activity to promote migration of dendritic cells to regional lymph nodes was an increase in migration counts of dendritic cells to the lower chamber by at least 5% in wells to which Sema3A was added as compared to wells to which Sema3A was not added. It means that the increase is preferably 10% or more, 20% or more, 30% or more, 50% or more, 75% or more, 80% or more, 90% or more, 95% or more.
  • Sema3A exerts an osteoprotective activity by acting on the activation of osteoblasts as well as suppressing the differentiation of osteoclasts through receptors expressed on osteoclasts and osteoblasts. It has been reported.
  • Osteoclast differentiation inhibitory activity There are various methods for measuring the activity, and it is evaluated by, for example, tartrate-resistant acid phosphatase (TRAP) staining as one example.
  • bone marrow cells are cultured in ⁇ -MEM medium containing M-CSF for 48 hours or more to prepare myeloid monocyte / macrophage precursor cells.
  • the medium is changed to a medium supplemented with RANKL and culture is continued for several days.
  • Sema3A the medium is changed to the medium to which Sema3A is added, and after 10 to 12 hours, RANKL is added and culture is continued for several days.
  • the medium is changed every few days, and when osteoclast formation is confirmed, TRAP staining and nuclear staining are performed.
  • TRAP staining and nuclear staining are performed.
  • Measure osteoclast differentiation inhibitory activity Specifically, the number of TRAP positive cells per well, or the number of TRAP positive and multinucleated cells are counted and compared.
  • the osteoclast differentiation inhibitory activity means that the number of TRAP-positive cells or the number of TRAP-positive and multinucleated cells is reduced by 5% or more in the wells to which Sema3A is added, as compared to the wells to which Sema3A is not added. Means a decrease of 10% or more, 20% or more, 30% or more, 50% or more, 75% or more, 80% or more, 90% or more, 95% or more.
  • Osteoblast activation activity There are various methods for measuring the activity, and for example, evaluation is made by, for example, alkaline phosphatase (ALP) staining, ALP activity measurement, and detection of calcification.
  • ALP alkaline phosphatase
  • calvarial cells and MC3T3-E1 cells are seeded on a collagen-coated plate and cultured in ⁇ -MEM medium containing ascorbic acid and ⁇ -glycerophosphate.
  • Sema3A culture is performed by adding Sema3A to the medium. The medium is changed every few days, and after completion of culture, ALP staining is performed to obtain an image by a microscope, or its ALP activity is measured using absorbance method.
  • Alizarin red staining is performed to detect calcification.
  • the stained image of cells to which Sema3A is added is obtained using a microscope, and the osteoblast activating action of Sema3A is evaluated by comparing with the stained image of cells to which Sema3A is not added. Specifically, cells after staining are photographed with a microscope, and the intensity of ALP staining and Alizarin red staining is visually compared to determine the presence or absence of activity.
  • the osteoblast activation activity means that the absorbance of ALP activity or the intensity of Alizarin Red staining is increased by 5% or more in the wells to which Sema 3A is added as compared with the wells to which Sema 3A is not added, preferably 10% or more 20% or more, 30% or more, 50% or more, 75% or more, 80% or more, 90% or more, 95% or more.
  • the term “antibody” is used in the broadest sense and, as long as it exhibits the desired antigen binding activity, it is not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. It includes various antibody structures, including bispecific antibodies) and antibody fragments.
  • the antibodies may be murine, human, humanized, chimeric or derived from other species.
  • the antibodies disclosed herein can be of any type (eg, IgG, IgE, IgM, IgD and IgA), class (eg, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule It can be.
  • the immunoglobulin may be from any species (eg, human, mouse or rabbit).
  • the terms “antibody”, “immunoglobulin” and “immunoglobulin” are used interchangeably in a broad sense.
  • antibody fragment refers to a molecule other than the complete antibody that comprises the portion of the complete antibody that binds to the antigen to which the complete antibody binds.
  • antibody fragments include, but are not limited to, Fv, Fab, Fab ', Fab'-SH, F (ab') 2, diabodies, linear antibodies, single chain antibody molecules (eg, scFv) And multispecific antibodies formed from antibody fragments.
  • Cross-over antibody is also referred to as cross-over antibody or cross-reacting antibody, and refers to an antibody that recognizes the same or similar epitopes in multiple antigens.
  • the plurality of antigens may be, for example, antigens of the same species, or antigens of different species.
  • the antibody in the present invention is preferably a recombinant antibody produced using genetic recombination technology.
  • the recombinant antibody may be cloned from antibody-producing cells such as hybridomas or sensitized lymphocytes producing the antibody, and then incorporated into a vector and introduced into a host (host cell) for production. It can be obtained by
  • the antibody of the present invention is not limited in its origin, such as human antibody, mouse antibody, rat antibody and the like.
  • genetically modified antibodies such as chimeric antibodies and humanized antibodies may be used.
  • a chimeric antibody is an antibody comprising the H chain and L chain variable regions of the antibody of the immunized animal and the H chain and L chain constant regions of a human antibody.
  • a chimeric antibody can be obtained by linking DNA encoding the variable region of an antibody derived from an immunized animal with DNA encoding the constant region of a human antibody, and incorporating this into an expression vector to introduce it into a host for production. .
  • a human antibody of interest can be obtained by immunizing a transgenic animal having a full repertoire of human antibody genes with an antigen of interest (WO 93 See WO / 12227, WO 92/03918, WO 94/02602, WO 94/25585, WO 96/34096, WO 96/33735).
