CN107840885B - GM-CSF antibodies and uses thereof - Google Patents

GM-CSF antibodies and uses thereof Download PDF

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CN107840885B
CN107840885B CN201610831525.9A CN201610831525A CN107840885B CN 107840885 B CN107840885 B CN 107840885B CN 201610831525 A CN201610831525 A CN 201610831525A CN 107840885 B CN107840885 B CN 107840885B
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antibody
csf
antigen
binding portion
seq
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CN107840885A (en
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王正毅
方磊
郭炳诗
臧敬五
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Tianjing Biotechnology Hangzhou Co ltd
Tianjing Biotechnology Hong Kong Ltd
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Tianjing Biotechnology Shanghai Co ltd
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Priority to BR112019005274A priority patent/BR112019005274A2/en
Priority to MYPI2019001075A priority patent/MY190021A/en
Priority to NZ750976A priority patent/NZ750976A/en
Priority to DK17840546.0T priority patent/DK3328887T3/en
Priority to AU2017328383A priority patent/AU2017328383B2/en
Priority to PE2019000664A priority patent/PE20190633A1/en
Priority to PT178405460T priority patent/PT3328887T/en
Priority to IL265331A priority patent/IL265331B2/en
Priority to MX2019003019A priority patent/MX2019003019A/en
Priority to EP17840546.0A priority patent/EP3328887B1/en
Priority to KR1020217000790A priority patent/KR102356187B1/en
Priority to KR1020187009572A priority patent/KR101953706B1/en
Priority to ES17840546T priority patent/ES2887343T3/en
Priority to PCT/CN2017/102057 priority patent/WO2018050111A1/en
Priority to PL17840546T priority patent/PL3328887T3/en
Priority to UAA201901976A priority patent/UA126565C2/en
Priority to JP2019511372A priority patent/JP7256116B2/en
Priority to CN201780003554.9A priority patent/CN109219616B/en
Priority to HUE17840546A priority patent/HUE056238T2/en
Priority to US15/744,734 priority patent/US10647767B2/en
Priority to SG11201901789WA priority patent/SG11201901789WA/en
Priority to CA3036509A priority patent/CA3036509C/en
Priority to KR1020197005546A priority patent/KR20190035790A/en
Publication of CN107840885A publication Critical patent/CN107840885A/en
Priority to US16/100,063 priority patent/US10889641B2/en
Priority to PH12019500434A priority patent/PH12019500434A1/en
Priority to CL2019000702A priority patent/CL2019000702A1/en
Priority to CONC2019/0002550A priority patent/CO2019002550A2/en
Priority to ZA2019/01835A priority patent/ZA201901835B/en
Priority to ZA2020/05792A priority patent/ZA202005792B/en
Publication of CN107840885B publication Critical patent/CN107840885B/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/243—Colony Stimulating Factors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/53—Colony-stimulating factor [CSF]
    • G01N2333/535—Granulocyte CSF; Granulocyte-macrophage CSF
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/06—Gastro-intestinal diseases
    • G01N2800/065—Bowel diseases, e.g. Crohn, ulcerative colitis, IBS
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/10—Musculoskeletal or connective tissue disorders
    • G01N2800/101—Diffuse connective tissue disease, e.g. Sjögren, Wegener's granulomatosis
    • G01N2800/102—Arthritis; Rheumatoid arthritis, i.e. inflammation of peripheral joints
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/10—Musculoskeletal or connective tissue disorders
    • G01N2800/101—Diffuse connective tissue disease, e.g. Sjögren, Wegener's granulomatosis
    • G01N2800/104—Lupus erythematosus [SLE]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/10—Musculoskeletal or connective tissue disorders
    • G01N2800/105—Osteoarthritis, e.g. cartilage alteration, hypertrophy of bone
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/12—Pulmonary diseases
    • G01N2800/122—Chronic or obstructive airway disorders, e.g. asthma COPD
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/16—Ophthalmology
    • G01N2800/164—Retinal disorders, e.g. retinopathy
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/20—Dermatological disorders
    • G01N2800/205—Scaling palpular diseases, e.g. psoriasis, pytiriasis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/24—Immunology or allergic disorders
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G01N2800/323—Arteriosclerosis, Stenosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/34—Genitourinary disorders
    • G01N2800/347—Renal failures; Glomerular diseases; Tubulointerstitial diseases, e.g. nephritic syndrome, glomerulonephritis; Renovascular diseases, e.g. renal artery occlusion, nephropathy

Abstract

The present invention discloses an isolated monoclonal antibody, or antigen binding portion thereof, that binds to human GM-CSF protein. The invention also discloses the application of the monoclonal antibody or the antigen binding part thereof in preparing a medicament for treating GM-CSF related diseases. The GM-CSF antibody of the invention can efficiently and specifically combine with human GM-CSF molecules, can treat autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, psoriasis and the like, and has good application prospect.

Description

GM-CSF antibodies and uses thereof
Technical Field
The invention relates to the technical field of genetic engineering antibodies, in particular to an anti-GM-CSF (granulocyte-macrophage colony stimulating factor) antibody and application thereof.
Background
Granulocyte-macrophage colony stimulating factor (GM-CSF) is a hematopoietic cell growth factor with pleiotropic effects, and has regulating and controlling effects on the development and maturation stages of Granulocyte lines and macrophages. In recent years, studies have shown that GM-CSF plays an important role in inflammatory responses and autoimmune diseases. GM-CSF can make macrophage differentiate into inflammatory macrophage under the stimulation of lipopolysaccharide, and the inflammatory macrophage can secrete a series of inflammatory factors of TNF-alpha, IL-6, IL-12p70 and IL-23; GM-CSF also has a key role in the differentiation of monocytes in synovial tissue into inflammatory dendritic cells, and is involved in the induction and maintenance of the development and progression of acute arthritis. During the inflammatory reaction, activated T cells secrete a large amount of GM-CSF factors, and since the T cells lack GM-CSF receptors, and antigen presenting cells such as macrophages and dendritic cells have the receptors on the surfaces, the GM-CSF derived from the T cells can stimulate the antigen presenting cells to secrete IL-1, IL-23 and the like to mediate inflammation, and the IL-1 and IL-23 promote the T cells to further secrete GM-CSF, so that a positive feedback is formed. The injection of GM-CSF in CIA, a mouse arthritis model, can lead to worsening of arthritis, whereas GM-CSF deficient mice are relatively less symptomatic than wild-type mice, suggesting that GM-CSF plays an important role in arthritis conditions. In contrast, in a CIA mouse model, the disease symptoms were significantly improved, the level of synovial inflammation and articular cartilage damage was reduced using GM-CSF monoclonal antibody to neutralize GM-CSF. Serum and synovial membrane tests of rheumatoid arthritis patients show that the level of GM-CSF is remarkably increased and is closely related to disease activities, mainly reflected in that bone erosion is worsened, and synovial lining cells and the lower layer thereof infiltrate a large number of macrophages.
