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WO2024113122A1 - Anticorps anti-il-8 - Google Patents

Anticorps anti-il-8 Download PDF

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Publication number
WO2024113122A1
WO2024113122A1 PCT/CN2022/134825 CN2022134825W WO2024113122A1 WO 2024113122 A1 WO2024113122 A1 WO 2024113122A1 CN 2022134825 W CN2022134825 W CN 2022134825W WO 2024113122 A1 WO2024113122 A1 WO 2024113122A1
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Prior art keywords
antibody
variable region
chain variable
heavy chain
light chain
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Inventor
Yingying YAO
Lin Chen
Liang Tang
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Suzhou Kaigene Biotechnology Co Ltd
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Suzhou Kaigene Biotechnology Co Ltd
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Priority to PCT/CN2022/134825 priority Critical patent/WO2024113122A1/fr
Priority to CN202280008469.2A priority patent/CN117203231B/zh
Publication of WO2024113122A1 publication Critical patent/WO2024113122A1/fr
Anticipated expiration legal-status Critical
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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
    • C07K16/244—Interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/10—Libraries containing peptides or polypeptides, or derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • 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
    • G01N2333/54—Interleukins [IL]
    • G01N2333/5421—IL-8

Definitions

  • the present invention relates to the field of biotechnology, and in particular to an anti-IL-8 antibody.
  • Interleukin 8 also known as CXCL8
  • CXCL8 monocyte-derived neutrophil chemotactic factor
  • NAP-1 neutrophil attractant/activation protein-1
  • IL-8 is a polypeptide and IL-8 can be secreted by fibroblasts, vascular endothelial cells, macrophages, dendric cells (DCs) , lymphocytes, keratinocytes, melanocytes, hepatocytes, and various tumor cells.
  • IL-8 is known to stimulate neutrophil chemotaxis and participates in the migration of neutrophils towards inflammatory sites through binding to its high-affinity receptors (CXCR1 and CXCR2) which are present on the surface of neutrophils.
  • CXCR1 and CXCR2 high-affinity receptors
  • IL-8 activates neutrophils by accelerating degranulation, reactive oxygen species (ROS) production and destroy the infiltrated tissue.
  • IL-8 is associated with rheumatoid arthritis, asthma, gout, inflammatory bowel disease (IBD) , and sepsis which are characterized by inflammation accompanied by neutrophil infiltration and tissue damage.
  • IL-8 is known to promote angiogenesis and growth of tumors. IL-8 can also attract myeloid-derived suppressor cells (MDSCs) traffic to the tumor microenvironment (TME) and contribute to tumor immune evasion.
  • MDSCs myeloid-derived suppressor cells
  • Human tumor cells such as, melanoma, breast cancer, hepatocellular carcinoma, castration resistant prostate cancer, colorectal cancer, glioma, constitutively express IL-8 which plays a role in tumor invasion and metastasis.
  • the inhibition of IL-8 could prevent inflammatory cells from infiltrating the tissue, decrease angiogenesis, MDSCs trafficking and ameliorate the diseases.
  • the object of the present invention is to provide an antibody which binds to IL-8 with high affinity and their use in blocking or inhibiting IL-8 induced activities.
  • the first aspect of the present invention provides a heavy chain variable region of an antibody, the heavy chain variable region comprises the following three complementary determining regions (CDRs) :
  • the antibody is anti-IL-8 antibody.
  • the IL-8 is human IL-8.
  • any one of the abovementioned amino acid sequences further comprises a derived sequence in which at least one amino acid (for example, 1-3, preferably 1-2, more preferably 1) is optionally added, deleted, modified and/or substituted.
  • at least one amino acid for example, 1-3, preferably 1-2, more preferably 1.
  • the derived sequence can retain the binding affinity of IL-8.
  • the derived sequence is anti-IL-8.
  • the heavy chain variable region further comprises a humanized FR region or a murine FR region.
  • the heavy chain variable region has an amino acid sequence as shown in SEQ ID NO: 7.
  • the second aspect of the present invention provides a heavy chain of an antibody, the heavy chain comprises the heavy chain variable region according to the first aspect of the present invention.
  • the heavy chain of the antibody further comprises a heavy chain constant region.
  • the heavy chain constant region is humanized, murine or rabbit origin.
  • the third aspect of the present invention provides a light chain variable region of an antibody, the light chain variable region comprises the following three complementary determining regions (CDRs) :
  • any one of the abovementioned amino acid sequences further comprises a derived sequence in which at least one amino acid (for example, 1-3, preferably 1-2, more preferably 1) is optionally added, deleted, modified and/or substituted.
  • at least one amino acid for example, 1-3, preferably 1-2, more preferably 1.
  • the derived sequence can retain the binding affinity of IL-8.
  • the derived sequence is anti-IL-8.
  • the light chain variable region further comprises a human FR region or a murine FR region.
  • the light chain variable region has an amino acid sequence as shown in SEQ ID NO: 8.
  • the fourth aspect of the present invention provides a light chain of an antibody, the light chain comprises the light chain variable region according to the third aspect of the present invention.
  • the light chain of the antibody further comprises a light chain constant region.
  • the light chain constant region is human, murine or rabbit origin.
  • the fifth aspect of the present invention provides an antibody, the antibody comprises:
  • the antibody comprises the heavy chain according to the second aspect of the present invention; and/or the light chain according to the fourth aspect of the present invention.
  • the antibody is anti-IL-8 antibody.
  • the IL-8 is human IL-8.
  • the antibody is selected from the group consisting of animal-derived antibodies, chimeric antibodies, humanized antibodies, and combinations thereof.
  • the antibody is a double-chain antibody or a single-chain antibody.
  • the antibody is a monoclonal antibody.
  • the antibody is a partially or fully humanized monoclonal antibody.
  • sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 7; and/or
  • sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 8.
