An anti-IL-8 antibody

Abstract

The present invention relates to an anti-IL-8 antibody. Specifically, the present invention provides an antibody heavy chain variable region, wherein the heavy chain variable region includes the following three complementary determining regions (CDRs): CDR1 as shown in SEQ ID NO: 1, CDR2 as shown in SEQ ID NO: 2, and CDR3 as shown in SEQ ID NO: 3. The antibody of the present invention can bind to IL-8 with high affinity and block or inhibit IL-8-induced activities, such as pro-inflammatory activity, chemotactic activity and angiogenesis, to treat immune, autoimmune, inflammatory or infectious diseases and cancers associated with IL-8.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an anti-IL-8 antibody. Background Art

[0002] Interleukin 8 (IL-8, also known as CXCL8), previously known as monocyte-derived neutrophil chemoattractant factor (MDNCF) or neutrophil induction / activation protein-1 (NAP-1), is an ELR + Chemokines display chemotactic activity towards specific types of leukocytes in inflammatory diseases.

[0003] IL-8 is a polypeptide that is secreted by fibroblasts, vascular endothelial cells, macrophages, dendritic cells (DCs), lymphocytes, keratinocytes, melanocytes, hepatocytes, and various tumor cells. IL-8 is known to stimulate neutrophil chemotaxis and participate in neutrophil migration to sites of inflammation by binding to high-affinity receptors (CXCR1 and CXCR2) on the neutrophil surface. IL-8 activates neutrophils by accelerating degranulation, the production of reactive oxygen species (ROS), and the destruction of infiltrated tissues.

[0004] Although neutrophil inflammatory responses are crucial for destroying invading pathogens, inappropriate neutrophil activation and infiltration may contribute to numerous diseases. It has been hypothesized that prolonged high expression of IL-8 may be associated with the development of autoimmune, inflammatory, or infectious diseases, as well as cancer. IL-8 has been implicated in rheumatoid arthritis, asthma, gout, inflammatory bowel disease (IBD), and sepsis, all of which are characterized by inflammation accompanied by neutrophil infiltration and tissue damage.

[0005] IL-8 is known to promote angiogenesis and tumor growth. IL-8 can also attract myeloid-derived suppressor cells (MDSCs) into the tumor microenvironment (TME) and contribute to tumor immune evasion.

[0006] Human tumor cells, such as melanoma, breast cancer, hepatocellular carcinoma, castration-resistant prostate cancer, colorectal cancer, and glioma, express IL-8, which plays a role in tumor invasion and metastasis. IL-8 inhibition can prevent inflammatory cell infiltration of tissues, reduce angiogenesis, and decrease the trafficking of MDSCs, thereby ameliorating disease.

[0007] Therefore, there is a need in the art to develop an antibody that can bind to IL-8 for use in diagnosing and treating IL-8-mediated diseases. Summary of the Invention

[0008] The object of the present invention is to provide an antibody that binds to IL-8 with high affinity and its use in blocking or inhibiting IL-8-induced activity.

[0009] The first aspect of the present invention provides an antibody heavy chain variable region, wherein the heavy chain variable region comprises the following three complementarity determining regions (CDRs):

[0010] CDR1 as shown in SEQ ID NO: 1,

[0011] CDR2 as shown in SEQ ID NO: 2, and

[0012] CDR3 as shown in SEQ ID NO:3.

[0013] In another preferred embodiment, the antibody is an anti-IL-8 antibody.

[0014] In another preferred embodiment, the IL-8 is human IL-8.

[0015] In another preferred embodiment, any one of the above amino acid sequences further includes a derivative sequence optionally obtained by adding, deleting, modifying and / or replacing at least 1 (such as 1-3, preferably 1-2, more preferably 1) amino acid.

[0016] In another preferred embodiment, the derived sequence retains the binding affinity for IL-8.

[0017] In another preferred embodiment, the derivative sequence is resistant to IL-8.

[0018] In another preferred embodiment, the heavy chain variable region further includes a human FR region or a mouse FR region.

[0019] In another preferred example, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 7.

[0020] The second aspect of the present invention provides an antibody heavy chain, wherein the heavy chain comprises the heavy chain variable region as described in the first aspect of the present invention.