In recent years, biomacromolecule antibody drug therapy has become a new trend for treating autoimmune diseases such as rheumatoid arthritis, psoriasis and the like, wherein various inflammatory cytokines are the first-choice targets of targeted antibody drug therapy, such as tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1), interleukin-6 (IL-6) and the like, and are also the hot spots of current research. The antibody medicine can be combined with other second-line medicines, and has the advantages of no generation of systemic immunosuppression and less adverse reaction. At present, such new drugs are on the market: IL-1 antibody (Anakinra), TNF-alpha antibody (Adalilimumab), TNF-alpha receptor fusion protein (Etanercept), and the like. However, such cytokine antibody drugs that have been marketed have limitations in therapeutic effects, and a considerable number of patients with rheumatoid arthritis are insensitive to such antibody drugs and are not responsive.
At present, there are many large pharmaceutical companies developing therapeutic monoclonal antibody drugs against GM-CSF in foreign countries, MOR-103 developed by GSK/Morphosys in combination, Namilumab developed by Takeda, and MORAB022 developed by Morphotek. These companies have entered into clinical trials with the above products, in which MOR-103 has completed clinical phase Ib and II trials for both rheumatoid arthritis and multiple sclerosis indications. However, since the import of macromolecular drugs requires high cost for patients, it is a problem to be solved urgently to develop a new monoclonal antibody against GM-CSF, which can reduce the burden on patients and the cost of treatment.
Disclosure of Invention
The invention aims to solve the technical problem of lack of GM-CSF antibody with low price and good effect clinically at present, and provides a novel GM-CSF antibody, which can be used for antibody drug treatment of inflammatory diseases and autoimmune diseases, and can also reduce the treatment cost burden of patients.
In order to solve the technical problems, the invention is realized by the following technical scheme:
in one aspect of the invention, there is provided an isolated monoclonal antibody or antigen binding portion thereof that binds human GM-CSF protein, the antibody or antigen binding portion thereof comprising heavy chain variable region CDR1, CDR2 and CDR3 and light chain variable region CDR1, CDR2 and CDR 3; wherein, the nucleotide sequence of the heavy chain variable region CDR1 is shown in SEQ ID NO.1, the nucleotide sequence of the CDR2 is shown in SEQ ID NO.2, and the nucleotide sequence of the CDR3 is shown in SEQ ID NO. 3; the nucleotide sequence of the light chain variable region CDR1 is shown in SEQ ID NO.4, the nucleotide sequence of the CDR2 is shown in SEQ ID NO.5, and the nucleotide sequence of the CDR3 is shown in SEQ ID NO. 6.
In the present invention, the term "antibody" should be construed to encompass any specific binding member having a binding domain with the desired specificity. Thus, this term encompasses antibody fragments, derivatives, and functional equivalents and homologues of antibodies homologous thereto, and also encompasses any polypeptide, whether natural or synthetically produced, that comprises an antigen-binding domain. Examples of antibodies are immunoglobulin subtypes (e.g., IgG, IgE, IgM, IgD and IgA) and subclasses thereof; it may also be a fragment comprising an antigen binding domain such as Fab, scFv, Fv, dAb or Fd, or diabodies (diabodies). Chimeric molecules comprising an antigen binding domain fused to another polypeptide or an equivalent are also included. Cloning and expression of chimeric antibodies is described in EP.A-0120694 and EP.A-0125023.
The monoclonal antibodies of the invention may be monovalent or single chain antibodies, diabodies, chimeric antibodies, as well as derivatives, functional equivalents and homologues of the above-mentioned antibodies, including antibody fragments and any polypeptides comprising an antigen binding domain.
Antibodies can be modified in a number of ways and recombinant DNA techniques can be used to produce other antibodies or chimeric molecules that retain the specificity of the original antibody. Such techniques may involve introducing DNA encoding the immunoglobulin variable regions or Complementarity Determining Regions (CDRs) of an antibody into the constant regions or constant region plus framework regions of different immunoglobulins. Genetic mutations or other changes may also be made to the hybridoma cells or other cells that produce the antibody, which may or may not alter the binding specificity of the produced antibody.
The monoclonal antibodies of the invention are framework regions in addition to the hypervariable regions CDR1, CDR2 and CDR3 and the linking sequences in the heavy and light chains. The framework regions can be replaced by other sequences under conditions where the three-dimensional structure required for binding is not affected, and the molecular basis for antibody specificity arises primarily from its highly variable regions CDR1, CDR2, and CDR3, which are key sites for antigen binding. To maintain preferred binding properties, the sequence of the CDRs should be preserved as much as possible, however, some amino acid changes may be required to optimize binding properties.
In one embodiment, the murine monoclonal antibody comprises the heavy chain variable region sequence shown in SEQ ID No.7 and the light chain variable region sequence shown in SEQ ID No. 18. Preferably, it is also subject to humanization.
In another embodiment, the monoclonal antibody comprises the heavy chain variable region sequence shown in SEQ ID No.8, or the light chain variable region sequence shown in SEQ ID No. 19.
In the humanization process of the monoclonal antibody, in order to maintain the binding activity of the antibody and GM-CSF, key amino acid sites in a framework region are reserved (namely, reversion sites), and the amino acids have important functions on the activity of the antibody after humanization.
Preferably, the heavy chain variable region further comprises any one or a combination of any two or more of the following back-mutation sites: 98R, 72S, 68, 70L, 48I, 26D, 29L.
The light chain variable region further comprises any one or a combination of any two or more of the following back-mutation sites: 1E, 46A, 60D, 70D, 43S, 87F.
Preferably, the amino terminus of the CDR1 sequence of the heavy chain variable region is linked to DYTLT (SEQ ID NO.38) or GYTFT (SEQ ID NO. 39).
Antibodies possessing any of these substitution positions and antibodies possessing all substitution positions also have binding activity to GM-CSF. Amino acid substitutions may be present in both the framework and CDR regions, or may be present in the framework or CDR regions separately.
Preferably, the antibody or antigen-binding portion thereof of the present invention comprises a heavy chain variable region sequence as set forth in any one of SEQ ID No.9 to SEQ ID No.17 and a light chain variable region sequence as set forth in any one of SEQ ID No.20 to SEQ ID No. 22.
In one embodiment of the invention, the antibody may comprise a) a heavy chain variable region having an amino acid sequence at least 90% identical to the sequence shown in SEQ ID No. 14; more preferably 95% identity and b) a light chain variable region having an amino acid sequence at least 90% identical to the sequence set forth in SEQ ID NO. 22; more preferably 95% identity. Thus, the antibody of interest may comprise a) a heavy chain variable region having an amino acid sequence at least about 90% identical to the sequence set forth in SEQ ID No. 14; more preferably 95%, 96%, 97%, 98%, 99% or 100% identity and b) a light chain variable region having an amino acid sequence at least about 90% identical to the sequence set forth in SEQ ID NO. 22; more preferably 95%, 96%, 97%, 98%, 99% or 100% identity. Preferably, the antibody comprises a) a heavy chain variable region having an amino acid sequence identical to the sequence set forth in SEQ ID No.14 and b) a light chain variable region having an amino acid sequence identical to the sequence set forth in SEQ ID No. 22.