  • the antibody is of an IgG, IgM, IgA, IgD, and IgE type.
  • the antibody is in the form of a drug conjugate.
  • the sixth aspect of the present invention provides a recombinant protein, the recombinant protein comprises:
  • the tag sequence comprises a 6His tag.
  • the recombinant protein comprises a fusion protein.
  • the recombinant protein is a monomer, dimer, or multimer.
  • the seventh aspect of the present invention provides an antibody-drug conjugate, the antibody-drug conjugate comprises:
  • an antibody portion is selected from the group consisting of the heavy chain variable region according to the first aspect of the present invention, the heavy chain according to the second aspect of the present invention, the light chain variable region according to the third aspect of the present invention, the light chain according to the fourth aspect of the present invention, or the antibody according to the fifth aspect of the present invention, and combinations thereof;
  • conjugating portion conjugated to the antibody portion
  • the conjugating portion is selected from the group consisting of detectable markers, drugs, toxins, cytokines, radionuclides, enzymes, and combinations thereof.
  • the antibody portion and the conjugating portion are conjugated through a chemical bond or linker.
  • the eighth aspect of the present invention provides a polynucleotide, the polynucleotide encodes polypeptides selected from the group consisting of:
  • the ninth aspect of the present invention provides a vector, the vector comprises the polynucleotide according to the eighth aspect of the present invention.
  • the vector comprises bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, or other vectors.
  • the tenth aspect of the present invention provides a genetically engineered host cell, the host cell comprises the vector according to the ninth aspect of the present invention or the polynucleotid according to the eighth aspect of the present invention is integrated into the genome of the host cell.
  • the eleventh aspect of the present invention provides a pharmaceutical composition
  • the pharmaceutical composition comprises the heavy chain variable region according to the first aspect of the present invention, the heavy chain according to the second aspect of the present invention, the light chain variable region according to the third aspect of the present invention, the light chain according to the fourth aspect of the present invention, or the antibody according to the fifth aspect of the present invention, the recombinant protein according to the sixth aspect of the present invention, the antibody-drug conjugate according to the seventh aspect of the present invention, the polynucleotide according to the eighth aspect of the present invention, the vector according to the ninth aspect of the present invention, and/or the genetically engineered host cell according to the tenth aspect of the present invention.
  • the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent or excipient.
  • the pharmaceutical composition is liquid preparation.
  • the preparation of the pharmaceutical composition is injection preparation.
  • the injection preparation is intravenous injection preparation.
  • the twelfth aspect of the present invention provides a uses of the heavy chain variable region according to the first aspect of the present invention, the heavy chain according to the second aspect of the present invention, the light chain variable region according to the third aspect of the present invention, the light chain according to the fourth aspect of the present invention, the antibody according to the fifth aspect of the present invention, and/or the recombinant protein according to the sixth aspect of the present for (i) preparing a drug or preparation for blocking or inhibiting IL-8 induced activities; (ii) preparing a drug or preparation for preventing and/or treating the diseases associated with the IL-8; and/or (iii) preparing a detection reagent or kit.
  • the IL-8 induced activities comprises proinflammatory activities, chemotactic activities, and/or angiogenesis.
  • the diseases associated with the IL-8 comprises autoimmune, inflammatory, infectious diseases and/or tumor.
  • the diseases associated with the IL-8 is the diseases associated with the elevated or unbalanced level of IL-8.
  • the diseases associated with the IL-8 is immune, autoimmune, inflammatory, infectious diseases or disorders characterised by elevated or unbalanced level of human IL-8, particularly rheumatoid arthritis, ulcerative colitis, asthma, chronic obstructive pulmonary disease (COPD) , gout, cancer, flu, acne, inflammatory bowel disease (IBD) , psoriasis, sepsis, osteoarthritis, erosive arthritis, atherosclerosis, transplant rejection, acute lung disease, acute lung injury, acute respiratory distress syndrome (ARDS) , Crohn's disease, peripheral artery disease, systemic sclerosis, deep vein thrombosis, meningitis, encephalitis, uveitis, endometriosis, cystic fibrosis, diffuse panbronchiolitis, reperfusion injury, cystic fibrosis, vasculitis, familial Mediterranean fever, nephritis, chronic renal failure, juvenile onset diabetes, purpura , acute pancre
  • the diseases associated with the IL-8 is inflammatory or skin disorders, such as psoriasis, pustulosis palmoplantaris (PPP) , bullous pemphigoid, pemphigus, contact dermatitis, eczema, erythematosus, and atopic dermatitis.
  • PPP pustulosis palmoplantaris
  • the diseases associated with the IL-8 is human virus associated diseases or infections, such as common cold as caused by human rhinovirus, coronavirus, other enterovirus, herpes virus, influenza virus, parainfluenza virus, respiratory syncytial virus or adenovirus infection and hepatitis C.
  • the detection reagent or kit is used to detect the IL-8 in a sample.
  • the detection reagent is a test chip.
  • the thirteenth aspect of the present invention provides a method for detecting the IL-8 in a sample in vitro, which comprises the following steps:
  • the method is non-diagnostic and non-therapeutic.
  • the fourteenth aspect of the present invention provides a test plate, the test plate comprises a substrate (support plate) and a test strip, the test strip comprises the antibody according to the fifth aspect of the present invention or the antibody-drug conjugate according to the seventh aspect of the present invention.
  • the fifteenth aspect of the present invention provides a method for blocking or inhibiting IL-8 induced activities; and/or preventing and/or treating the diseases associated with the IL-8, which comprises administering the antibody according to the fifth aspect of the present invention, the recombinant protein according to the sixth aspect of the present invention, the antibody-drug conjugate according to the seventh aspect of the present invention, the polynucleotide according to the eighth aspect of the present invention, the vector according to the ninth aspect of the present invention, the genetically engineered host cell according to the tenth aspect of the present invention, and/or the pharmaceutical composition according to the eleventh aspect of the present invention to a subject in need, thereby blocking or inhibiting IL-8 induced activities; and/or preventing and/or treating the diseases associated with the IL-8.