  • human antibodies can be produced by hybridoma-based methods.
  • Human myeloma and mouse-human hetero cell lines for the production of human monoclonal antibodies are available (Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp 51-63. (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991)).
  • Human antibodies generated through human B cell hybridoma technology are also known (see Li et al., Proc. Natl. Acad. Sci. USA, 103: 3557-3562 (2006)).
  • Trioma technology Human hybridoma technology (trioma technology) is also available (Vallmers and Brandlein, Histology and Histopathology, 20 (3): 927-937 (2005) and Vallmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27 (3) ): 185-91 (2005)).
  • Human antibodies can also be generated by isolating Fv clone variable domain sequences selected from phage display libraries of human origin. Such variable domain sequences may be combined with the desired human constant domain. Techniques for selecting human antibodies from antibody libraries are described below. The present technology is not limited to human antibodies.
  • variable region of a human antibody can be expressed as a single chain antibody (scFv) on the surface of phage by phage display method, and phage that bind to an antigen can be selected.
  • scFv single chain antibody
  • phage that bind to an antigen can be selected.
  • the DNA sequence encoding the variable region of a human antibody that binds to the antigen is clarified, an appropriate expression vector can be produced based on the sequence and a human antibody can be obtained.
  • Antibodies or antibody fragments isolated from human antibody libraries are considered herein as human antibodies or fragments of human antibodies.
  • Humanized antibodies are modified antibodies, also referred to as reshaped human antibodies. Humanized antibodies are constructed by grafting the CDRs of the antibody derived from the immunized animal into the complementarity determining regions of human antibodies. The general genetic recombination method is also known (European Patent Application Publication No. 239400, WO 96/02576, Sato K et al, Cancer Research 1993, 53: 851-856, WO 99 / 51743).
  • the anti-plexin A1 antibody is an antibody described in any of the following (1) to (4) or an antibody fragment thereof, and can be used, for example, for the prophylaxis and / or treatment of ALS.
  • SEQ ID NO: 4 An antibody or an antibody fragment thereof comprising a light chain variable region comprising CDR2 of 5: and CDR3 of SEQ ID NO: 6;
  • H chain variable region comprising CDR1 of SEQ ID NO: 7, CDR2 of SEQ ID NO: 8, and CDR3 of SEQ ID NO: 9, and CDR1 of SEQ ID NO: 10
  • the anti-plexin A1 antibody is the antibody or antibody fragment thereof according to any one of the following (5) to (8).
  • An antibody or antibody fragment thereof comprising the H chain variable region described in SEQ ID NO: 25 and the L chain variable region described in SEQ ID NO: 26;
  • An antibody or antibody fragment thereof comprising the H chain variable region of SEQ ID NO: 27 and the L chain variable region of SEQ ID NO: 28;
  • An antibody or antibody fragment thereof comprising the H chain variable region described in SEQ ID NO: 29 and the L chain variable region described in SEQ ID NO: 30;
  • An antibody or antibody fragment thereof comprising the H chain variable region shown in SEQ ID NO: 31 and the L chain variable region shown in SEQ ID NO: 32.
  • the anti-plexin A1 antibody is the antibody or antibody fragment thereof according to any of the following (9) to (12).
  • amino acid modifications known to those skilled in the art can be added to the aforementioned antibody or antibody fragment thereof as appropriate.
  • the amino acid contained in the amino acid sequence of the antibody or its antibody fragment may also be post-translationally modified (eg, modification to pyroglutamic acid by pyroglutamylation of N-terminal glutamine is a modification well known to those skilled in the art).
  • modification to pyroglutamic acid by pyroglutamylation of N-terminal glutamine is a modification well known to those skilled in the art.
  • an antibody or an antibody fragment thereof is naturally included in the antibody or an antibody fragment thereof.
  • the antibody or antibody fragment thereof is an antibody or antibody fragment thereof that inhibits the biological activity of semaphorin 3A. In a particular aspect, the antibody or antibody fragment thereof is an antibody or antibody fragment thereof that inhibits the regression of cell morphology.
  • the antibody or antibody fragment thereof is an anti-human mouse cross plexin A1 antibody.
  • the term "monoclonal antibody” refers to a population of substantially homogeneous antibodies, ie, the individual antibodies that make up the population are homogeneous except for small naturally occurring variants that may occur in nature. Refers to an antibody obtained from an antibody population. Monoclonal antibodies are highly specific, generally acting against a single antigenic site. In addition, monoclonal antibodies are directed to a single antigenic determinant on an antigen, as compared to conventional polyclonal antibody preparations that typically include different antibodies to different antigenic determinants (epitopes). In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized by hybridoma cultures etc. that are not contaminated with other antibodies.
  • the modifier "monoclonal” indicates the properties of the antibody obtained from a substantially homogeneous population of antibodies, and does not require that the antibody be produced by a particular method.
  • the monoclonal antibodies used in the present invention may be produced, for example, by the hybridoma method (e.g. Kohler and Milstein, Nature 256: 495 (1975)), or by a recombinant method (e.g. U.S. Pat. No. 4,816,567). .
  • the monoclonal antibodies used in the present invention may also be isolated from phage antibody libraries (e.g. Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222). : 581-597 (1991)).