In another embodiment of the invention, the antibody may comprise a) a heavy chain having an amino acid sequence at least 90% identical to the sequence shown in SEQ ID NO. 17; more preferably 95% identity and b) a light chain having an amino acid sequence at least 90% identical to the sequence as set forth in SEQ ID NO. 19; more preferably 95% identity. Thus, the antibody of interest may comprise a) a heavy chain having an amino acid sequence at least about 90% identical to the sequence shown in SEQ ID NO. 17; more preferably 95%, 96%, 97%, 98%, 99% or 100% identity and b) a light chain having an amino acid sequence at least about 90% identical to the sequence shown in SEQ ID NO. 19; more preferably 95%, 96%, 97%, 98%, 99% or 100% identity. Preferably, the antibody comprises a) a heavy chain variable region having an amino acid sequence identical to the sequence set forth in SEQ ID No.17 and b) a light chain variable region having an amino acid sequence identical to the sequence set forth in SEQ ID No. 19.
In addition to the amino acid substitutions described above, the antibody of interest may have additional amino acids at either end of the heavy or light chain. For example, the antibody of interest may comprise at least 1, 2,3, 4, 5 or 6 or more additional amino acids at the C-or N-terminus of the heavy and/or light chain, respectively. In certain embodiments, the antibody of interest may be shorter than the exemplary amino acids described herein, with the primary difference being 1, 2,3, 4, 5, or 6 amino acids less than the exemplary amino acids at either end of the heavy and light chains, respectively.
In another aspect of the invention, there is also provided an immunoconjugate comprising said monoclonal antibody, or antigen-binding portion thereof; and a functional molecule attached thereto; the functional molecule is selected from: a second antibody or antigen-binding portion thereof, a cytokine or other protein, a detectable label, a toxin (e.g., a tumor-inhibiting toxin). Preferably, the detectable label comprises: fluorescent markers, chromogenic markers.
Such detectable labels include, but are not limited to: fluorescent markers, chromogenic markers; such as: enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals, and nonradioactive paramagnetic metal ions. More than one label may also be included. The label used to label the antibody for detection and/or analysis and/or diagnostic purposes depends on the particular detection/analysis/diagnostic technique and/or method used, e.g., immunohistochemical staining of (tissue) samples, flow cytometry, etc. Suitable labels are well known to those skilled in the art for detection/analysis/diagnostic techniques and/or methods known in the art.
Bispecific antibodies possess two specific antigen binding sites and can interact with both target cells and functional cells (typically T cells) to enhance killing of the target cells.
In another aspect of the present invention, there is also provided a pharmaceutical composition comprising the monoclonal antibody or antigen-binding portion thereof described above, and a pharmaceutically acceptable carrier or diluent. By "pharmaceutically acceptable" is meant that the molecular entities and compositions do not produce adverse, allergic, or other untoward reactions when properly administered to an animal or human. Specific examples of some substances that may serve as pharmaceutically acceptable carriers or components thereof are sugars, such as lactose, glucose and sucrose; also for example, starch, cellulose and its derivatives, polyols, alginic acid, emulsifiers, colorants, flavors, stabilizers, antioxidants, preservatives, isotonic saline solutions, phosphate buffers, and the like. The composition of the present invention can be prepared into various dosage forms according to requirements.
In another aspect of the invention, there is also provided an isolated nucleic acid encoding the heavy or light chain variable region of the monoclonal antibody or antigen binding portion thereof described above.
In another aspect of the invention, there is also provided a recombinant expression vector comprising the above nucleic acid.
In another aspect of the invention, a host cell comprising the recombinant expression vector is also provided.
The monoclonal antibody of the invention can be prepared by a hybridoma method or a genetic engineering antibody method. The DNA sequence encoding the humanized antibody of the present invention can be artificially synthesized based on the amino acid sequence disclosed in the present invention or amplified by PCR to obtain the DNA sequence of the humanized antibody, and then the sequence is ligated into an appropriate expression vector.
The monoclonal antibody of the invention can also be prepared by a stable cell strain method, such as artificially synthesizing plasmid transfected cells according to the amino acid sequence disclosed by the invention, and screening to obtain a cell strain of a stably expressed antibody.
Once the antibody molecule of the invention is prepared, it can be purified by any method known in the art for purifying immunoglobulin molecules, for example, by chromatography (e.g., ion exchange chromatography, affinity chromatography, particularly by affinity chromatography of protein a for a specific antigen and other column chromatography), centrifugation, use of solubility differences, or by any other standard technique for purifying proteins. In many embodiments, the antibody is secreted from the cell into the culture medium, and the antibody is obtained by collecting the culture medium and purifying it.
In another aspect of the present invention, there is also provided a use of the above monoclonal antibody or an antigen binding portion thereof for the preparation of a medicament for the treatment of a GM-CSF related disease.
The GM-CSF related diseases include inflammatory diseases and tumor diseases. Among the inflammatory diseases are: autoimmune diseases such as rheumatoid arthritis, asthma, multiple sclerosis, chronic obstructive pulmonary disease, acute respiratory distress syndrome, idiopathic pulmonary fibrosis, inflammatory bowel disease, Crohn's disease, uveitis, macular Degeneration, colitis, psoriasis, Wallerian Degeneration, antiphospholipid syndrome, acute coronary syndrome, vascular restenosis, arteriosclerosis, recurrent polychondritis, acute or chronic hepatitis, glomerulonephritis, and lupus. The tumor diseases include various cancers, such as leukemia, multiple myeloma, gastric cancer, skin cancer, etc.
The antibody can also be used for scientific research related to GM-CSF, such as scientific research in multiple fields of developmental biology, cell biology, metabolism, structural biology, functional genomics and the like, or medical and pharmaceutical application research of tumors, systemic autoimmune diseases and the like.
In another aspect of the present invention, there is also provided a use of the monoclonal antibody or an antigen-binding portion thereof for the preparation of a medicament for diagnosing a GM-CSF related disease.
In another aspect of the present invention, there is also provided a kit for treating a GM-CSF-related disease, comprising said monoclonal antibody, or antigen binding portion thereof; or a pharmaceutical composition comprising the same.
In another aspect of the present invention, there is also provided a detection chip comprising the monoclonal antibody or the antigen-binding portion thereof described above.
In another aspect of the present invention, there is also provided a kit for detecting GM-CSF factor, comprising the monoclonal antibody or antigen-binding portion thereof described above; or the detection chip is included therein.
In another aspect of the present invention, there is also provided a method for preventing, alleviating or treating a disease associated with the GM-CSF factor GM-CSF, the method comprising: administering to a subject in need of prevention, amelioration, or treatment an effective amount of the monoclonal antibody, or antigen binding portion thereof; or the pharmaceutical composition. The dosage to be administered to a patient may be determined by the clinician to be beneficial depending on such factors as the type, age, weight and general condition of the patient, the mode of administration, and the like. Administration may be by injection or other therapeutic means, for example.
In another aspect of the present invention, there is also provided a method of detecting GM-CSF factor, the method comprising: and (3) coupling the monoclonal antibody or the antigen binding part thereof with a detectable marker, and detecting a sample to be detected so as to determine whether the GM-CSF factor exists or not or the quantity exists in the sample to be detected. The method may be a diagnostic method for a disease, or a non-diagnostic method.
The GM-CSF antibody of the invention can efficiently and specifically bind to human GM-CSF factors, can treat inflammatory diseases such as rheumatoid arthritis, multiple sclerosis and the like and tumor diseases by effectively neutralizing GM-CSF, and has good application prospects in the aspects of treatment, diagnosis and detection of GM-CSF related diseases.