  • the subject comprises human and non-human mammal.
  • the non-human mammal comprises cattle, horse, sheep, dog, cat or mice.
  • FIG. 1 shows the experimental results of ELISA analysis of anti-IL-8 antibodies of the present invention binding to human IL-8.
  • FIG. 2 shows the experimental results of biolayer interferometry (BLI) binding analysis using GATOR instrument.
  • the data of dissociation (KD or kd) and association (ka) rate constants were obtained using GATOR software.
  • the equilibrium dissociation constants (K D ) were calculated from the ratio of kd over ka.
  • FIG. 3 shows the results of Differential Scanning Fluorimetry (DSF) analysis in a ABI7500 Fast Real-Time PCR instrument.
  • FIG. 4 shows the results of inhibitions of IL-8 mediated CXCR1 + cells chemotaxis by anti-IL-8 antibodies as determined by a transmigration assay using a Boyden chamber, wherein, the “B30” refers to the antibody LJH001-B30, the “Positive” refers to the Monoclonal Mouse IgG1 Clone # 6217.
  • the present invention provides an antibody which binds to IL-8 with high affinity and their use in blocking or inhibiting IL-8 induced activities, e.g., proinflammatory activities, chemotactic activities and angiogenesis, to treat immune, autoimmune, inflammatory or infectious diseases and cancer associated with IL-8.
  • IL-8 induced activities e.g., proinflammatory activities, chemotactic activities and angiogenesis
  • the terms “comprise” , “comprising” , and “containing” are used interchangeably, which not only comprise closed definitions, but also semi-closed and open definitions. In other words, the term comprises “consisting of” and “essentially consisting of” .
  • IL-8 interleukin 8
  • domain refers to a region in a polypeptide that is independent of other regions and folded into a specific structure.
  • single-chain variable fragment refers to a single-chain polypeptide derived from an antibody, which retains the ability to bind to an antigen.
  • An example of ScFv includes an antibody polypeptide formed by recombinant DNA technology, and Fv regions of immunoglobulin heavy chain (H chain) and light chain (L chain) fragments therein are connected via a spacer sequence.
  • H chain immunoglobulin heavy chain
  • L chain light chain
  • the terms “administration” refer to the application of exogenous drugs, therapeutic agents, diagnostic agents or compositions to animals, humans, subjects, cells, tissues, organs, or biological fluids.
  • the “administration” and “treatment” can refer to treatment, pharmacokinetics, diagnosis, research, and experimental methods.
  • the treatment of cells includes contact between reagents and cells, contact between reagents and fluids, and contact between fluids and cells.
  • the “administration” and “treatment” also mean treatment by reagents, diagnosis, binding compositions, or by another cells in vitro and ex vivo.
  • the “treatment” is applied to humans, animals or research subjects, it refers to treatment, prevention or preventive measures, research and diagnosis; including contact between anti-human IL-8 antibodies and humans or animals, subjects, cells, tissues, physiological compartments or physiological fluids.
  • treatment refers to the administration of an internal or external therapeutic agent, including any one of the anti-human IL-8 antibodies of the present invention and a composition thereof, to a patient who has one or more disease symptoms, and it is known that the therapeutic agent has a therapeutic effect on these symptoms.
  • the patient is administered in an amount (therapeutically effective dose) of a therapeutic agent effective to alleviate one or more disease symptoms.
  • the term “optional” or “optionally” means that the event or situation described later can occur but does not have to occur.
  • “optionally including 1-3 antibody heavy chain variable regions” means that the antibody heavy chain variable region of a specific sequence may have but does not have to be, and it can be 1, 2, or 3.
  • sequence identity in the present invention refers to when optimally aligned and compared, the degree of identity between two nucleotide sequences or two amino acid sequences with appropriate mutations such as substitutions, insertions or deletions.
  • sequence identity between the sequence described in the present invention and identical sequence thereof may be at least 85%, 90%or 95%, preferably at least 95%. Non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%.
  • amino acid sequence is from N-terminal to C-terminal.
  • Interleukin 8 also known as CXCL8
  • CXCL8 monocyte-derived neutrophil chemotactic factor
  • NAP-1 neutrophil attractant/activation protein-1
  • IL-8 is a polypeptide and IL-8 can be secreted by fibroblasts, vascular endothelial cells, macrophages, dendric cells (DCs) , lymphocytes, keratinocytes, melanocytes, hepatocytes, and various tumor cells.
  • fibroblasts vascular endothelial cells
  • macrophages macrophages
  • dendric cells DCs
  • lymphocytes keratinocytes
  • melanocytes melanocytes
  • hepatocytes and various tumor cells.
  • the term “antibody” refers to an immunoglobulin, with a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds.
  • the amino acid composition and sequence of heavy chain constant regions of immunoglobulins are different, so their antigenicities are also different. Accordingly, the immunoglobulins can be divided into five classes, or isotypes of the immunoglobulins, namely IgM, IgD, IgG, IgA and IgE.
  • the corresponding heavy chains are ⁇ chain, ⁇ chain, ⁇ chain, ⁇ chain, and ⁇ chain, respectively.
  • the same class of Ig can be divided into different subclasses according to the difference in the amino acid composition of hinge regions and the number and position of heavy chain disulfide bonds.
  • IgG can be divided into IgG1, IgG2, IgG3, and IgG4.
  • the light chain is divided into a ⁇ chain or a ⁇ chain according to the difference of the constant region.
  • Each of the five classes of Ig can have a ⁇ chain or a ⁇ chain.
  • the subunit structures and three-dimensional structures of different classes of immunoglobulins are well known to a person skilled in the art.