  • the antibodies of the present invention can be produced by methods known to those skilled in the art. Specifically, DNA encoding a target antibody is incorporated into an expression vector. At that time, it is incorporated into an expression vector to be expressed under the control of an expression control region, such as an enhancer or a promoter. Next, host cells are transformed with this expression vector to express an antibody. At that time, a combination of appropriate host and expression vector can be used.
  • an expression control region such as an enhancer or a promoter.
  • vectors examples include M13 vectors, pUC vectors, pBR322, pBluescript, pCR-Script and the like.
  • pGEM-T for the purpose of subcloning and excision of cDNA, for example, pGEM-T, pDIRECT, pT7 and the like can be used in addition to the above vectors.
  • expression vectors are useful when using vectors to produce antibodies.
  • an expression vector for example, when the host is E. coli such as JM109, DH5 ⁇ , HB101, XL1-Blue, a promoter that can be efficiently expressed in E. coli, for example, lacZ promoter (Ward et al., Nature (1989) 341) , 542-546; FASEB J. (1992) 6, 2422-2427), araB promoter (Better et al., Science (1988) 240, 1041-1043), or having a T7 promoter or the like is essential.
  • pGEX-5X-1 manufactured by Pharmacia
  • QIAexpress system manufactured by QIAGEN
  • pEGFP pEGFP
  • pET in this case, the host expresses T7 RNA polymerase
  • the vector may also contain a signal sequence for polypeptide secretion.
  • a signal sequence for polypeptide secretion for example, pelB signal sequence (Lei, SP et al J. Bacteriol. (1987) 169, 4397) may be used when producing in the periplasm of E. coli.
  • the introduction of the vector into host cells can be performed, for example, using the calcium chloride method or electroporation.
  • vectors for producing the antibody of the present invention other than E. coli expression vectors include, for example, mammalian-derived expression vectors (for example, pcDNA3 (manufactured by Invitrogen) and pEGF-BOS (Nucleic Acids. Res. 1990, 18).
  • mammalian-derived expression vectors for example, pcDNA3 (manufactured by Invitrogen) and pEGF-BOS (Nucleic Acids. Res. 1990, 18).
  • a promoter necessary for intracellular expression for example, the SV40 promoter (Mulligan et al., Nature (1979) 277, 108), It is essential to have MMTV-LTR promoter, EF1 ⁇ promoter (Mizushima et al., Nucleic Acids Res. (1990) 18, 5322), CAG promoter (Gene. (1991) 108, 193), CMV promoter, etc. It is more preferable to have a gene for selecting transformed cells. Examples of genes for selecting transformed cells include drug resistant genes that can be distinguished by drugs (neomycin, G418, etc.). Examples of vectors having such characteristics include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, pOP13 and the like.
  • a vector having a DHFR gene that complements the CHO cell deficient in the nucleic acid synthesis pathway for example, , PCHOI etc.
  • MTX methotrexate
  • COS having a gene expressing SV40 T antigen on the chromosome COS having a gene expressing SV40 T antigen on the chromosome.
  • a method of transforming cells using a vector (such as pcD) having an SV40 origin of replication can be mentioned.
  • a replication origin those derived from polyoma virus, adenovirus, bovine papilloma virus (BPV) and the like can also be used.
  • the expression vector is used as a selection marker for selection of aminoglycoside transferase (APH) gene, thymidine kinase (TK) gene, E. coli xanthine guanine phosphoribosyltransferase (Ecogpt) gene, dihydrofolate reductase ( dhfr) gene etc. can be included.
  • APH aminoglycoside transferase
  • TK thymidine kinase
  • Ecogpt E. coli xanthine guanine phosphoribosyltransferase
  • dhfr dihydrofolate reductase
  • the antibody of the present invention thus obtained can be isolated from the inside or outside of the host cell (such as medium) and purified as a substantially pure homogeneous antibody.
  • the separation and purification of the antibody may be carried out using the separation and purification methods used in the conventional purification of antibodies, and is not limited in any way. For example, chromatography column, filter, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing method, dialysis, recrystallization etc. are appropriately selected, In combination, antibodies can be separated and purified.
  • the chromatography includes, for example, affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, adsorption chromatography, etc. (Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press, 1996). These chromatographies can be performed using liquid phase chromatography, for example, liquid phase chromatography such as HPLC or FPLC. Examples of columns used for affinity chromatography include Protein A column and Protein G column. For example, as columns using Protein A, Hyper D, POROS, Sepharose FF (GE Amersham Biosciences), etc. may be mentioned. The present invention also encompasses highly purified antibodies using these purification methods.
  • the resulting antibodies can be purified to homogeneity.
  • separation and purification methods used for ordinary proteins may be used.
  • separation and purification of antibodies can be achieved by appropriately selecting and combining chromatography columns such as affinity chromatography, filters, ultrafiltration, salting out, dialysis, SDS polyacrylamide gel electrophoresis, isoelectric focusing, etc.
  • chromatography columns such as affinity chromatography, filters, ultrafiltration, salting out, dialysis, SDS polyacrylamide gel electrophoresis, isoelectric focusing, etc.
  • affinity chromatography such as affinity chromatography, filters, ultrafiltration, salting out, dialysis, SDS polyacrylamide gel electrophoresis, isoelectric focusing, etc.
  • examples of columns used for affinity chromatography include Protein A column, Protein G column and the like.
  • Aptamer refers to a nucleic acid molecule that specifically binds to a target molecule, such as a polypeptide.
  • the aptamer of the present invention can be an RNA aptamer that can specifically bind to plexin A1.