[0021] In another preferred embodiment, the heavy chain of the antibody further includes a heavy chain constant region.

[0022] In another preferred embodiment, the heavy chain constant region is of human, mouse or rabbit origin.

[0023] A third aspect of the present invention provides a light chain variable region of an antibody, wherein the light chain variable region comprises the following three complementarity determining regions (CDRs):

[0024] CDR1' as shown in SEQ ID NO:4,

[0025] CDR2' as shown in SEQ ID NO: 5, and

[0026] CDR3' as shown in SEQ ID NO:6.

[0027] In another preferred embodiment, any one of the above amino acid sequences further includes a derivative sequence optionally obtained by adding, deleting, modifying and / or replacing at least 1 (such as 1-3, preferably 1-2, more preferably 1) amino acid.

[0028] In another preferred embodiment, the derived sequence retains the binding affinity for IL-8.

[0029] In another preferred embodiment, the derivative sequence is resistant to IL-8.

[0030] In another preferred embodiment, the light chain variable region further includes a human FR region or a mouse FR region.

[0031] In another preferred example, the light chain variable region has the amino acid sequence shown in SEQ ID NO: 8.

[0032] The fourth aspect of the present invention provides a light chain of an antibody, wherein the light chain comprises the light chain variable region as described in the third aspect of the present invention.

[0033] In another preferred embodiment, the light chain of the antibody further includes a light chain constant region.

[0034] In another preferred embodiment, the light chain constant region is of human, mouse or rabbit origin.

[0035] A fifth aspect of the present invention provides an antibody comprising:

[0036] (1) the heavy chain variable region as described in the first aspect of the present invention; and / or

[0037] (2) The light chain variable region as described in the third aspect of the present invention.

[0038] In another preferred embodiment, the antibody comprises the heavy chain as described in the second aspect of the present invention; and / or the light chain as described in the fourth aspect of the present invention.

[0039] In another preferred embodiment, the antibody is an anti-IL-8 antibody.

[0040] In another preferred embodiment, the IL-8 is human IL-8.

[0041] In another preferred embodiment, the antibody is selected from the following group: animal-derived antibodies, chimeric antibodies, humanized antibodies, or a combination thereof.

[0042] In another preferred embodiment, the antibody is a double-chain antibody or a single-chain antibody.

[0043] In another preferred embodiment, the antibody is a monoclonal antibody.

[0044] In another preferred embodiment, the antibody is a partially or fully humanized monoclonal antibody.

[0045] In another preferred embodiment, the heavy chain variable region sequence of the antibody is as shown in SEQ ID NO: 7; and / or

[0046] The light chain variable region sequence of the antibody is shown in SEQ ID NO: 8.

[0047] In another preferred embodiment, the antibody is of IgG, IgM, IgA, IgD or IgE type.

[0048] In another preferred embodiment, the antibody is in the form of a drug conjugate.

[0049] A sixth aspect of the present invention provides a recombinant protein, comprising:

[0050] (i) the heavy chain variable region of the first aspect of the invention, the heavy chain of the second aspect of the invention, the light chain variable region of the third aspect of the invention, the light chain of the fourth aspect of the invention, or the antibody of the fifth aspect of the invention; and

[0051] (ii) optionally a tag sequence to facilitate expression and / or purification.

[0052] In another preferred embodiment, the tag sequence includes a 6His tag.

[0053] In another preferred embodiment, the recombinant protein includes a fusion protein.

[0054] In another preferred embodiment, the recombinant protein is a monomer, a dimer, or a multimer.

[0055] A seventh aspect of the present invention provides an antibody-drug conjugate, comprising:

[0056] (a) an antibody portion selected from the group consisting of the heavy chain variable region of the first aspect of the invention, the heavy chain of the second aspect of the invention, the light chain variable region of the third aspect of the invention, the light chain of the fourth aspect of the invention, or the antibody of the fifth aspect of the invention, or a combination thereof; and

[0057] (b) a conjugated moiety conjugated to the antibody portion, wherein the conjugated moiety is selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.

[0058] In another preferred embodiment, the antibody portion and the coupling portion are coupled via a chemical bond or a linker.