  • the antibody light chain of the present invention may further include a light chain constant region, and the light chain constant region includes a humanized or murine ⁇ or ⁇ chain or variant thereof.
  • the antibody heavy chain of the present invention may further include a heavy chain constant region, and the heavy chain constant region includes humanized or murine IgG1, IgG2, IgG3, IgG4 or variant thereof.
  • the sequence of about 110 amino acids near the N-terminals of the antibody heavy and light chains varies greatly and is a variable region (Fv region) ; and the sequence of remaining amino acids near the C-terminal is relatively stable and is a constant region.
  • the variable region includes 3 hypervariable regions (HVR) and 4 framework regions (FR) with relatively conservative sequences.
  • the three hypervariable regions determine the specificity of the antibody, and are also known as complementary determining regions (CDR) .
  • Each light chain variable region (LCVR) and heavy chain variable region (HCVR) is composed of 3 CDR regions and 4 FR regions, and their sequence from the amino terminal to the carboxy terminal is: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.
  • the 3 CDR regions of the light chain refer to CDR1', CDR2 'and CDR3'; and the 3 CDR regions of the heavy chain refer to CDR1, CDR2 and CDR3.
  • the antibodies of the present invention include murine antibodies, chimeric antibodies, and humanized antibodies, preferably humanized antibodies.
  • the term “murine antibody” in the present invention is an anti-human IL-8 monoclonal antibody prepared according to the knowledge and skills in the art. During the preparation, the test subject is injected with the IL-8 antigen, and then hybridomas expressing antibodies with the desired sequence or functional properties are isolated.
  • the murine IL-8 antibody or antigen binding fragment thereof may further include a light chain constant region of a murine ⁇ or ⁇ chain or variant thereof, or further include a heavy chain constant region of murine IgG1, IgG2, IgG3 or variant thereof.
  • chimeric antibody is an antibody formed by fusing a variable region of a murine antibody with a constant region of a human antibody, which can reduce the immune response induced by the murine antibody.
  • humanized antibody also known as CDR-grafted antibody, refers to the transplantation of a mouse CDR sequence into a human antibody variable region framework, that is, an antibody produced in different types of human germline antibody framework sequences.
  • the humanized antibody can overcome the heterogeneous reaction induced by the chimeric antibody carrying a large amount of murine protein components.
  • framework sequences can be obtained from public DNA databases or published references that include antibody gene sequences in various germ lines.
  • the human antibody variable region framework sequence can be subjected to minimal reverse mutations or back mutations to maintain its activity.
  • antigen binding fragment of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (for example, IL-8) . It has been shown that full-length fragments of an antibody can be used to perform the antigen binding function of the antibody. Examples of the binding fragment contained in the term “antigen binding fragment of an antibody” include:
  • Fab fragment a monovalent fragment consisting of V L , V H , CL and CH1 domains
  • F (ab') 2 fragment a bivalent fragment containing two Fab fragments connected by a disulfide bridge on a hinge region;
  • the Fv antibody contains a heavy chain variable region and a light chain variable region, but does not have a constant region, and has a minimum antibody fragment with all antigen binding sites. Generally, the Fv antibody further contains a polypeptide linker between the V H and V L domains, and can form a structure required for antigen binding.
  • CDR refers to one of the six hypervariable regions in the variable domain of an antibody that mainly contribute to antigen binding.
  • epitope or “antigenic determinant” refers to a site on an antigen where an immunoglobulin or antibody specifically binds (for example, a specific site on a IL-8 molecule) .
  • the epitope usually includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 continuous or discontinuous amino acids in a unique spatial conformation.
  • binding refers to the binding of an antibody to an epitope on a predetermined antigen.
  • competitive binding refers to an antibody that recognizes the same epitope (also called an antigenic determinant) or a part of the same epitope on the extracellular region of human IL-8 as the monoclonal antibody of the present invention and binds to the antigen.
  • the antibody that binds to the same epitope as the monoclonal antibody of the present invention refers to an antibody that recognizes and binds to the amino acid sequence of human IL-8 recognized by the monoclonal antibody of the present invention.
  • KD or “Kd” refers to a dissociation equilibrium constant of a specific antibody-antigen interaction.
  • antigenic determinant refers to three-dimensional sites that are discontinuous on the antigen and are recognized by the antibody or antigen binding fragment of the present invention.
  • the present invention includes not only complete antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies and other sequences. Therefore, the present invention further includes fragments, derivatives and analogs of the antibodies.
  • the antibodies include murine, chimeric, humanized, or fully human antibodies prepared by the technologies well known to a person skilled in the art.
  • Recombinant antibodies such as chimeric and humanized monoclonal antibodies, including human and non-human parts, can be prepared using DNA recombinant technology well known in the art.
  • the term “monoclonal antibody” refers to an antibody secreted by a clone derived from a single cell.
  • the monoclonal antibody is highly specific and is directed against a single epitope.
  • the cells may be eukaryotic, prokaryotic, or phage clonal cell strains.
  • the antibodies may be monospecific, bispecific, trispecific, or more multispecific.
  • the antibody of the present invention further includes its conservative variants, which means that compared with the amino acid sequence of the antibody of the present invention, at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids are substituted by amino acids with similar or similar properties to form a polypeptide.
  • conservative variant polypeptides are best produced by amino acid substitution according to Table A.
  • the present invention provides an anti-human IL-8 antibody (hereinafter referred to as IL-8 antibody) .
  • IL-8 antibody an anti-human IL-8 antibody
  • the present invention provides a high-specificity and high-affinity antibody against IL-8, including a heavy chain and a light chain, the heavy chain containing a heavy chain variable region (V H ) amino acid sequence, and the light chain containing a light chain variable region (V L ) amino acid sequence.
  • the heavy chain variable region comprises the following three complementary determining regions (CDRs) :
  • the heavy chain variable region has an amino acid sequence as shown in SEQ ID NO: 7.