  • the generation and therapeutic use of aptamers is well established in the art.
  • the aptamer can be obtained by using the SELEX method (see US Patent Nos. 5475096, 5580737, 5657588, 57077796, 5765177 etc.).
  • an "antisense nucleic acid” is an oligonucleotide which is an oligoribonucleotide, an oligodeoxyribonucleotide, a modified oligoribonucleotide, or a modified oligodeoxyribonucleotide, and which is under physiological conditions and contains mRNA of a specific gene or mRNA of that gene. Refers to said oligonucleotides which hybridize to a substance and thereby inhibit transcription of its gene and / or translation of its mRNA.
  • Antisense RNA hybridizes to mRNA in vivo to inhibit translation of the mRNA molecule (Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression (CRC Press: Boca Raton, FL, 1988)). Methods for obtaining antisense nucleic acids are well established in the art.
  • Ribozyme nucleic acids are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. For details, see, for example, Rossi, Current Biology 4: 469-471 (1994). Ribozymes act by sequence specific hybridization to a complementary target RNA, followed by cleavage of the nucleotide strand.
  • the composition of the ribozyme nucleic acid preferably comprises one or more sequences complementary to the target mRNA and a known or functionally equivalent sequence involved in mRNA cleavage (see, eg, US Pat. No. 5,093,246).
  • RNAi activity is a molecule that degrades mRNA in a sequence specific manner or inhibits its translation. Examples of this molecule include double stranded RNA (dsRNA), siRNA, miRNA, short hairpin RNA (shRNA).
  • dsRNA double stranded RNA
  • siRNA siRNA
  • miRNA miRNA
  • shRNA short hairpin RNA
  • a list of known miRNA sequences can be found in databases maintained by research institutions such as Wellcome Trust Sanger Institute, Penn Center for Bioinformatics, Memorial Sloan Kettering Cancer Center, and the European Molecule Biology Laboratory, among others.
  • Known effective siRNA sequences are also well shown in the relevant literature. Methods for making and using nucleic acids having RNAi activity are well known to those skilled in the art.
  • there are computational tools that increase the opportunity to discover effective and specific sequence motifs (Pei et al 2006, Reynolds et al 2004, Khvorova et al 2003, Schwarz et al 2003, Ui-Tei et al 2004, Heale et al 2005, Chalk et al 2004 , Amarzguioui et al. 2004).
  • the pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carriers include sterile water and saline, stabilizers, excipients, antioxidants (such as ascorbic acid), buffers (such as phosphoric acid, citric acid, and other organic acids) And preservatives, surfactants (PEG, Tween etc.), chelating agents (EDTA etc.), binders and the like.
  • polypeptides such as serum albumin, gelatin or immunoglobulin, amino acids such as glycine, glutamine, asparagine, arginine and lysine, saccharides and carbohydrates such as polysaccharides and monosaccharides, mannitol, sorbitol and the like It may contain a sugar alcohol.
  • aqueous solution for injection for example, physiological saline, isotonic solution containing glucose and other adjuvants, such as D-sorbitol, D-mannose, D-mannitol, sodium chloride, etc. may be mentioned, and appropriate dissolution You may use together with adjuvants, such as alcohol (ethanol etc.), polyalcohol (propylene glycol, PEG etc.), nonionic surfactant (polysorbate 80, HCO-50) etc.
  • adjuvants such as alcohol (ethanol etc.), polyalcohol (propylene glycol, PEG etc.), nonionic surfactant (polysorbate 80, HCO-50)
  • the pharmaceutical composition of the present invention may, if necessary, be encapsulated in microcapsules (microcapsules such as hydroxymethylcellulose, gelatin, poly [methyl methacrylate], etc.) or a colloid drug delivery system (liposomes, albumin microspheres, It may also be an emulsion, nanoparticles, nanocapsule etc.) (see “Remington's Pharmaceutical Science 16th edition", Oslo Ed., 1980 etc.).
  • methods of making the drug a sustained release drug are also known and may be applied to the present invention (Langer et al., J. Biomed. Mater. Res. 1981, 15: 167-277; Langer, Chem. Tech 1982, 12: 98-105; US3773919; EP58481; Sidman et al., Biopolymers 1983, 22: 547-556; EP 133988).
  • Administration to patients can be either oral or parenteral administration, but is preferably parenteral administration.
  • the form (dosage form) of the pharmaceutical composition of the present invention is not particularly limited, and injection, nasal administration, transpulmonary administration, transdermal administration, lyophilization, solution administration Etc.
  • Lyophilization can be carried out by methods well known to the person skilled in the art (Pharm Biotechnol, 2002, 13, 109-33, Int J Pharm. 2000, 203 (1-2), 1-60, Pharm Res. 1997, 14 (8). ), 969-975).
  • the solution is dispensed in an appropriate amount into a container such as a vial used for lyophilization and carried out in a freezing or lyophilizing chamber or immersed in a refrigerant such as acetone / dry ice and liquid nitrogen.
  • a refrigerant such as acetone / dry ice and liquid nitrogen.
  • it can implement by the method known to those skilled in the art. For example, as described in J. Pharm. Sc, 2004, 93 (6), 1390-1402, a membrane concentration method using a TFF membrane is used.
  • the injection form can be administered systemically or locally, for example, by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection and the like.
  • the administration method can be appropriately selected according to the age and symptoms of the patient.