[0059] The eighth aspect of the present invention provides a polynucleotide encoding a polypeptide selected from the group consisting of:

[0060] (1) the heavy chain variable region of the first aspect of the present invention, the heavy chain of the second aspect of the present invention, the light chain variable region of the third aspect of the present invention, the light chain of the fourth aspect of the present invention, or the antibody of the fifth aspect of the present invention; or

[0061] (3) The recombinant protein as described in the sixth aspect of the present invention.

[0062] The ninth aspect of the present invention provides a vector comprising the polynucleotide as described in the eighth aspect of the present invention.

[0063] In another preferred embodiment, the vector includes: bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, or other vectors.

[0064] The tenth aspect of the present invention provides a genetically engineered host cell, which comprises the vector described in the ninth aspect of the present invention or the polynucleotide described in the eighth aspect of the present invention is integrated into the genome of the host cell.

[0065] The eleventh aspect of the present invention provides a pharmaceutical composition, comprising:

[0066] The heavy chain variable region as described in the first aspect of the present invention, the heavy chain as described in the second aspect of the present invention, the light chain variable region as described in the third aspect of the present invention, the light chain as described in the fourth aspect of the present invention, or the antibody as described in the fifth aspect of the present invention, the recombinant protein as described in the sixth aspect of the present invention, the antibody-drug conjugate as described in the seventh aspect of the present invention, the polynucleotide as described in the eighth aspect of the present invention, the vector as described in the ninth aspect of the present invention and / or the genetically engineered host cell as described in the tenth aspect of the present invention.

[0067] In another preferred embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent or excipient.

[0068] In another preferred embodiment, the pharmaceutical composition is a liquid preparation.

[0069] In another preferred embodiment, the dosage form of the pharmaceutical composition is an injection.

[0070] In another preferred embodiment, the injection is an intravenous injection.

[0071] The twelfth aspect of the present invention provides a use of the heavy chain variable region as described in the first aspect of the present invention, the heavy chain as described in the second aspect of the present invention, the light chain variable region as described in the third aspect of the present invention, the light chain as described in the fourth aspect of the present invention, the antibody as described in the fifth aspect of the present invention, and / or the recombinant protein as described in the sixth aspect of the present invention, for (i) preparing a drug or preparation for blocking or inhibiting IL-8-induced activity; (ii) preparing a drug or preparation for preventing and / or treating diseases related to IL-8; and / or (iii) preparing a detection reagent or kit.

[0072] In another preferred embodiment, the IL-8-induced activity includes pro-inflammatory activity, chemotactic activity and / or angiogenesis.

[0073] In another preferred embodiment, the diseases associated with IL-8 include autoimmunity, inflammation, infectious diseases and / or tumors.

[0074] In another preferred embodiment, the disease associated with IL-8 is a disease associated with increased or unbalanced IL-8 levels.

[0075] In another preferred embodiment, the disease associated with IL-8 is an immune, autoimmune, inflammatory, infectious disease or a disease characterized by increased or unbalanced human IL-8 levels, in particular rheumatoid arthritis, ulcerative colitis, asthma, chronic obstructive pulmonary disease (COPD), gout, cancer, influenza, 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 arterial 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 diabetes, purpura, and acute pancreatitis.

[0076] In another preferred embodiment, the disease associated with IL-8 is an inflammatory or skin disease, such as psoriasis, palmoplantar pustulosis (PPP), bullous pemphigoid, pemphigus, contact dermatitis, eczema, lupus erythematosus and atopic dermatitis.

[0077] In another preferred embodiment, the disease associated with IL-8 is a human virus-related disease or infection, such as the common cold caused by human rhinovirus, coronavirus, other enterovirus, herpes virus, influenza virus, parainfluenza virus, respiratory syncytial virus or adenovirus infection and hepatitis C.

[0078] In another preferred embodiment, the detection reagent or kit is used to detect IL-8 in a sample.

[0079] In another preferred embodiment, the detection reagent is a test chip.

[0080] A thirteenth aspect of the present invention provides a method for detecting IL-8 in a sample in vitro, the method comprising the steps of:

[0081] (1) contacting the sample with the antibody according to the fifth aspect of the present invention in vitro;

[0082] (2) Detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of IL-8 in the sample.