  • the light chain variable region comprises the following three complementary determining regions (CDRs) :
  • the light chain variable region has an amino acid sequence as shown in SEQ ID NO: 8.
  • Any one of the abovementioned amino acid sequences includes a sequence in which at least one (for example, 1-5, 1-3, preferably 1-2, more preferably 1) amino acid is added, deleted, modified and/or substituted and that has IL-8 binding affinity.
  • sequence in which at least one (for example, 1-5, 1-3, preferably 1-2, more preferably 1) amino acid is added, deleted, modified and/or substituted is preferably an amino acid sequence having a homology of at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95%.
  • the antibody of the present invention may be a double-chain or single-chain antibody, and may be selected from animal antibodies, chimeric antibodies and humanized antibodies, more preferably humanized antibodies and human-animal chimeric antibodies, and more preferably fully humanized antibodies.
  • the antibody derivatives of the present invention may be single-chain antibodies and/or antibody fragments, such as: Fab, Fab', (Fab') 2 , or other antibody derivatives known in the art, as well as IgA, IgD, IgE, IgG and IgM antibodies or any one or more of antibodies of other subtypes.
  • the animal is preferably a mammal, such as murine.
  • the antibody of the present invention may be a murine antibody, chimeric antibody, humanized antibody, or CDR grafted and/or modified antibody targeting human IL-8.
  • any one or more of the sequences shown in SEQ ID NOs: 1, 2 and 3, or sequences thereof in which at least one amino acid is added, deleted, modified and/or substituted and that have IL-8 binding affinity, are within the CDR region of the heavy chain variable region (V H ) .
  • any one or more of the sequences shown in SEQ ID NOs: 4, 5 and 6, or sequences thereof in which at least one amino acid is added, deleted, modified and/or substituted and that have IL-8 binding affinity, are within the CDR region of the light chain variable region (V L ) .
  • V H CDR1, CDR2, and CDR3 are independently selected from any one or more of the sequences shown in SEQ IDs NOs: 1, 2, and 3, or sequences thereof in which at least one amino acid is added, deleted, modified and/or substituted and that have IL-8 binding affinity; and V L , CDR1', CDR2', and CDR3' are independently selected from any one or more of the sequences shown in SEQ ID NOs: 4, 5 and 6, or sequences thereof in which at least one amino acid is added, deleted, modified and/or substituted and that have IL-8 binding affinity.
  • the number of the added, deleted, modified and/or substituted amino acids is preferably not more than 40%of the total number of amino acids in the initial amino acid sequence, more preferably not more than 35%, more preferably 1-33%, more preferably 5-30%, more preferably 10-25%, and more preferably 15-20%.
  • the number of the added, deleted, modified and/or substituted amino acids is usually 1, 2, 3, 4 or 5, preferably 1-3, more preferably 1-2, and most preferably 1.
  • the antibody or antigen binding fragment thereof of the invention comprises the heavy chain variable region sequence comprising any amino acid sequence selected from the group consisting of SEQ ID NO: 7, and an amino acid sequence having more than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%sequence identity with any one of SEQ ID NO: 7.
  • the antibody or antigen binding fragment thereof of the invention comprises the light chain variable region sequence comprising any amino acid sequence selected from the group consisting of SEQ ID NO: 8, and an amino acid sequence having more than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99% sequence identity with any one of SEQ ID NO: 8.
  • the heavy chain variable region sequence comprises amino acid sequence of SEQ ID NO: 7 and the light chain variable region sequence comprises amino acid sequence of SEQ ID NO: 8.
  • any method suitable for producing monoclonal antibodies can be used to produce the IL-8 antibody of the present invention.
  • animals can be immunized with linked or natural IL-8 proteins or fragments thereof.
  • Suitable immunization methods can be used, including adjuvant, immunostimulant, and repeated booster immunizations, and one or more of the methods can be used.
  • IL-8 can be used as an immunogen (antigen) to produce non-human antibodies specific to IL-8 and screen the biological activity of the antibodies.
  • the immunogen can be used alone or used with one or more immunogenicity enhancers known in the art.
  • the immunogen can be purified from natural sources or produced in genetically modified cells.
  • DNA encoding the immunogen may be genomic or non-genomic (e.g., cDNA) in source.
  • a suitable genetic vector can be used to express the DNA encoding the immunogen, and the vector includes but is not limited to adenoviral vectors, baculoviral vectors, plasmids, and non-viral vectors.
  • the humanized antibody may be selected from any kind of immunoglobulin, including IgM, IgD, IgG, IgA, and IgE.
  • the antibody is an IgG antibody of an IgG1 or IgG4 subtype.
  • any type of light chain can be used in the compound and method herein.
  • the ⁇ or ⁇ chain or variant thereof can be used in the compound and method of the present invention.
  • the sequence of the DNA molecule of the antibody or fragment thereof of the present invention can be obtained by conventional technology, such as PCR amplification or genomic library screening etc.
  • the coding sequences of different light chains and heavy chains can be fused together in different combinations to form single-chain antibodies.
  • Optimized single-chain antibodies can be obtained by testing and analyzing the functions of single-chain antibodies modified by different combinations or different linkages.
  • relevant sequences can be obtained on a large scale by recombination. This usually involves cloning into a vector, then transferring to a cell, and then isolating from the proliferated host cell by a conventional method to obtain the relevant sequences.
  • the relevant sequences can also be synthesized artificially, especially in the case that the fragments are short. Usually, a plurality of small fragments are first synthesized and then joined to obtain very long fragments. The DNA sequence can then be introduced into various existing DNA molecules (or such as vectors) and cells known in the art.
  • nucleic acid molecule refers to DNA molecules and RNA molecules.
  • the nucleic acid molecule may be single-stranded or double-stranded, but preferably double-stranded DNA.