Drawings
The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.
FIG. 1 is a graph showing the results of the indirect ELISA screening by subclones according to example 1 of the present invention;
FIG. 2 is a graph showing the results of screening TF-1 cell proliferation response induced by inhibition of GM-CSF in example 1 of the present invention;
FIG. 3 is a graph showing the results of the binding activity of the 23F4 antibody to human GM-CSF antigen in example 2 of the present invention;
FIG. 4 is a graph showing the results of the binding activity of the 23F4 antibody of example 3 of the present invention to a cynomolgus monkey GM-CSF antigen;
FIG. 5 is a graph showing the results of the 23F4 antibody competing for the binding of human GM-CSF to the receptor GMCSFR-alpha according to example 4 of the present invention;
FIG. 6 is a graph showing the results of the intracellular signaling pathway response induced by blocking GM-CSF by the 23F4 antibody according to example 5 of the present invention;
FIG. 7 is a graph showing the results of inhibition of GM-CSF-induced TF-1 proliferation response by the 23F4 antibody of example 6 of the present invention;
FIG. 8 is a graph showing the results of the affinity assay for the 23F4 antibody in example 7 of the present invention;
FIG. 9 is a graph showing the results of an experiment for the antigen-binding activity of the humanized antibody of example 9 of the present invention; units of EC50 values in the graph: ng/ml.
FIG. 10 is a graph showing the results of the GM-CSF-induced TF-1 proliferation response inhibited by the humanized antibody of example 11 of the present invention;
FIG. 11 is a graph showing the results of the intracellular signaling pathway response induced by blocking GM-CSF by the humanized antibody of example 12 of the present invention;
FIG. 12 is a graph showing the results of the binding activity of the humanized antibody of example 13 of the present invention to a cynomolgus GM-CSF antigen.
Detailed Description
The invention utilizes the monoclonal antibody preparation technology to screen and obtain a mouse anti-human GM-CSF monoclonal antibody which can efficiently combine with human GM-CSF and is named as 23F4 antibody. Next, 3 Complementarity Determining Regions (CDRs) CDR1, CDR2 and CDR3 sequences in the VL of the 23F4 antibody were grafted onto framework region sequences (framework sequences) of the best-matching human immunoglobulin germline gene L8/Jk4 by humanization design experiments. Meanwhile, the CDR1, CDR2 and CDR3 sequences of the 23F4 antibody VH were grafted onto the framework region sequences of the best matching human immunoglobulin germline gene VH1-F/JH 6. Then, the humanized VH and VL genes were cloned into expression vectors containing human heavy chain IgG1 and light chain kappa chain constant regions, respectively, and recombinant expression was performed to obtain tens of humanized GM-CSF antibodies. Through a series of experimental verification of antigen binding activity detection, affinity determination, inhibition of GM-CSF-induced TF-1 proliferation reaction, blocking of GM-CSF-induced intracellular signal pathway reaction and the like, the humanized GM-CSF antibody disclosed by the invention can be efficiently and specifically bound with human GM-CSF molecules, is suitable for treating inflammatory diseases such as rheumatoid arthritis, multiple sclerosis and the like, is also suitable for treating various tumor diseases, and has a very good market application prospect.
EXAMPLE 1 Generation and screening of mouse monoclonal antibodies against human GM-CSF
Antigen: recombinant human GM-CSF protein (Genscript)
The immunization method comprises the following steps: C57/BL6 mice were bled from the orbit 4 days before immunization at 6-8 weeks, and 15-30ul serum was obtained and stored at-20 ℃ as a pre-immunization serum sample. 20ug of recombinant human GM-CSF protein was injected subcutaneously into C57/BL6 mice. 5ug of recombinant human GM-CSF protein was boosted by intraperitoneal injection 14 days after the primary immunization. Immunization was further boosted 28 days post-priming with a further subcutaneous injection of 5ug of recombinant human GM-CSF protein. A third booster immunization was performed 42 days after priming with intraperitoneal injection of 5ug recombinant human GM-CSF protein. One week after the third booster immunization, 15-30ul of orbital blood was collected and stored at-20 ℃ for testing serum titer. Mice with higher titer were selected on day 60 after priming and were injected intraperitoneally with 25ug of recombinant human GM-CSF protein for final immunization. Cell fusion was performed with SP20 cells after 3 days, in a quantity ratio of 10: 1 to 5: 1, the number of splenocytes laid in each hole is not more than 1 × 105A cell. The medium was changed 7 days after the fusion to supplement nutrients and reduce the detection background. Screening positive hybridoma cells by an indirect ELISA method and a reaction for inhibiting the proliferation function of the GM-CSF-induced TF-1 cell line, carrying out amplification culture on the selected hybridoma cells, carrying out subcloning for 3 times, carrying out ELISA and inhibition function screening on each subcloning to identify positive monoclonal antibody, and finally obtaining an optimal mouse anti-human GM-CSF monoclonal antibody 23F 4.
The subclone indirect ELISA screening detection method comprises the following steps:
1. the recombinant human GM-CSF protein coated ELISA plate with 1ug/ml coating amount per well at 4 deg.C overnight.
2. Washed 3 times with PBST containing 0.5% tween-20, 300 ul/well.
3. Blocking was performed for 1 hour by adding PBS containing 1% BSA.
4. Washed 3 times with PBST containing 0.5% tween-20, 300 ul/well.
5. Cell culture supernatant was added at 100ul per well and incubated at 37 ℃ for 1 hour.
6. Washed 3 times with PBST containing 0.5% tween-20, 300 ul/well.
7. Adding goat anti-mouse-HRP, diluting by 10000, and incubating at 37 deg.C for 1 hr.
8. Washed 3 times with PBST containing 0.5% tween-20, 300 ul/well.
9. Adding 100ul of TMB developing solution into each well, and adding 2% H after 10min2SO4And stopping the solution.
10. OD450 values were measured by a microplate reader.
Subclone ELISA screening results: as shown in fig. 1, 23a12, 23F4, 26E2, 28G2, 31a10, 32C4, 32C6, 33C3, 34C11, 34H6, 35B5, 35C12, 35E10, 36E6, 38a4, 39F7, 40B7, 43E6, 43G6, 43G11, 44F9, 45F12, 46B3, 49B3, 50C5 showed strong binding activity.
The screening method for inhibiting GM-CSF from inducing TF-1 cell proliferation reaction includes:
1. cell culture of human erythroleukemia cell line TF-1 GM-CSF 2ng/mL was added in RPMI1640+ 10% serum + 1% double antibody medium (standard medium, CM) at 37 deg.C and 5% CO2Cultured in an incubator. And (4) once passage is carried out for 2-3 days, and cells which are in a good cell state and are in a logarithmic growth phase are taken for inoculation.
2. Starvation of cells in the first day, about 3 pm, cells in good growth state were starved. The cells were centrifuged to remove CM, washed once with RPMI1640+ 1% double antibody medium (basal medium, BCM) without serum and GM-CSF, and then resuspended in BCM at 37 deg.C and 5% CO2Was cultured overnight in an incubator.
3. Cell inoculation, about 10 am the next day after cell starvation, the starved cells were centrifuged, the medium removed, and resuspended to 1.67X 10 using CM5One cell/mL, at 90. mu.L/well, and 1.5 ten thousand cells/well in 96-well plates. Or resuspended to 3X 10 with CM5One cell/mL, 50. mu.L/well, and 1.5 ten thousand cells/well in a 96-well plate.