[0083] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0084] The fourteenth aspect of the present invention provides a detection plate, which comprises: a substrate (support plate) and a test strip, wherein the test strip comprises the antibody as described in the fifth aspect of the present invention or the antibody-drug conjugate as described in the seventh aspect of the present invention.

[0085] In a fifteenth aspect, the present invention provides a method for blocking or inhibiting IL-8-induced activity; and / or preventing and / or treating diseases associated with IL-8, the method comprising administering to the subject 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, thereby blocking or inhibiting IL-8-induced activity; and / or preventing and / or treating diseases associated with IL-8.

[0086] In another preferred embodiment, the subject includes humans and non-human mammals.

[0087] In another preferred embodiment, the non-human mammal includes cows, horses, sheep, dogs, cats or mice.

[0088] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0089] Figure 1 The figures show the results of ELISA analysis of the binding of the anti-IL-8 antibody of the present invention to human IL-8.

[0090] Figure 2Results of biolayer interferometry (BLI) binding assays performed using a GATOR instrument. Dissociation (KD or kd) and association (ka) rate constants were obtained using GATOR software. The equilibrium dissociation constant (K D ) is calculated from the ratio of kd to ka.

[0091] Figure 3 The results of differential scanning fluorimetry (DSF) analysis were performed in an ABI7500 fast real-time PCR instrument.

[0092] Figure 4 The results of the inhibition of IL-8-mediated CXCR1+ cell chemotaxis by anti-IL-8 antibodies were determined by migration assay using a Boyden chamber. “B30” refers to the antibody LJH001-B30, and “positive” refers to the monoclonal mouse IgG1 clone #6217. DETAILED DESCRIPTION

[0093] The present invention provides an antibody that binds to IL-8 with high affinity, and its use in blocking or inhibiting IL-8-induced activities, such as pro-inflammatory activity, chemotactic activity and angiogenesis, to treat IL-8-related immune, autoimmune, inflammatory or infectious diseases and cancers.

[0094] the term

[0095] As used herein, the terms "include," "comprise," and "contain" are used interchangeably to encompass not only open definitions but also semi-closed and closed definitions. In other words, the terms encompass "consisting of," "consisting essentially of."

[0096] As used herein, the term "interleukin 8" is abbreviated as IL-8.

[0097] As used herein, "domain" refers to a region of a polypeptide that is independent of other regions and folds into a specific structure.

[0098] As used herein, "single-chain variable region fragment (ScFv)" refers to a single-chain polypeptide derived from an antibody that retains the ability to bind to an antigen. Examples of ScFv include antibody polypeptides formed by recombinant DNA technology, in which the Fv regions of immunoglobulin heavy chain (H chain) and light chain (L chain) fragments are linked via a spacer sequence. Various methods for improving ScFv are known to those skilled in the art.

[0099] As used herein, the term "administer" refers to the application of an exogenous drug, therapeutic agent, diagnostic agent, or composition to an animal, human, subject, cell, tissue, organ, or biological fluid. "Administer" can refer to therapy, pharmacokinetics, diagnosis, research, and experimental methods. Treatment of cells includes contact of an agent with a cell, as well as contact of an agent with a fluid, or contact of a fluid with a cell. "Administer" also means in vitro and ex vivo treatment with an agent, diagnostic, binding composition, or with another cell. "Administer" when applied to a human, animal, or research subject refers to treatment, prophylaxis, or preventative measures, research, and diagnosis; including contact of an anti-human IL-8 antibody with a human or animal, subject, cell, tissue, physiological compartment, or physiological fluid.

[0100] As used herein, the term "treatment" refers to administering an internal or external therapeutic agent, including any of the anti-human IL-8 antibodies and compositions of the present invention, to a patient experiencing one or more symptoms of a disease for which the therapeutic agent is known to have therapeutic effects. Generally, the therapeutic agent is administered to the patient in an amount effective to alleviate one or more symptoms of the disease (a therapeutically effective amount).

[0101] As used herein, the term "optional" or "optionally" means that the subsequently described event or circumstance may occur but need not occur. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that the antibody heavy chain variable regions of a specific sequence may have but need not have, and may have 1, 2, or 3.

[0102] As used herein, "sequence identity" refers to the degree of identity between two nucleic acid or amino acid sequences when optimally aligned and compared with appropriate mutations such as substitutions, insertions, or deletions. The sequence identity between a sequence described herein and a sequence to which it is identical 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%.