  • the nucleic acid is “effectively linked. ” For example, if a promoter or enhancer affects the transcription of a coding sequence, the promoter or enhancer is effectively linked to the coding sequence.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • the vector is a “plasmid” , which refers to a circular double-stranded DNA loop to which an additional DNA fragment can be linked.
  • the present invention further relates to vectors containing the above-mentioned suitable DNA sequence and suitable promoter or control sequence. These vectors can be used to transform appropriate host cells to enable the host cells to express proteins.
  • the term “host cell” refers to a cell into which an expression vector has been introduced.
  • the host cells may be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as plant or animal cells (e.g., mammalian cells) .
  • the step of transforming host cells with recombinant DNA in the present invention can be performed by the technologies well known in the art.
  • the obtained transformants can be cultured by conventional methods, and the transformants express polypeptides encoded by the genes of the present invention. Culture is carried out in a conventional medium under suitable conditions according to the used host cells.
  • the transformed host cells are cultured under conditions suitable for expression of the antibodies of the present invention.
  • a conventional immunoglobulin purification step proceeds, to obtain the antibodies of the present invention by conventional isolation and purification means well known to a person skilled in the art, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography.
  • the obtained monoclonal antibodies can be identified by conventional means.
  • the binding specificity of the monoclonal antibodies can be measured by immunoprecipitation or binding assay in vitro (e.g., radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA) ) .
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunosorbent assay
  • a nucleotide sequence encoding a desired molecule can be obtained by a recombination method known in the art, for example, by screening a library from cells expressing genes, and obtaining a gene from a vector known to include the gene, or by directly isolating a gene from the cell and tissue containing the gene through a standard technology.
  • the gene of interest can be produced synthetically.
  • the present invention further provides a vector into which an expression cassette of the present invention is inserted.
  • Vectors derived from retroviruses such as lentiviruses are suitable tools to achieve long-term gene delivery, because they allow long-term and stable integration of transgenes and propagation in daughter cells.
  • the lentiviral vectors have advantages over vectors derived from oncogenic retroviruses such as murine leukemia viruses, because they can transduce non-proliferating cells such as hepatocytes. They also have the advantage of low immunogenicity.
  • the expression cassette or nucleotide sequence of the present invention is usually operably linked to a promoter and incorporated into an expression vector.
  • the vector is suitable for replication and integration of eukaryotic cell.
  • a typical cloning vector contains a transcription and translation terminator that can be used to regulate the expression of a desired nucleotide sequence, an initial sequence, and a promoter.
  • the expression construct of the present invention can also be used for nucleic acid immunization and gene therapy by using standard gene delivery schemes. Methods of gene delivery are known in the art. See, for example.
  • the present invention provides a gene therapy vector.
  • the nucleic acid can be cloned into many types of vectors.
  • the nucleic acid can be cloned into such vectors, which include, but are not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids.
  • Specific vectors of interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.
  • the expression vectors can be provided to cells in the form of viral vectors.
  • Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses.
  • a suitable vector contains an origin of replication that functions in at least one organism, a promoter sequence, a convenient restriction enzyme site, and one or more optional markers.
  • retroviruses provide a convenient platform for gene delivery systems.
  • the selected gene can be inserted into a vector and packaged into a retrovirus particle by using a technology known in the art.
  • the recombinant virus can then be isolated and transferred to an object cell in vivo or ex vivo.
  • retrovirus systems are known in the art.
  • adenoviral vectors are used.
  • Many adenoviral vectors are known in the art.
  • lentiviral vectors are used.
  • promoter components can regulate the frequency of transcription initiation. Generally, these are located in a 30-110 bp region upstream of the initiation site, although it has recently been shown that many promoters also contain functional components downstream of the initiation site. The spacing between the promoter components is often flexible in order to maintain promoter functions when one component is inverted or moved relative to the other one. In a thymidine kinase (tk) promoter, the activity does not begin to decrease until that the spacing between promoter components can be increased by 50 bp. Depending on the promoter, it appears that individual components can act cooperatively or independently to initiate transcription.
  • tk thymidine kinase
  • a suitable promoter is an immediate early cytomegalovirus (CMV) promoter sequence.
  • the promoter sequence is a strong constitutive promoter sequence capable of driving high-level expression of any polynucleotide sequence operably linked thereto.
  • Another example of a suitable promoter is an elongation growth factor-1 ⁇ (EF-1 ⁇ ) .
  • constitutive promoter sequences can also be used, including but not limited to simian virus 40 (SV40) early promoters, mouse mannary tumour virus (MMTV) and human immunodeficiency virus (HIV) long terminal repeat (LTR) promoters, MoMuLV promoters, avian leukemia virus promoters, Epstein-Barr virus immediate early promoters, Ruth sarcoma virus promoters, and human gene promoters, such as but not limited to actin promoters, myosin promoters, heme promoters and creatine kinase promoters.
  • the present invention should not be limited to the application of constitutive promoters. Inducible promoters are also considered part of the present invention.
  • an inducible promoter provides a molecular switch that can turn on expression of a polynucleotide sequence operably linked to the inducible promoter when such expression is desired, or turn off the expression when the expression is not desired.
  • the inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters and tetracycline promoters.
  • the vector can be easily introduced into a host cell, for example, a mammalian, bacteria, yeast, or insect cell, by any method in the art.
  • the expression vector can be transferred into the host cell by physical, chemical or biological means.
  • Physical methods of introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Methods of producing cells including vectors and/or exogenous nucleic acids are well known in the art.
  • the preferred method of introducing polynucleotides into host cells is calcium phosphate transfection.
  • Biological methods for introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors.
  • Viral vectors especially retroviral vectors, have become the most widely used method of inserting genes into mammalian cells such as human cells.
  • Other viral vectors can be derived from lentivirus, poxvirus, herpes simplex virus I, adenovirus, adeno-associated virus, etc.
  • Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, and beads; and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, and beads
  • lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • Exemplary colloidal systems used as delivery vehicles in vitro and in vivo are liposomes (e.g., artificial membrane vesicles) .
  • an exemplary delivery vehicle is liposome.
  • Lipid preparation are considerable to introduce nucleic acids into host cells (in vitro, ex vivo or in vivo) .
  • the nucleic acids can be associated with lipids.
  • a lipid-associated nucleic acid can be encapsulated in the aqueous interior of a liposome, dispersed in the lipid bilayer of the liposome, attached to the liposome via a linker molecule associated with both the liposome and an oligonucleotide, trapped in the liposome, complexed with the liposome, dispersed in a lipid-containing solution, mixed with the lipid, combined with the lipid, contained in the lipid as a suspension, contained in micelles or complexed with micelles, or associated with the lipid in other way.
  • the lipid, lipid/DNA or lipid/expression vector associated with a composition is not limited to any specific structure in the solution.
  • Lipids are fatty substances, which can be naturally occurring or synthetic lipids.
  • the lipids include fat droplets, which occur naturally in cytoplasm and in such compounds containing long-chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols and aldehydes.
  • the vector is a lentiviral vector.
  • the present invention provides a preparation containing the heavy chain variable region according to the first aspect of the present invention, the heavy chain according to the second aspect of the present invention, the light chain variable region according to the third aspect of the present invention, the light chain according to the fourth aspect of the present invention, or the antibody according to the fifth aspect of the present invention, the recombinant protein according to the sixth aspect of the present invention, the antibody-drug conjugate according to the seventh aspect of the present invention, the polynucleotide according to the eighth aspect of the present invention, the vector according to the ninth aspect of the present invention, and/or genetically engineered host cell according to the tenth aspect of the present invention, and a pharmaceutically acceptable carrier, diluent or excipient.
  • the preparation is a liquid preparation.
  • the preparation is an injection.
  • the preparation may include a buffer such as neutral buffered saline, or sulfate buffered saline etc.; a carbohydrate such as glucose, mannose, sucrose or dextran, or mannitol; a protein; a polypeptide or amino acid such as glycine; an antioxidant; a chelating agent such as EDTA or glutathione; an adjuvant (for example, aluminum hydroxide) ; and a preservative.
  • the preparation of the present invention is preferably administered for intravenous or intraperitoneal administration.
  • the antibodies of the present invention can be used in test applications, for example, to test samples, thereby providing diagnostic information.
  • the samples (specimens) used include cells, tissue samples and biopsy samples.
  • the term “biopsy” used in the present invention shall include all kinds of biopsy known to a person skilled in the art. Therefore, the biopsy used in the present invention may include, for example, tissue samples prepared by endoscopic methods or organ puncture or needle biopsy.
  • the samples used in the present invention include immobilized or preserved cell or tissue samples.
  • the present invention also provides a kit containing the antibody (or fragment thereof) and scFv of the present invention.
  • the kit further includes a vessel, an instruction for use, a buffer, etc.
  • the antibody of the present invention can be immobilized on a test plate.
  • the main advantages of the present invention comprise:
  • the present invention provides an antibody which binds to IL-8 with high affinity and their use in blocking or inhibiting IL-8 induced activities, e.g., proinflammatory activities, chemotactic activities and angiogenesis, to treat immune, autoimmune, inflammatory or infectious diseases and cancer associated with IL-8.
  • IL-8 induced activities e.g., proinflammatory activities, chemotactic activities and angiogenesis
  • phage human anti-IL-8 Fab-antibodies were obtained from the combinatorial phage Fab-display library which created by Sanyou Biopharmaceuticals Co. Ltd. The selection was performed on human IL-8 by phage display method [Science. 1985 Jun 14; 228 (4705) : 1315-7; Nature. 1991 Aug 15; 352 (6336) : 624-8] but with the use of magnetic particles and the KingFisher instrument and immunotube-based panning approach.
  • antigen biotinylated IL-8
  • streptavidin magnetic particles were immobilized on the surface of streptavidin magnetic particles by incubating the protein with particles for one hour at room temperature on a rotator. The particles were then washed with PBS (pH 7.4) , then the particles were blocked with 2.5%BSA solution on PBS (pH 7.4) for one hour. Then the phage solution in PBS (pH 7.4) with 2.5%BSA was added to antigen-bound magnetic particles. The mixture was incubated for 40 minutes with stirring at room temperature. Washings the magnetic particles with PBST (PBS with 0.05%tween-20) for several times to remove unbound phages.
  • PBST PBS with 0.05%tween-20
  • Phages that remained bound with the antigen on the surface of magnetic particles were eluted from the particles with trypsin solution during 15 min under stirring.
  • E. coli (SS320) bacteria were infected with phages obtained; phages were produced therein and isolated and used in the next selection round.
  • DNAs (phagemids) were isolated from the phages, and the antibody variable domain genes were cloned into expression vectors for the production of Fab in E. coli cells.
  • the phage display derived anti-IL-8 antibody had been used in the following examples.
  • ELISA was used to search for Fab binding to human IL-8. All subsequent steps were carried out according to the standard ELISA protocol. ELISA plate wells were coated with 30 ⁇ l of human IL-8 (2 ⁇ g/ml) , hermetically sealed and incubated overnight at 4 °C. Washings the plates with PBST (PBS with 0.05%tween-20) for three times. To block non-specific binding, a blocking buffer of 20 ⁇ L of PMSM (5%skimmed milk in PBS) was added. The plates were incubated on a shaker for an hour at room temperature.
  • PBST PBS with 0.05%tween-20
  • the plates were shaken on a rotary shaker (50 min, room temperature) and washed for nine times with PBST (PBS with 0.05%tween-20) buffer.