GM-CSF was formulated using 0.05ng/mL as the final concentration, based on previous results. GM-CSF was formulated at a concentration of 0.2ng/mL (4X), or at a concentration of 1ng/mL (20X), using CM as a diluent.
5. Reference Ab preparation-based on the previous results, the final concentration of reference Ab (R & D MAB215) was chosen to correspond to IC80, and 0.2. mu.g/mL. Positive antibody stock solutions (100. mu.g/mL) were prepared as working solutions (4X) at a concentration of 0.8. mu.g/mL using CM containing 4 XHCM as a diluent. Or, a positive antibody stock solution (100. mu.g/mL) was prepared as a working solution (20X) at a concentration of 4. mu.g/mL using 100% HCM as a diluent.
6. And (4) preparing a sample, wherein the final concentration of the culture supernatant of the hybridoma cells is 5%.
7. Pre-incubation, mixing the positive antibody prepared in the step 4) and the positive antibody prepared in the step 5), 6) and 7) respectively according to the volume ratio of 1:1, and incubating for 30min at 37 ℃.
8. Adding the sample, namely adding the mixed solution prepared in the step 8) into each hole of the 96-hole plate prepared in the step 3), putting the culture plate into an incubator, and continuing to culture for 72 hours. The control group of cells without GM-CSF and the control group of cells plus GM-CSF were also set.
9. Cell viability assay in accordance
Figure BDA0001116520550000091
Preparing a detection solution according to the Luminecent Cell visual Assay specification, adding 100 mu L of the detection solution into the detection solution per hole, slightly shaking and uniformly mixing the detection solution, and detecting the detection solution on a microplate reader after 10 minutes.
As shown in FIG. 2, the three clones, 23F4,32C4 and 50C5, were able to significantly inhibit GM-CSF from inducing the TF-1 cell proliferation response, with 23F4 being the most desirable.
Example 223F 4 antibody binding Activity with human GM-CSF antigen
Human GM-CSF protein coated at 1ug/ml was applied to an Elisa plate overnight at 4 ℃. The next day after PBST washing 2 times, add 1% BSA blocking solution, 37 degrees C were incubated for 1 hours. After 2 washes, a gradient of 23F4 antibody, starting at 1ug/ml, 3-fold dilutions, 10 concentrations total, was added and incubated for 1 hour at 37 ℃. After washing 3 times, goat anti mouse-HRP was added at a dilution of 10000 and incubated at 37 ℃ for 1 hour. After 3 washes, TMB was developed for 10min and 2% H was used2SO4And stopping the solution. After the microplate reader was preheated for 15min, the OD450 readings were taken and the data were fitted. As a result, as shown in FIG. 3, antibody 23F4 capable of efficiently binding to human GM-CSF with antigen-binding activity of EC was obtained50＝18.11ng/ml。
Example 323F 4 antibody binding Activity with cynomolgus GM-CSF antigen
Macaque GM-CSF protein coated at 1ug/ml was plated on an Elisa plate overnight at 4 ℃. The next day after PBST washing 2 times, add 1% BSA blocking solution, 37 degrees C were incubated for 1 hours. After 2 washes, a gradient of 23F4 antibody, starting at 1ug/ml, 3-fold dilutions, 10 concentrations total, was added and incubated for 1 hour at 37 ℃. After washing 3 times, goat anti mouse-HRP was added at a dilution of 10000 and incubated at 37 ℃ for 1 hour. After 3 washes, TMB was developed for 10min and 2% H was used2SO4And stopping the solution. After the microplate reader was preheated for 15min, the OD450 readings were taken and the data were fitted as follows. As a result, as shown in FIG. 4, antibody 23F4 was obtained which was capable of binding cynomolgus monkey GM-CSF efficiently with an antigen-binding activity of EC50＝10.17ng/ml。
Example 423F 4 antibody competes for binding of human GM-CSF to the receptor GMCSFR-alpha
Recombinant human GMCSFR-alpha (CD116) protein coated at 2ug/ml was plated on an Elisa plate overnight at 4 ℃. The next day after PBST washing 2 times, add 1% BSA blocking solution, 37 degrees C were incubated for 1 hours. After 2 washes, a gradient of 23F4 antibody was added, starting at 30ug/ml, diluted 3-fold, and incubated at 37 ℃ for 1 hour for a total of 11 concentrations and biotin-labeled GMCSFR-alpha protein (0.05 ug/ml). After washing 3 times, Streptavidin-HRP secondary antibody was added at a dilution of 10000, and incubated at 37 ℃ for 1 hour. After 3 washes, TMB was developed for 10min and 2% H was used2SO4And stopping the solution. After the microplate reader was preheated for 15min, the OD450 readings were taken and the data were fitted as follows. The results are shown in FIG. 5, where the 23F4 antibody was effective in antagonizing the binding of human GM-CSF to the receptor GMCSFR-alpha, the IC50Reaching 1.1 ug/ml.
Example 523F 4 antibody blocks the intracellular signaling pathway response elicited by GMCSF
Human CD14 magnetic beads were used to separate CD14 positive monocytes from human peripheral blood mononuclear cells, stimulated with human GMCSF (0.2ng/ml) with various dilutions of 23F4 antibody at a gradient and incubated for 30min at 37 ℃. After the cells were collected, they were washed once with PBS containing 2% FBS, then fixed with 2% PFA and icebound methanol in order, and then membrane-ruptured, and then the phosphorylated STAT5 level inside the cells was stained with PE fluorescently labeled anti-human phosphorylated STAT5 antibody (pY694, BD pharmingen) and detected by flow cytometry. % of inhibition ═ 100% [1- (PE fluorescence MFI of wells to be detected)/(PE fluorescence MFI of control no-antibody group) ].
The results are shown in FIG. 6, where the 23F4 antibody inhibited intracellular levels of phosphorylated STAT5 by 80% at concentrations of 0.1ug/ml and 1 ug/ml.
Example 623F 4 antibody inhibits GM-CSF-induced TF-1 proliferative response
Human erythroleukemia cell line TF-1 was cultured in RPMI1640+ 10% serum + 1% double antibody medium (standard medium, CM) with 2ng/mL GM-CSF at 37 deg.C and 5% CO2Cultured in an incubator. And (4) once passage is carried out for 2-3 days, and cells which are in a good cell state and are in a logarithmic growth phase are taken for inoculation. The cells in good growth state were starved about 3 pm the day before the cell plate. The cells were centrifuged to remove CM, washed once with RPMI1640+ 1% double antibody medium (basal medium, BCM) without serum and GM-CSF, and then resuspended in BCM at 37 deg.C and 5% CO2Was cultured overnight in an incubator. The next day after cell starvation, about 10 am, starved cells were centrifuged, the medium removed, resuspended to 1.67 × 105 cells/mL with CM, and seeded at 90 μ L/well, i.e., 1.5 ten thousand/well, in 96-well plates. Alternatively, CM was resuspended to 3X 105 cells/mL and seeded at 50. mu.L/well, and 1.5 ten thousand cells/well, in a 96-well plate. GM-CSF used 0.05ng/mL as the final concentration. GM-CSF was formulated at a concentration of 0.2ng/mL (4X), or at a concentration of 1ng/mL (20X), using CM as a diluent. Reference Ab (R) according to previous results&D MAB215) the final concentration was selected to correspond to the concentration of IC80, and 0.2. mu.g/mL. Positive antibody stock solutions (100. mu.g/mL) were prepared as working solutions (4X) at a concentration of 0.8. mu.g/mL using CM containing 4 XHCM as a diluent. Or, a positive antibody stock solution (100. mu.g/mL) was prepared as a working solution (20X) at a concentration of 4. mu.g/mL using 100% HCM as a diluent. Diluting 23F4 antibody according to different concentration gradients, adding into 96-well plate paved with TF-1 cells, placing the culture plate into an incubator, culturing for 72h, and then using
Figure BDA0001116520550000111
Luminescent Cell Viability Assay detects Cell proliferation. The results are shown in FIG. 7, 23F4 resistantThe body can remarkably inhibit GM-CSF-induced TF-1 cell proliferation response and IC thereof5010.9ng/ml is reached.