[0103] In the present invention, the amino acid sequence is from N-terminus to C-terminus.

[0104] IL-8

[0105] Interleukin 8 (IL-8, also known as CXCL8), previously known as monocyte-derived neutrophil chemoattractant factor (MDNCF) or neutrophil induction / activation protein-1 (NAP-1), is an ELR + Chemokines display chemotactic activity towards specific types of leukocytes in inflammatory diseases.

[0106] IL-8 is a polypeptide that can be secreted by fibroblasts, vascular endothelial cells, macrophages, dendritic cells (DCs), lymphocytes, keratinocytes, melanocytes, hepatocytes and various tumor cells.

[0107] Antibody

[0108] As used herein, the term "antibody" refers to an immunoglobulin, a tetrapeptide structure composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds. Immunoglobulins differ in their antigenicity due to the amino acid composition and order of their constant regions. Consequently, immunoglobulins can be divided into five classes, or isotypes, namely IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε, respectively. Within the same class, Igs are further divided into subclasses based on the amino acid composition of their hinge regions and the number and location of heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are classified as either kappa or lambda chains, depending on the constant region. Each of the five classes of Ig can have either kappa or lambda chains. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known in the art.

[0109] The antibody light chain of the present invention may further comprise a light chain constant region, wherein the light chain constant region comprises a human or murine κ, λ chain or a variant thereof.

[0110] In the present invention, the antibody heavy chain described in the present invention may further comprise a heavy chain constant region, wherein the heavy chain constant region comprises human or mouse IgG1, IgG2, IgG3, IgG4 or variants thereof. The sequence of approximately 110 amino acids near the N-terminus of the antibody heavy chain and light chain varies greatly and is the variable region (Fv region); the remaining amino acid sequence near the C-terminus is relatively stable and is the constant region. The variable region includes three hypervariable regions (HVRs) and four framework regions (FRs) with relatively conserved sequences. The three hypervariable regions determine the specificity of the antibody and are also called complementarity determining regions (CDRs). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of three CDR regions and four FR regions, and the order arranged from amino terminus to carboxyl terminus is: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The three CDR regions of the light chain are referred to as CDR1', CDR2' and CDR3'; the three CDR regions of the heavy chain are referred to as CDR1, CDR2 and CDR3.

[0111] The antibodies of the present invention include murine antibodies, chimeric antibodies, and humanized antibodies, with humanized antibodies being preferred. The term "murine antibody" as used herein refers to monoclonal antibodies against human IL-8 prepared according to the knowledge and skill in the art. During preparation, a test subject is injected with an IL-8 antigen, and hybridomas expressing antibodies with the desired sequence or functional properties are then isolated. In a preferred embodiment of the present invention, the murine IL-8 antibody or antigen-binding fragment thereof may further comprise a light chain constant region of a murine kappa or lambda chain, or variants thereof, or a heavy chain constant region of a murine IgG1, IgG2, IgG3, or variants thereof.

[0112] The term "chimeric antibody" refers to an antibody formed by fusing the variable region of a mouse antibody with the constant region of a human antibody, which can reduce the immune response induced by the mouse antibody.

[0113] The term "humanized antibody," also known as a CDR-grafted antibody, refers to an antibody produced by grafting murine CDR sequences onto a human antibody variable region framework, i.e., different types of human germline antibody framework sequences. Humanized antibodies can overcome the heterologous reactions induced by chimeric antibodies due to the large number of murine protein components. Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. To avoid a decrease in immunogenicity and a resulting decrease in activity, minimal reverse mutations or back mutations can be performed on the human antibody variable region framework sequences to maintain activity.

[0114] The term "antigen-binding fragment of an antibody" (or simply "antibody fragment") refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., IL-8). It has been shown that fragments of a full-length antibody can be used to perform the antigen-binding function of an antibody. Examples of binding fragments encompassed by the term "antigen-binding fragment of an antibody" include

[0115] (i) Fab fragment, consisting of V L 、V H , CL and CH1 domains;

[0116] (ii) F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region;

[0117] (iii) By V H and the Fd fragment consisting of the CH1 domain;

[0118] (iv) V of a single arm of the antibody H and V L Fv fragment composed of structural domains.