  • PBST PBS with 0.05%tween-20
  • the colourimetric signal was developed by adding TMB (30 ⁇ L/well) until saturation (average 5-10 min) , then the development was quenched by adding stop solution (30 ⁇ L/well, 2M sulphuric acid) .
  • Colour signal was measured at a wavelength of 450 nm using a suitable plate reader (from Molecular Device) .
  • the cloned V L and V H regions from Fab sequence in “1.1 Selection of Phage Antibody Fab Libraries” were subcloned into the expression cassettes of an immunoglobulin expression vector (pcDNA3.4) .
  • the expression vector was transfected into Chinese hamster ovary (CHO-s) cells. The cultures were incubated on an orbital shaker platform rotating at 135 rpm. The culture medium was harvested after 4-7 days. After purified by Protein-A affinity chromatography, the affinity of recombinant antibody produced from CHO cells was assessed by ELISA and kinetic analyses using biolayer interferometry (BLI) technology in a GATOR instrument.
  • ELISA analysis was used to test anti-IL-8 antibodies binding to human IL-8.
  • ELISA plate wells were coated with 30 ⁇ l of human IL-8 (2 ⁇ g/ml) , hermetically sealed and incubated overnight at 4 °C. Washings the plates with PBST for three times. Blocking buffer of 20 ⁇ L of PMSM was added and the plates were incubated on a shaker for an hour at room temperature. After washing with PBST three times, 30 ⁇ l per well of the test anti-IL-8 mAbs mixed with an equal volume of PMSM was added. The plates were again incubated, shaking for one hour at room temperature, followed by washing for three times each plate well with PBST buffer to eliminate un-bounded antibodies.
  • anti-human-Kappa+Lambda-HRP (30 ⁇ L/well) were added at 1: 5000 ratio in PBSM and incubated for one hour. The plates were washed for six times with PBST buffer. TMB (30 ⁇ L/well) was added for color rendering and then the development was quenched by adding stop solution (30 ⁇ L/well, 2M sulphuric acid) . Optical density was measured at a wavelength of 450 nm using a suitable plate reader (from Molecular Device) . As shown in FIG. 1, the antibodies binded to human IL-8 with high affinity.
  • BLI was a label free biomolecular detection method and biomolecular interactions were detected by measuring the interference pattern of white light reflected from the surface of a biosensor.
  • the data of dissociation (kd) and association (ka) rate constants were obtained using GATOR software.
  • the equilibrium dissociation constants (K D ) were calculated from the ratio of kd over ka. As shown in the FIG. 2 and Table 1, all the anti-IL-8 mAbs bind to IL-8 with high affinity.
  • the heavy chain variable region of the antibody LJH001-B30 had the following three complementary determining regions (CDRs) :
  • the heavy chain variable region of the antibody LJH001-B30 had an amino acid sequence as shown in SEQ ID NO: 7 (QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCASPRYFDWSEDHYDAFDIWGQGTMVTVSS) ;
  • the light chain variable region of the antibody LJH001-B30 had the following three complementary determining regions (CDRs) :
  • the light chain variable region of the antibody LJH001-B30 had an amino acid sequence as shown in SEQ ID NO: 8 (QSALTQPPSASGTPGQRVTISCSGSSSNIGSYTVNWFQQLPGTAPKLLIYSNTQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLHGAVFGGGTQLAVL) .
  • the thermal stability of anti-IL-8 antibody was measured by Differential Scanning Fluorimetry (DSF) technology in a ABI7500 Fast Real-Time PCR instrument. DSF could be conveniently used to determining a protein’s melting temperature (Tm) .
  • DSF also known as Protein Thermal Shift Assay
  • DSF monitored thermally induced protein denaturation by measuring changes in fluorescence of a dye that binds preferentially to unfolded protein (such as Sypro Orange, which binded to hydrophobic regions of proteins exposed by unfolding) which typically performed by using a real-time PCR instrument.
  • the stability of a protein was related to its Gibbs free energy of unfolding which was temperature-dependent. Briefly, 20 ⁇ l of 0.2mg/ml anti-IL-8 antibody sample in PBS (pH 7.4) and containing 100 ⁇ SYPRO Orange was heated from 25 to 99 °C. in 1 °C.
  • IL-8 antibody LJH001-B30 to inhibit IL-8 induced Jurkat-CXCR1 (Jurkat cell over expressing human CXCR1) migration was evaluated utilizing a Boyden chamber chemotaxis assay.
  • Human IL-8 (20ng/ml) was incubated with varying concentrations of anti-IL-8 antibody LJH001-B30 in the lower compartment of the Boyden chamber.
  • Jurkat-CXCR1 (4 ⁇ 10 5 cells) were incubated in the upper compartment. The assay was incubated at 37 °C for 4-6 hours.
  • the data depicted in FIG. 4 showed that Jurkat-CXCR1 chemotaxis was inhibited in a dose-dependent manner by anti-IL-8 antibody.

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Abstract

La présente invention concerne un anticorps anti-IL-8. Plus particulièrement, la présente invention concerne une région variable de chaîne lourde d'un anticorps, la région variable de chaîne lourde comprenant les trois régions déterminant la complémentarité (CDR) suivantes : CDR1 telle que représentée dans SEQ ID NO : 1, CDR2 telle que représentée dans SEQ ID NO : 2, et CDR3 tel que représenté dans SEQ ID NO : 3. L'anticorps selon la présente invention peut se lier à IL-8 avec une affinité élevée et bloquer ou inhiber des activités induites par IL-8, par exemple, des activités pro-inflammatoires, des activités chimiotactiques et l'angiogenèse, pour traiter des maladies immunitaires, auto-immunes, inflammatoires ou infectieuses et un cancer associé à l'IL-8.
PCT/CN2022/134825 2022-11-28 2022-11-28 Anticorps anti-il-8 Ceased WO2024113122A1 (fr)

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