Example 723F 4 antibody affinity assays
Using a CM5 chip coupled with anti-mouse Fc fragment antibody and HBS-EP + as experimental buffer, the binding between three concentrations of antigen and captured antibody was determined for each injection cycle, each cycle comprising injection and regeneration of the captured antibody, antigen at different concentrations. Three antibodies were captured in 2,3 and 4 channels, respectively, at a flow rate of 10. mu.L/min. The samples diluted step by step were sequentially injected at a flow rate of 30. mu.L/min for binding for 3 minutes and dissociation for 20 minutes. For stronger antigen-antibody binding, only the dissociation of the highest concentration of antigen was detected for 60 minutes using the SNL method. Regeneration was then performed with Glycine pH 1.5 at a flow rate of 10 μ L/min to remove captured antibody and antigen. Data analysis data were analyzed using Biacore T200 analysis software (version number 1.0). The model used for software analysis was 1:1 binding. 23F4 antibody affinity KD ═ 4.17x10-11M。
As a result, as shown in FIG. 8 and Table 1 below, the 23F4 antibody was able to bind human GM-CSF efficiently with an Affinity (Affinity) of 4.17X10-11M。
TABLE 123F 4 antibody affinity detection data
Antibodies ka(1/Ms) kd(1/s) KD(M)
23F4 8.723E+05 3.635E‐05 4.168E‐11
Example 823F 4 antibody humanization design
The information on the heavy and light chain variable regions of the murine anti-human GM-CSF monoclonal antibody was determined to be shown in SEQ ID NO.7 and SEQ ID NO.18 (Table 5).
The heavy chain variable region and light chain variable region sequences of murine anti-human GM-CSF monoclonal antibody 23F4 were obtained and humanized. The VH of the 23F4 antibody was first aligned and searched with the amino acid sequence of VL in the human immunoglobulin (human Ig) amino acid sequence database to find the best matching human immunoglobulin germline gene sequences (human Ig germline gene sequences). The human immunoglobulin germline gene most similar to the VL protein sequence of the 23F4 antibody is L8/Jk 4. The human immunoglobulin germline gene that most closely resembles the VH protein sequence of the 23F4 antibody is VH1-F/JH 6. The 23F4 antibody VH was humanized by grafting the CDR1, CDR2 and CDR3 sequences of the 23F4 antibody VH of Table 2 below onto the framework region sequences of the VH1-F/JH6 genes. Humanized design of 23F4 antibody VL 3 Complementarity Determining Regions (CDRs) CDR1, CDR2 and CDR3 sequences in the 23F4 antibody VL of table 3 below were grafted onto framework sequences of the L8/Jk4 gene.
CDR1, CDR2 and CDR3 sequences of the VH of the 223F 4 antibody
CDR1 SHYLH(SEQ ID NO.1)
CDR2 WIFPGDDKTKYNEKFKG(SEQ ID NO.2)
CDR3 GTKYLNWNFDV(SEQ ID NO.3)
CDR1, CDR2 and CDR3 sequences of the VL antibody of Table 323F 4
CDR1 KANQNVGTTLA(SEQ ID NO.4)
CDR2 SASYRYS(SEQ ID NO.5)
CDR3 HQYTTYPLT(SEQ ID NO.6)
Through antibody 3D modeling, it was found that: some of the framework amino acids in the murine anti-23F 4 variable region sequence were bound to the CDR regions and even involved in the formation of the CDR regions. These amino acids are important for maintaining the activity of the humanized antibody, and thus should be retained, i.e., Back-mutated, during the humanization process. As shown in Table 4, these amino acids in VH were 98R (i.e., R at position 98, the same below), 72S, 68A, 70L, 48I, 26D, 29L; the amino acids in VL are 1E (E at position 1 (Kabat nomenclature system), the same below) and 46A, 60D, 70D, 43S, 87F. The specific amino acid sequences of the humanized design are shown in table 5, and the corresponding nucleic acid sequences are respectively: 23F4 VH (SEQ ID NO.23), 23F4 VH.1(SEQ ID NO.24), 23F4 VH.1a (SEQ ID NO.25), 23F4 VH.1b (SEQ ID NO.26), 23F4 VH.1c (SEQ ID NO.27), 23F4 VH.1d (SEQ ID NO.28), 23F4 VH.2(SEQ ID NO.29), 23F4 VH.2a (SEQ ID NO.30), 23F4 VH.2b (SEQ ID NO.31), 23F4 VH.2c (SEQ ID NO.32), 23F4 VH.2d (SEQ ID NO.33), 23F4 VK (SEQ ID NO.34), 23F4 VK.1(SEQ ID NO.35), 23F4 VK.1a (SEQ ID NO.36), 23F4 VK.1b (SEQ ID NO.37), and 23F4 VK.1c (SEQ ID NO. 38).
Humanized VH and VL genes were synthesized and cloned into pcDNA3.1 vector containing human heavy chain IgG1 and light chain kappa chain constant regions, expressed on 293T cells and purified by protein A/Gc to give humanized antibodies. The heavy and light chains of these antibodies were combined separately into 38 humanized GM-CSF antibodies (table 6).
Note: the antibodies in which the VH and VL of 23F4 were directly cloned into an expression vector containing human heavy chain IgG1 and light chain kappa chain constant regions were chimeric antibodies (Chimera).
TABLE 4 humanization design
Figure BDA0001116520550000131
Figure BDA0001116520550000141
TABLE 5 specific amino acid sequences for humanization design (wherein CDR regions are underlined)
Figure BDA0001116520550000142
Figure BDA0001116520550000151
TABLE 638 Synthesis protocol and nomenclature for humanized antibodies
Figure BDA0001116520550000152
Example 9 antigen binding Activity of humanized antibodies
Human GM-CSF protein coated at 1ug/ml was applied to an Elisa plate overnight at 4 ℃. Next timeAfter 2 washes of daily PBST, 1% BSA blocking solution was added and incubated at 37 ℃ for 1 hour. After 2 washes, a gradient of diluted humanized antibody, starting at 1ug/ml, 3-fold dilutions, at a total of 10 concentrations, was added and incubated for 1 hour at 37 ℃. After washing for 3 times, anti-human IgG-HRP was added at a dilution of 10000, and incubation was carried out at 37 ℃ for 1 hour. After 3 washes, TMB was developed for 10min and 2% H was used2SO4And stopping the solution. After the microplate reader is preheated for 15min, OD450 is read. The results are shown in FIG. 9, where all humanized antibodies were very similar to the chimeric antibody (23F4 Chimera) in ELISA binding activity, indicating that these humanized antibodies were able to bind GM-CSF antigen efficiently.