  • the dose can be selected, for example, in the range of 0.0001 mg to 1000 mg per kg body weight. Alternatively, for example, the dose can be selected in the range of 0.001 to 100,000 mg / body per patient.
  • the present invention is not limited to these dosages and administration methods.
  • a therapeutically and / or prophylactically effective amount of a plexin A1 antagonist is that amount of the antagonist that, when administered to a subject, is effective to prevent, delay, reduce or inhibit a symptom or biological activity associated with a disease or disorder.
  • Means Administration may consist of a single dose or multiple doses and may be given in combination with other pharmaceutical compositions.
  • kits The present invention provides a kit comprising at least the pharmaceutical composition for the prevention and / or treatment of ALS of the present invention.
  • the kit can also be packaged with syringes, injection needles, pharmaceutically acceptable vehicles, alcohol cotton cloths, bandages, instructions describing the method of use, and the like.
  • the invention relates to a method for the prophylaxis and / or treatment of ALS, comprising the step of administering to a patient a plexin A1 antagonist.
  • the invention relates to plexin A1 antagonists for use in a method of preventing and / or treating ALS.
  • the invention relates to the use of a plexin A1 antagonist in the manufacture of a medicament for the prevention and / or treatment of ALS.
  • the present invention relates to a pharmaceutical composition for suppressing muscle weakness or muscle atrophy comprising a plexin A1 antagonist.
  • muscle weakness or muscle atrophy is muscle weakness or muscle atrophy due to ALS.
  • muscle weakness or muscle atrophy is extremity, muscle weakness or muscle atrophy associated with breathing, speech or swallowing.
  • muscle weakness or muscle atrophy is ALS-related limb weakness, muscle weakness or muscle atrophy related to speech or speech.
  • the present invention relates to a pharmaceutical composition for suppressing degeneration or loss of motor nerves containing a plexin A1 antagonist.
  • degeneration or loss of motor nerves is degeneration or loss of motor nerves by ALS.
  • the present invention is a pharmaceutical composition for use in the prevention and / or treatment of a disease associated with biological activity of semaphorin 3A, which comprises a plexin A1 antagonist.
  • a disease associated with biological activity of semaphorin 3A is ALS.
  • Example 1 Preparation of Semaphorin 3A Protein
  • Human semaphorin 3A protein performs gene synthesis based on the sequence of NP — 006071.1 (SEQ ID NO: 41), and arginine residues (552, 555, 757, 759, 760) of the protease recognition site Converted to alanine.
  • the signal peptide (the 20th alanine from the N terminal) is replaced with the artificial signal peptide HMM + 38 (MWRWRWWLLLLLLLLWPMVWA, SEQ ID NO: 42), and a spacer of glutamate-aspartate-arginine is interposed between the signal peptide and the asparagine 21st.
  • the His tag sequence was inserted.
  • a FLAG tag sequence (DYKDDDDK, SEQ ID NO: 43) was inserted at the C-terminus.
  • the generated amino acid sequence is shown in SEQ ID NO: 44.
  • the prepared gene was incorporated into an expression vector, introduced into Invitrogen's FreeStyle 293 cells for expression, and semaphorin 3A protein was purified from the culture supernatant by affinity purification using HisTrap excel (GE Healthcare) and gel filtration chromatography.
  • the secreted protein mouse semaphorin 3A (mSema 3A) was prepared as follows.
  • the prepared gene was incorporated into a vector for animal cell expression, and was transfected into FreeStyle 293 cells (Invitorgen) using 293Fectin (Invitorgen). At this time, a gene expressing EBNA1 was simultaneously introduced to improve the expression efficiency of the target gene product.
  • the transfected cells were cultured at 37 ° C., 8% CO 2 for 5 days to secrete the desired protein into the culture supernatant.
  • Example 2 Preparation of Anti-Human Mouse Cross Plexin A1 Antibody An anti-human mouse cross Plexin A1 antibody was generated from a naive human antibody library and a synthetic human antibody library by the following method.
  • the gene encoding human plexin A1 (hPlexin A1) (SEQ ID NO: 49) thus prepared was incorporated into an expression vector for animal cells, and was introduced into FreeStyle 293 cells (Invitorgen) using 293 Fectin (Invitorgen). At this time, in order to improve the expression efficiency of the target gene, a gene expressing EBNA1 (SEQ ID NO: 50) was simultaneously introduced.
  • the cells transfected according to the above-mentioned procedure were cultured at 37 ° C., 8% CO 2 for 6 days to secrete the desired protein into the culture supernatant.
  • a cell culture solution containing the target hPlexin A1 was filtered with a 0.22 ⁇ m bottle top filter to obtain a culture supernatant.
  • the fraction containing hPlexin A1 was fractionated by gel filtration chromatography using Superdex 200 (GE Healthcare) equilibrated with D-PBS ( ⁇ ).
  • Biotin-labeled hPlexin A1 was prepared by using EZ-Link NHS-PEG4-Biotin (Thermo SCIENTIFIC) against hPlexin A1 prepared as described above.
  • the linker of the phagemid Fab and the phage pIII protein, and the sequence of the phage display library in which the trypsin cleavage sequence is inserted between the N2 domain and the CT domain of the helper phage pIII protein gene was used.
  • a synthetic human antibody phage display library was constructed using 10 types of heavy chain germline sequences and 7 types of light chain germline sequences by methods known to those skilled in the art.