[0119] Fv antibody contains the variable region of the heavy chain and the variable region of the light chain, but no constant region, and is the smallest antibody fragment with all antigen binding sites. H and V L The polypeptide linker between the domains can form the structure required for antigen binding.

[0120] The term "CDR" refers to one of the six hypervariable regions within the variable domain of an antibody that primarily contributes to antigen binding.

[0121] The term "epitope" or "antigenic determinant" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds (e.g., a specific site on an IL-8 molecule). An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or non-consecutive amino acids in a unique spatial conformation.

[0122] The terms "specific binding," "selective binding," "selectively binds," and "specifically binds" refer to the binding of an antibody to a predetermined epitope on an antigen.

[0123] The term "competitive binding" refers to an antibody that recognizes the same epitope (also referred to as an antigenic determinant) or a portion 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. An 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.

[0124] The term "KD" or "Kd" refers to the dissociation equilibrium constant for a specific antibody-antigen interaction.

[0125] As used herein, the term "antigenic determinant" refers to a discrete three-dimensional site on an antigen that is recognized by the antibodies or antigen-binding fragments of the present invention.

[0126] The present invention includes not only complete antibodies, but also fragments of antibodies with immunological activity or fusion proteins formed by antibodies and other sequences. Therefore, the present invention also includes fragments, derivatives and analogs of the antibodies.

[0127] In the present invention, antibodies include murine, chimeric, humanized, or fully human antibodies prepared using techniques well known to those skilled in the art. Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, including human and non-human portions, can be prepared using recombinant DNA techniques well known in the art.

[0128] As used herein, the term "monoclonal antibody" refers to an antibody secreted by a clone derived from a single cell. Monoclonal antibodies are highly specific, being directed against a single antigenic epitope. The cell may be a eukaryotic, prokaryotic, or phage clone.

[0129] In the present invention, the antibodies can be monospecific, bispecific, trispecific, or more multispecific.

[0130] In the present invention, the antibodies of the present invention also include conservative variants thereof, which refer to polypeptides in which no more than 10, preferably no more than 8, more preferably no more than 5, and most preferably no more than 3 amino acids are replaced with amino acids having similar or similar properties, compared to the amino acid sequence of the antibodies of the present invention. These conservative variant polypeptides are preferably generated by making amino acid substitutions according to Table A.

[0131] Table A

[0132]

[0133] IL-8-specific antibodies

[0134] The present invention provides an anti-human IL-8 antibody (hereinafter referred to as IL-8 antibody). Specifically, the present invention provides an antibody with high specificity and high affinity for IL-8, which comprises a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region (V H ) amino acid sequence, the light chain contains a light chain variable region (V L ) amino acid sequence.

[0135] In a preferred embodiment of the present invention, the heavy chain variable region comprises the following three complementarity determining regions (CDRs):

[0136] CDR1 as shown in SEQ ID NO: 1,

[0137] CDR2 as shown in SEQ ID NO: 2, and

[0138] CDR3 as shown in SEQ ID NO:3.

[0139] Preferably, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO:7.

[0140] In a preferred embodiment of the present invention, the light chain variable region comprises the following three complementarity determining regions (CDRs):

[0141] CDR1' as shown in SEQ ID NO:4,

[0142] CDR2' as shown in SEQ ID NO: 5, and

[0143] CDR3' as shown in SEQ ID NO:6.

[0144] Preferably, the light chain variable region has the amino acid sequence shown in SEQ ID NO:8.

[0145] Any of the above amino acid sequences includes a sequence having IL-8 binding affinity after addition, deletion, modification and / or substitution of at least 1 (such as 1-5, 1-3, preferably 1-2, more preferably 1) amino acid.

[0146] In another preferred embodiment, the sequence formed by adding, deleting, modifying and / or replacing at least 1 (such as 1-5, 1-3, preferably 1-2, more preferably 1) amino acid sequence is preferably an amino acid sequence with a homology of at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95%.

[0147] The antibody of the present invention may be a double-chain or single-chain antibody, and may be selected from animal-derived antibodies, chimeric antibodies, and humanized antibodies, more preferably humanized antibodies, human-animal chimeric antibodies, and even more preferably fully humanized antibodies.