Example 10 humanized antibody affinity assay
The results of affinity assay using Biacore T200 for humanized candidate antibodies are shown in Table 7 below, in which three antibodies Hu23F4-13, Hu23F4-27 and Hu23F4-36 have higher affinity and are closest to the chimeric antibody.
TABLE 7 antigen affinity of humanized antibodies
Figure BDA0001116520550000161
Example 11 humanized antibodies inhibit GM-CSF-induced TF-1 proliferation response
Human erythroleukemia cell line TF-1 was cultured in RPMI1640+ 10% serum + 1% double antibody medium (standard medium, CM) with 2ng/mL GM-CSF at 37 deg.C and 5% CO2Cultured in an incubator. And (4) once passage is carried out for 2-3 days, and cells which are in a good cell state and are in a logarithmic growth phase are taken for inoculation. The cells in good growth state were starved about 3 pm the day before the cell plate. The cells were centrifuged to remove CM, washed once with RPMI1640+ 1% double antibody medium (basal medium, BCM) without serum and GM-CSF, and then resuspended in BCM at 37 deg.C and 5% CO2Was cultured overnight in an incubator. The next day after cell starvation, about 10 am, starved cells were centrifuged, the medium removed, resuspended to 1.67 × 105 cells/mL with CM, and seeded at 90 μ L/well, i.e., 1.5 ten thousand/well, in 96-well plates. OrOne was resuspended to 3X 105 cells/mL with CM and seeded at 50. mu.L/well, and 1.5 ten thousand cells/well, on a 96-well plate. GM-CSF used 0.05ng/mL as the final concentration. GM-CSF was formulated at a concentration of 0.2ng/mL (4X), or at a concentration of 1ng/mL (20X), using CM as a diluent. Reference Ab (R) according to previous results&D MAB215) the final concentration was selected to correspond to the concentration of IC80, and 0.2. mu.g/mL. Positive antibody stock solutions (100. mu.g/mL) were prepared as working solutions (4X) at a concentration of 0.8. mu.g/mL using CM containing 4 XHCM as a diluent. Or, a positive antibody stock solution (100. mu.g/mL) was prepared as a working solution (20X) at a concentration of 4. mu.g/mL using 100% HCM as a diluent. Diluting humanized antibody according to different concentration gradient, adding into 96-well plate paved with TF-1 cells, placing the culture plate into incubator, culturing for 72 hr, and then using
Figure BDA0001116520550000171
Luminescent Cell Viability Assay detects Cell proliferation.
As shown in FIG. 10 and Table 8 below, the humanized antibodies tested all inhibited GM-CSF-induced TF-1 cell proliferation, wherein the three antibodies Hu23F4-13, Hu23F4-27 and Hu23F4-36 had the strongest inhibitory effect, respectively reaching IC50＝4.95ng/ml,IC503.95ng/ml and IC50＝3.30ng/ml。
TABLE 8 data for the inhibition of GM-CSF-induced TF-1 proliferation response by humanized candidate antibodies
Antibody numbering Inhibition of TF-1 cell proliferation activity (IC50)
Hu23F4-chimera 8.55ng/ml
Hu23F4-5 15.58ng/ml
Hu23F4-6 7.87ng/ml
Hu23F4-13 4.95ng/ml
Hu23F4-20 8.77ng/ml
Hu23F4-23 9.08ng/ml
Hu23F4-25 12.22ng/ml
Hu23F4-27 3.95ng/ml
Hu23F4-29 27.44ng/ml
Hu23F4-36 3.30ng/ml
Example 12 blocking of intracellular signaling pathway responses by GM-CSF by humanized antibodies
Human CD14 magnetic beads were used to separate CD14 positive monocytes from human peripheral blood monocytes, stimulated with human GM-CSF (0.2ng/ml) while varying gradients of diluted humanized antibody were added and incubated at 37 ℃ for 30 min. After the cells were collected, they were washed once with PBS containing 2% FBS, then fixed with 2% PFA and icebound methanol in order, and then membrane-ruptured, and then the phosphorylated STAT5 level inside the cells was stained with PE fluorescently labeled anti-human phosphorylated STAT5 antibody (pY694, BD pharmingen) and detected by flow cytometry. % of inhibition ═ 100% [1- (PE fluorescence MFI of wells to be detected)/(PE fluorescence MFI of control no-antibody group) ].
As shown in FIG. 11, the three antibodies Hu23F4-13, Hu23F4-27 and Hu23F4-36 all significantly inhibited GM-CSF-induced intracellular phosphorylation STAT5 levels by approximately 80% at concentrations of 0.1ug/ml and 1 ug/ml.
Example 13 binding Activity of humanized antibodies to cynomolgus GM-CSF antigen
Macaque GMCSF protein coated at 1ug/ml was applied to an Elisa plate overnight at 4 ℃. The next day after PBST washing 2 times, add 1% BSA blocking solution, 37 degrees C were incubated for 1 hours. After 2 washes, a gradient of diluted humanized antibody, starting at 1ug/ml, 3-fold dilutions, at a total of 10 concentrations, was added and incubated for 1 hour at 37 ℃. After washing 3 times, goat anti mouse-HRP was added at a dilution of 10000 and incubated at 37 ℃ for 1 hour. After 3 washes, TMB was developed for 10min and 2% H was used2SO4And stopping the solution. After the microplate reader is preheated for 15min, OD450 is read.
As shown in FIG. 12 and Table 9 below, the Hu23F4-13, Hu23F4-27 and Hu23F4-36 antibodies were able to effectively bind to cynomolgus monkey GM-CSF protein with binding activities reaching EC respectively50＝7.44ng/ml，EC506.25ng/ml and EC50＝7.75ng/ml。
TABLE 9 humanized antibody binding Activity data with cynomolgus GM-CSF antigen
Antibody numbering Binding to macaque GMCSF Activity (EC50)
Hu23F4-13 7.44ng/ml
Hu23F4-27 6.25ng/ml
Hu23F4-36 7.75ng/ml
The above-mentioned embodiments only express the embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Figure IDA0001116520630000011
Figure IDA0001116520630000021
Figure IDA0001116520630000031
Figure IDA0001116520630000041
Figure IDA0001116520630000051
Figure IDA0001116520630000061
Figure IDA0001116520630000071
Figure IDA0001116520630000081
Figure IDA0001116520630000091
Figure IDA0001116520630000101
Figure IDA0001116520630000111
Figure IDA0001116520630000121
Figure IDA0001116520630000131
Figure IDA0001116520630000141
Figure IDA0001116520630000151
Figure IDA0001116520630000161
Figure IDA0001116520630000171

Claims (19)

1. An isolated monoclonal antibody or antigen binding portion thereof that binds to human GM-CSF protein, the antibody or antigen binding portion thereof comprising heavy chain variable region CDR1, CDR2 and CDR3 and light chain variable region CDR1, CDR2 and CDR 3; wherein, the amino acid sequence of the heavy chain variable region CDR1 is shown in SEQ ID NO.1, the amino acid sequence of the CDR2 is shown in SEQ ID NO.2, and the amino acid sequence of the CDR3 is shown in SEQ ID NO. 3; the amino acid sequence of the light chain variable region CDR1 is shown in SEQ ID NO.4, the amino acid sequence of the CDR2 is shown in SEQ ID NO.5, and the amino acid sequence of the CDR3 is shown in SEQ ID NO. 6.