  • the Germline sequence used is based on the frequency of appearance in the human B cell repertoire and the physicochemical properties of the variable region family as indexes, and VH1-2, VH1-69, VH3-23, VH3-66, VH3-72, VH4-59. , VH4-61, VH4-b, VH5-51, VH6-1, V ⁇ 1-39, V ⁇ 2-28, V ⁇ 3-20, V ⁇ 1-40, V ⁇ 1-44, V ⁇ 2-14, V ⁇ 3-21.
  • the antigen recognition site of the synthetic antibody library was diversified.
  • Phage production was carried out from E. coli carrying the constructed phagemid for phage display.
  • a phage library solution was obtained by adding 2.5 M NaCl / 10% PEG to a culture solution of phage-produced E. coli to dilute the precipitated phage population with TBS.
  • the final concentration of 4% BSA, 1.2 mM calcium ion was prepared by adding BSA and CaCl 2 to the phage library solution.
  • the phage library solution was brought into contact with the antigen for 60 minutes at room temperature by adding 250 pmol of the biotin-labeled hPlexin A1 described above to the prepared phage library solution.
  • BSA-blocked magnetic beads were added, and the complex of antigen and phage was allowed to bind to the magnetic beads for 15 minutes at room temperature.
  • TBS TBS
  • TBS TBS
  • the recovered phage solution was added to 10 mL of E. coli strain ER2738 in the logarithmic growth phase (OD600 of 0.4 to 0.7).
  • the phage was infected with E. coli by stirring the E. coli gently at 37 ° C. for 1 hour.
  • the infected E. coli was plated on a 225 mm x 225 mm plate.
  • the phage library solution was prepared by recovering the phage from the culture solution of the inoculated E. coli and used for the second panning.
  • the culture supernatant containing the skimmed milk and the phage to which CaCl 2 was added was subjected to ELISA in the following procedure. StreptaWell 96 microtiter plates (Roche) were coated overnight with 100 ⁇ L PBS containing biotin-labeled antigen. After removing the antigen by washing each well of the plate with PBST, the well was blocked with 250 ⁇ L of 0.02% skim milk-TBS for 1 hour or more. The plate-displayed plate containing the prepared culture supernatant was allowed to stand at 37 ° C. for 1 hour in each well from which 0.02% skim milk-TBS had been removed, whereby the antibody displayed on the phage was used as an antigen present in each well. It was combined.
  • Phagemid extraction was performed from NucleoBond Xtra Midi Plus (MACHEREY-NAGEL, 74041.50) from E. coli collected at the time of conversion from antibody fragment to full-length antibody, expression and purification at the third panning. Thereafter, the base sequence of the variable region of the antibody was excised by restriction enzyme treatment.
  • the antibody constant region (the constant region of the H chain is human IgG1 (hG1d) (SEQ ID NO: 51), L chain, and the vector carrying the excision antibody variable region partial base sequence to the EF1 promoter and OriP that is the replication origin of EBNA1.
  • the ligation product was used to transform E. coli DH5 ⁇ (TOYOBO, DNA-903), and extraction of a full-length antibody plasmid for animal cell expression was performed from the obtained single colony.
  • the expression of the antibody was performed using the following method. Human fetal kidney cell-derived FreeStyle 293-F strain (Invitrogen) was suspended in FreeStyle 293 Expression Medium (Invitrogen), and 190 ⁇ L each was seeded at a cell density of 5.0 ⁇ 10 4 cells / well in each well of a 96-well plate. Each prepared plasmid was introduced into cells by lipofection. The cells were cultured for 5 days in a CO 2 incubator (37 ° C., 8% CO 2 ) to secrete the antibody into the culture supernatant.
  • a CO 2 incubator 37 ° C., 8% CO 2
  • the secreted antibody in the culture supernatant was filtered using Multi screen HTS GV (Millipore, MSGVN 2250), and the obtained culture supernatant was subjected to cell ELISA.
  • the antibody was expressed in FreeStyle 293-F strain, and the antibody was affinity purified from the culture supernatant with a Protein A column, and used for evaluation of biological activity and evaluation of drug efficacy.
  • Antigen expressing cell lines were constructed in the following manner.
  • the Myc tag SEQ ID NO: 55
  • a plasmid was prepared in which the cDNA constructed so as to express the fused protein was inserted.
  • the prepared plasmid was digested with restriction enzyme PvuI to linearize it, and then introduced into Ba / F3 cells by electroporation using GenePulser X cell (Bio-Rad).
  • the cells were seeded on a 96 well plate by limiting dilution and subjected to selection with G418. After 1 to 2 weeks, visually check whether the selected cells form single colonies in the wells, collect some cells from those forming single colonies, and use Myc tag antibody Western blot analysis and ELISA to determine whether the Myc tag antibody reacts with 4% paraformaldehyde, acetone and methanol immobilized on a 96-well plate and fixed with 4% paraformaldehyde, acetone and methanol, human and mouse Plexin A1 was confirmed to be expressed in Ba / F3 cells.
  • the antibody supernatant obtained above was subjected to cell ELISA in the following procedure. First, a 384 well plate was prepared, and human PlexinA1 and mouse PlexinA1 expressing Ba / F3 cells were captured on the bottom of separate wells. After washing each well of the plate with PBS, the prepared antibody supernatant was added at 20 ⁇ L / well and allowed to stand at room temperature for 1 hour. After that, each well was washed with 1 M Hepes (pH 7.4), HRP-labeled anti-human IgG antibody (Invitrogen, AHI0304) diluted 5000 times with TBS was added at 20 ⁇ L / well each, and left at room temperature for 1 hour.