[0148] The antibody derivatives of the present invention can 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 any one or more of IgA, IgD, IgE, IgG, IgM antibodies or other subtypes of antibodies.

[0149] The animal is preferably a mammal, such as a mouse.

[0150] The antibody of the present invention may be a murine antibody, a chimeric antibody, a humanized antibody, or a CDR-grafted and / or modified antibody targeting human IL-8.

[0151] In a preferred embodiment of the present invention, any one or more of the sequences in SEQ ID NO: 1, 2 and 3, or sequences thereof having IL-8 binding affinity after addition, deletion, modification and / or substitution of at least one amino acid, is located in the heavy chain variable region (V H )'s CDR region.

[0152] In a preferred embodiment of the present invention, any one or more of the sequences in SEQ ID NO: 4, 5 and 6, or sequences thereof having IL-8 binding affinity after addition, deletion, modification and / or substitution of at least one amino acid, is located in the light chain variable region (V L )'s CDR region.

[0153] In a preferred embodiment of the present invention, V HCDR1, CDR2, and CDR3 are independently selected from any one or more of SEQ ID NOs: 1, 2, and 3, or sequences thereof having IL-8 binding affinity after addition, deletion, modification, and / or substitution of at least one amino acid; V L CDR1', CDR2', and CDR3' are independently selected from any one or more sequences of SEQ ID NO: 4, 5, and 6, or sequences thereof having IL-8 binding affinity after addition, deletion, modification, and / or substitution of at least one amino acid.

[0154] In the above content of the present invention, the number of 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%, more preferably 15-20%.

[0155] In the present invention, the number of amino acids added, deleted, modified and / or substituted is usually 1, 2, 3, 4 or 5, preferably 1-3, more preferably 1-2, and most preferably 1.

[0156] In certain embodiments, an antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region sequence comprising an amino acid sequence selected from SEQ ID NO:7, and an amino acid sequence having greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:7.

[0157] In certain embodiments, an antibody or antigen-binding fragment thereof of the invention comprises a light chain variable region sequence comprising an amino acid sequence selected from SEQ ID NO:8, and an amino acid sequence having greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:8.

[0158] In certain embodiments, the heavy chain variable region sequence comprises the amino acid sequence of SEQ ID NO:7, and the light chain variable region sequence comprises the amino acid sequence of SEQ ID NO:8.

[0159] Antibody preparation

[0160] Any method suitable for preparing monoclonal antibodies can be used to generate the IL-8 antibodies of the present invention. For example, animals can be immunized with linked or naturally occurring IL-8 proteins or fragments thereof. Suitable immunization methods, including adjuvants, immunostimulants, and repeated booster immunizations, can be used, and one or more routes of administration can be used.

[0161] Any suitable form of IL-8 can be used as an immunogen (antigen) to produce non-human antibodies specific for IL-8 and screen the biological activity of the antibodies. The immunogen can be used alone or in combination with one or more immunogenicity enhancers known in the art. The immunogen can be purified from a natural source or produced in genetically modified cells. The DNA encoding the immunogen can be genomic or non-genomic (e.g., cDNA) in origin. The DNA encoding the immunogen can be expressed using a suitable genetic vector, including but not limited to adenoviral vectors, baculoviral vectors, plasmids, and non-viral vectors.

[0162] The humanized antibody can be selected from any class of immunoglobulins, including IgM, IgD, IgG, IgA and IgE. In the present invention, the antibody is an IgG antibody, and IgG1 or IgG4 subtype is used.

[0163] Likewise, any type of light chain can be used in the compounds and methods herein. Specifically, kappa, lambda chains, or variants thereof can be used in the compounds and methods of the invention.

[0164] The DNA sequences of the antibodies or fragments thereof of the present invention can be obtained using conventional techniques, such as PCR amplification or genomic library screening. Furthermore, the coding sequences of different light and heavy chains can be fused together in various combinations to form single-chain antibodies. Optimized single-chain antibodies can be obtained by testing and analyzing the functions of single-chain antibodies with different combinations or linkage modifications.

[0165] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.