2. The monoclonal antibody, or antigen-binding portion thereof, of claim 1, wherein the antibody, or antigen-binding portion thereof, comprises the heavy chain variable region sequence of SEQ ID No.7 and the light chain variable region sequence of SEQ ID No. 18.
3. The monoclonal antibody or antigen-binding portion thereof of claim 1, wherein the antibody or antigen-binding portion thereof comprises the heavy chain variable region sequence of SEQ ID No.8 or the light chain variable region sequence of SEQ ID No. 19.
4. The monoclonal antibody, or antigen-binding portion thereof, of claim 3, wherein the heavy chain variable region further comprises any one or a combination of any two or more of the following back-mutation sites: 98R, 72S, 68A, 70L, 48I, 26D, 29L; or
The light chain variable region further comprises any one or a combination of any two or more of the following back-mutation sites: 1E, 46A, 60D, 70D, 43S, 87F.
5. The monoclonal antibody or antigen binding portion thereof of claim 1, wherein the amino terminus of the CDR1 sequence of the heavy chain variable region is linked to DYTLT or GYTFT.
6. The monoclonal antibody or antigen-binding portion thereof according to claim 1, wherein the antibody or antigen-binding portion thereof comprises a heavy chain variable region sequence as set forth in any one of SEQ ID No. 9-17 and a light chain variable region sequence as set forth in any one of SEQ ID No. 20-22.
7. An immunoconjugate, wherein said conjugate comprises:
the monoclonal antibody or antigen binding portion thereof of any one of claims 1-6; and
a functional molecule attached thereto; the functional molecule is selected from: a second antibody or antigen-binding portion thereof, a cytokine, a detectable label, a toxin.
8. The immunoconjugate of claim 7, wherein the detectable label comprises: fluorescent markers, chromogenic markers.
9. A pharmaceutical composition comprising the monoclonal antibody or antigen-binding portion thereof of any one of claims 1-6, and a pharmaceutically acceptable carrier or diluent.
10. An isolated nucleic acid encoding the heavy chain variable region and the light chain variable region of the monoclonal antibody, or antigen binding portion thereof, of any one of claims 1-6.
11. A recombinant expression vector comprising the nucleic acid of claim 10.
12. A host cell comprising the recombinant expression vector of claim 11.
13. Use of a monoclonal antibody or antigen binding portion thereof according to any one of claims 1-6 in the manufacture of a medicament for the treatment of a GM-CSF related disorder; the GM-CSF related diseases include inflammatory diseases and tumor diseases; the inflammatory disease is selected from: rheumatoid arthritis, asthma, multiple sclerosis, inflammatory bowel disease, colitis, psoriasis; or the neoplastic disease is leukemia.
14. The use of claim 13, wherein the inflammatory bowel disease is regional enteritis.
15. A kit for treating a GM-CSF-related disease comprising the monoclonal antibody or antigen binding portion thereof of any one of claims 1-6; or a pharmaceutical composition according to claim 9; the GM-CSF related diseases include inflammatory diseases and neoplastic diseases, the inflammatory diseases being selected from the group consisting of: rheumatoid arthritis, asthma, multiple sclerosis, inflammatory bowel disease, colitis, psoriasis; or the neoplastic disease is leukemia.
16. The kit of claim 15, wherein the inflammatory bowel disease is regional enteritis.
17. A detection chip comprising the monoclonal antibody or the antigen-binding portion thereof according to any one of claims 1 to 6.
18. A kit for detecting GM-CSF factor, comprising the monoclonal antibody or antigen-binding portion thereof of any one of claims 1-6; or comprising therein the detection chip of claim 17.
19. A method of detecting GM-CSF factor, the method comprising: detecting a test sample by conjugating the monoclonal antibody or antigen-binding portion thereof of any one of claims 1-6 to a detectable label, thereby determining the presence or amount of GM-CSF factor in the test sample; the method is a non-diagnostic method.
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KR1020187009572A KR101953706B1 (en) 2016-09-19 2017-09-18 Anti-GM-CSF antibody and its use
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BR112019005274A BR112019005274A2 (en) 2016-09-19 2017-09-18 isolated antibody or fragment thereof, composition, isolated cell, and methods for treating an inflammatory or autoimmune disease or condition, treating cancer, reducing or alleviating pain, and detecting gm-csf expression in a sample.
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Families Citing this family (3)

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Publication number Priority date Publication date Assignee Title
WO2020078270A1 (en) * 2018-10-15 2020-04-23 Elixiron Immunotherapeutics (hong Kong) Limited Antibodies to granulocyte-macrophage colony stimulating factor and uses thereof
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010071923A1 (en) * 2008-12-22 2010-07-01 The University Of Melbourne Pain treatment
CN101861336A (en) * 2007-09-18 2010-10-13 安姆根有限公司 Human GM-CSF antigen binding proteins
WO2010124163A2 (en) * 2009-04-23 2010-10-28 Theraclone Sciences, Inc. Granulocyte-macrophage colony-stimulating factor (gm-csf) neutralizing antibodies
WO2014138862A1 (en) * 2013-03-14 2014-09-18 John Schrader Human monoclonal antibodies that neutralize bioactivity of granulocyte macrophage colony-stimulating factor and methods and uses thereof
WO2015028657A1 (en) * 2013-08-30 2015-03-05 Takeda Gmbh Antibodies neutralizing gm-csf for use in the treatment of rheumatoid arthritis or as analgesics

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110044996A1 (en) * 2007-05-23 2011-02-24 Charles Mackay Neutralizing antibodies

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101861336A (en) * 2007-09-18 2010-10-13 安姆根有限公司 Human GM-CSF antigen binding proteins
WO2010071923A1 (en) * 2008-12-22 2010-07-01 The University Of Melbourne Pain treatment
WO2010124163A2 (en) * 2009-04-23 2010-10-28 Theraclone Sciences, Inc. Granulocyte-macrophage colony-stimulating factor (gm-csf) neutralizing antibodies
WO2014138862A1 (en) * 2013-03-14 2014-09-18 John Schrader Human monoclonal antibodies that neutralize bioactivity of granulocyte macrophage colony-stimulating factor and methods and uses thereof
WO2015028657A1 (en) * 2013-08-30 2015-03-05 Takeda Gmbh Antibodies neutralizing gm-csf for use in the treatment of rheumatoid arthritis or as analgesics

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Functions of granulocyte-macrophage colony-stimulating factor;Fleetwood AJ et al.;《Crit Rev Immunol》;20051231;第405-428页 *
GM-CSF抗体在免疫正常格特隐球菌感染中的作用研究;金怡等;《临床军医杂志》;20140630;第611-613页 *

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