  • HRP-labeled anti-human IgG antibody Invitrogen, AHI0304
  • Each well of the plate is washed with 1 M Hepes (pH 7.4), 20 ⁇ L / well of substrate solution (ABTS peroxidase substrate system) is dispensed, color is allowed to develop for 1 hour at room temperature, and then SpectraMax by Molecular Device is used. Binding to human PlexinA1 and binding to mouse PlexinA1 were confirmed by measuring absorbance at 405 nm.
  • An antibody having an absorbance value of 0.15 or more for human Plexin A1 expressing Ba / F3 cells and an absorbance value for mouse Plexin A1 expressing Ba / F3 cells was selected and subjected to the in vitro biological activity evaluation assay described later.
  • the antibodies showing the absorbance values shown in “Table 1” are shown as anti-human mouse crossover Plexin A1 antibodies (hPANK # 016; amino acid sequences of H chain CDRs 1 to 3 in SEQ ID NOs: 1 to 3, SEQ ID NOs: 4 to 6)
  • Amino acid sequences of L chain CDRs 1 to 3; amino acid sequences of H chain variable region, amino acid sequences of L chain variable region, SEQ ID NOs: 33 and 34 amino acid sequence of H chain, amino acid sequence of L chain HPANK # 135; amino acid sequences of H chain CDR1 to 3 in SEQ ID NO: 7-9, amino acid sequences of L chain CDR1 to 3 in SEQ ID NO: 10 to 12, amino acid sequences of H chain variable region in SEQ ID NO: 27;
  • the amino acid sequence of the chain variable region, the amino acid sequence of the H chain in SEQ ID NOs: 35 and 36, and the amino acid sequence of the L chain are shown hPANK # 354; the amino acid sequences of the H chain C
  • Example 3 Evaluation system construction of mouse semaphorin 3A biological activity on mouse bone marrow-derived dendritic cells using xCELLigence system
  • an assay using the xCELLigence system was performed. The assay was performed by the following method.
  • Mouse bone marrow-derived dendritic cells have been described in MB Luntz et al. J Immunol Methods. 223 (1): 77-92 (1999) and Kushimoto Hiroki et al. Journal of Japanese Society for Emergency Medicine 27 (4): 557-562 (2007) It prepared according to the method as described in 2.).
  • mice bone marrow-derived dendritic cells are suspended in RPMI medium containing mouse GM-CSF and FBS, and seeded in wells of the plate. Culture overnight at 37 ° C. After culture, mouse semaphorin 3A was diluted to an appropriate concentration with RPMI medium containing FBS and added to the cell culture solution, and the Cell index value of each well was measured with xCELLigence system (ACEA). The cell index value about one hour after addition of mouse semaphorin 3A is shown in FIG. There was a concentration-dependent decrease in Cell index value by mouse semaphorin 3A, confirming mouse semaphorin 3A biological activity against mouse bone marrow-derived dendritic cells.
  • E-plate 96 E-plate 96
  • Example 4 Evaluation system construction of human semaphorin 3A biological activity on human monocyte-derived dendritic cells using xCELLigence system
  • an assay using the xCELLigence system was performed.
  • the assay was performed by the following method.
  • Human monocyte-derived dendritic cells were prepared by the following method. Peripheral blood mononuclear cells were isolated and collected from human heparinized peripheral blood, and only monocyte fraction was obtained using Monocyte Isolation Kit II, human (Mirteni Biotech Co., Ltd.).
  • the monocyte fraction obtained was cultured in RPMI medium containing FBS supplemented with human IL-4 and human GM-CSF (both from R & D systems).
  • AIM-V medium containing human GM-CSF into E-plate 96 (ACEA), which is a plate exclusively for the xCELLigence system
  • human monocyte-derived dendritic cells are resuspended in AIM-V medium containing human GM-CSF.
  • the suspension was seeded in a well of a plate and cultured overnight at 37 ° C.
  • Human semaphorin 3A was diluted with AIM-V medium to an appropriate concentration and added to the cell culture solution, and Cell index value of each well was measured with xCELLigence system (ACEA).
  • the cell index value about one hour after the addition of human semaphorin 3A is shown in FIG. There was a concentration-dependent decrease in Cell index value due to human semaphorin 3A, confirming the human semaphorin 3A biological activity against human monocyte-derived dendritic cells.
  • Example 5 Evaluation of Antagonist Activity of Anti-Human Mouse Cross Plexin A1 Antibody
  • the biological activity of the anti-human mouse crossover Plexin A1 antibody prepared in Example 2 was evaluated using the human semaphorin 3A activity measurement system by the xCELLigence system described in Example 4.
  • Human monocyte-derived dendritic cells were seeded on E-plate 96 (ACEA), a plate exclusively for xCELLigence system, and cultured overnight at 37 ° C.
  • Anti-human mouse crossover Plexin A1 antibody and anti-Keyhole limpet hemocyanin (KLH) antibody as a control were diluted to appropriate concentrations in AIM-V medium and added to the wells.
  • KLH Keyhole limpet hemocyanin
  • Example 6 Evaluation of Antagonist Activity of Anti-Human Mouse Cross Plexin A1 Antibody
  • Anti-human mouse crossover PlexinA1 antibody hPASK # 188 H chain constant region is mouse IgG2a variant (mIgG2a silent) (SEQ ID NO: 2) prepared in Example 2 using the mouse Semaphorin 3A activity measurement system according to the xCELLigence system described in Example 3. : 56), L chain constant region evaluated the antagonist activity of mouse IgIg (mk1) (SEQ ID NO: 57).
  • Mouse bone marrow-derived dendritic cells were suspended in RPMI medium containing mouse GM-CSF and FBS, seeded on E-plate 96 (ACEA), which is a plate exclusively for xCELLigence system, and cultured overnight at 37 ° C.
  • Anti-human mouse cross Plexin A1 antibody was diluted to the appropriate concentration in RPMI medium containing FBS and added to the wells. Thereafter, mouse semaphorin 3A diluted to an appropriate concentration with FBS-containing RPMI medium was added to each well, and the cell index value of each well was measured.
  • the cell index value of wells added with mouse Semaphorin 3A is 0%, and the Cell index value of non-added wells is 100%, the value of anti-human mouse crossed Plexin A1 antibody and mouse Semaphorin 3A added wells (%) was calculated as antagonist activity (Percent inhibition (%)).
  • the results are shown in FIG.
  • the obtained anti-human mouse crossover Plexin A1 antibody inhibited the decrease of Cell index by mouse semaphorin 3A, and it was confirmed that there is an antagonist activity to mouse semaphorin 3A.
  • Transgenic mice B6SJL-TgN (SOD1-G93A) 1 Gurd / J, hereinafter referred to as mSOD1 mice
  • mSOD1 mice B6SJL-TgN (SOD1-G93A) 1 Gurd / J
  • mSOD1 mice overexpressing human SOD1 G93A mutant purchased from Jackson Laboratories were bred in an experimental animal storage facility at Osaka University. The colonies were maintained.
  • mSOD1 mice female mice that had been backcrossed for at least 10 generations with wild-type mice (C57BL / 6) were used.
  • the genotype of the transgenic mice was determined using polymerase chain reaction.
  • ex4Pla 5'-CATCAGCCCTAATCCATCTGA-3 '(SEQ ID NO: 58) and ex4P2a 5'-TGGACTCTTAGAATTCGCGAC-3' SEQ ID NO: 59
  • reaction conditions 94 ° C. for 30 seconds, 60 ° C. for 30 seconds, and 72 ° C. for 1 minute.
  • the mice were observed daily, and once a week, the time to drop of the mice was measured by the rotarod test.
  • the rotarod test is a test in which a mouse is placed on a rotating rotor and the time until it does not stay on the rotor and falls is reflected, and the time spent on the rotor reflects the muscle strength.
  • Anti-Plexin A1 antagonist antibody hPASK # 188 H chain constant region is mouse IgG2a variant (mIgG2a silent) (SEQ ID NO: 56), L chain constant region is mouse Ig ⁇ (mk1) (SEQ ID NO: 57) in ALS model mice
  • Fig. 5 Survival time of mice treated with anti-PlexinA1 antagonist antibody hPASK # 188 and control antibody in ALS model mice (11 mice in hPASK # 188 administration group, 6 mice in anti-KLH antibody administration group), rotarod test (hPASK # The time to drop of the mice was shown in 8 groups administered in 188 and 6 in group administered with anti-KLH antibody. This result indicates that the anti-Plexin A1 antagonist antibody hPASK # 188 showed a significant prolongation of the survival time as compared to the control antibody, and further that the rotarod test extended the time until the mouse fell.

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Abstract

L'invention concerne une composition médicinale pour la prévention et/ou le traitement de la sclérose latérale amyotrophique (SLA), ladite composition contenant un antagoniste de plexine A1.
PCT/JP2017/036585 2017-10-10 2017-10-10 Composition médicinale contenant un antagoniste de plexine a1 pour la prévention et/ou le traitement de la sclérose latérale amyotrophique WO2019073507A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007308465A (ja) * 2006-05-15 2007-11-29 Boehringer Ingelheim Internatl Gmbh 炎症性疾患、自己免疫疾患又は骨吸収異常の治療方法
WO2017002919A1 (fr) * 2015-06-30 2017-01-05 国立大学法人大阪大学 Anticorps agoniste d'antiplexine a1

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007308465A (ja) * 2006-05-15 2007-11-29 Boehringer Ingelheim Internatl Gmbh 炎症性疾患、自己免疫疾患又は骨吸収異常の治療方法
WO2017002919A1 (fr) * 2015-06-30 2017-01-05 国立大学法人大阪大学 Anticorps agoniste d'antiplexine a1

Non-Patent Citations (3)

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Title
ANTIPENKO A. ET AL.: "Structure of the Semaphorin- 3A Receptor Binding Module", NEURON, vol. 39, no. 4, 2003, pages 589 - 598, XP055302516 *
KORNER S. ET AL.: "The Axon Guidance Protein Semaphorin 3A is Increased in the Motor Cortex of Patients With Amyotrophic Lateral Sclerosis", J NEUROPATHOL EXP NEUROL., vol. 75, no. 4, 2016, pages 326 - 333, XP055593487, ISSN: 0022-3069, DOI: 10.1093/jnen/nlw003 *
PASTERKAMP R. J. ET AL.: "Emerging roles for semaphorins in neural regeneration", BRAIN RES BRAIN RES REV., vol. 35, no. 1, 2001, pages 36 - 54, XP055593489, ISSN: 0165-0173, DOI: 10.1016/S0165-0173(00)00050-3 *

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