[0166] In addition, artificial synthesis methods can also be used to synthesize relevant sequences, especially when the fragment length is relatively short. Generally, by first synthesizing multiple small fragments and then connecting them, very long fragments of sequence can be obtained. The DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art.

[0167] The term "nucleic acid molecule" refers to DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, but are preferably double-stranded DNA. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.

[0168] The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. In one embodiment, the vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated.

[0169] The present invention also relates to vectors comprising the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins.

[0170] The term "host cell" refers to a cell into which an expression vector has been introduced. The host cell can be a prokaryotic cell, such as a bacterial cell, a lower eukaryotic cell, such as a yeast cell, or a higher eukaryotic cell, such as a plant or animal cell (e.g., a mammalian cell).

[0171] The steps of transforming host cells with recombinant DNA described in the present invention can be carried out using techniques well known in the art. The transformants obtained can be cultured using conventional methods, and the transformants express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, conventional culture medium is used under appropriate conditions.

[0172] Typically, the transformed host cells are cultured under conditions suitable for expression of the antibodies of the present invention. The antibodies of the present invention are then purified using conventional immunoglobulin purification procedures, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, or affinity chromatography, among other conventional separation and purification methods well known to those skilled in the art.

[0173] The resulting monoclonal antibodies can be characterized by conventional means. For example, the binding specificity of the monoclonal antibodies can be determined by immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

[0174] carrier

[0175] The nucleic acid sequence encoding the desired molecule can be obtained using recombinant methods known in the art, such as, for example, by screening libraries from cells expressing the gene, by obtaining the gene from a vector known to include the gene, or by directly isolating from cells and tissues containing the gene using standard techniques. Alternatively, the gene of interest can be produced synthetically.

[0176] The present invention also provides vectors into which the expression cassettes of the present invention are inserted. Vectors derived from retroviruses, such as lentiviruses, are suitable tools for achieving long-term gene transfer because they allow for long-term, stable integration of transgenes and their proliferation in daughter cells. 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.

[0177] In brief summary, the expression cassette or nucleic acid sequence of the present invention is generally operably linked to a promoter and incorporated into an expression vector. Such vectors are suitable for replication and integration into eukaryotic cells. Typical cloning vectors contain transcriptional and translational terminators, initiation sequences, and promoters that can be used to regulate expression of the desired nucleic acid sequence.

[0178] The expression constructs of the present invention can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods of gene delivery are known in the art. In another embodiment, the present invention provides gene therapy vectors.

[0179] The nucleic acid can be cloned into many types of vectors. For example, 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.

[0180] Furthermore, the expression vector can be provided to the cell in the form of a viral vector. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. Typically, a suitable vector comprises an origin of replication that functions in at least one organism, a promoter sequence, convenient restriction enzyme sites, and one or more selectable markers.

[0181] Many virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The selected gene can be inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to the subject's cells in vivo or in vitro. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In one embodiment, a lentiviral vector is used.

[0182] Additional promoter elements, such as enhancers, can regulate the frequency of transcription initiation. Typically, these are located in the 30-110 bp region upstream of the start site, although recently it has been shown that many promoters also contain functional elements downstream of the start site. The spacing between promoter elements is often flexible so that when an element is inverted or moved relative to another, promoter function is maintained. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased by 50 bp before activity begins to decline. Depending on the promoter, it has been shown that individual elements can work together or independently to initiate transcription.

[0183] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence that can drive any polynucleotide sequence operably connected thereto for high-level expression. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including but not limited to simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr (Epstein-Barr) virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters, such as but not limited to actin promoter, myosin promoter, heme promoter, and creatine kinase promoter. Further, the present invention should not be limited to the application of constitutive promoters. Inducible promoters are also considered to be part of the present invention. The use of 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 expression when expression is undesirable. Examples of inducible promoters include, but are not limited to, the metallothionein promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter.

[0184] Methods for introducing genes into cells and expressing genes in cells are known in the art. In the context of expression vectors, the vectors can be readily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by any method known in the art. For example, expression vectors can be transferred into host cells by physical, chemical, or biological means.

[0185] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. A preferred method for introducing polynucleotides into host cells is calcium phosphate transfection.

[0186] Biological methods for introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human, cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, among others.

[0187] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and 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).

[0188] In the case of using non-viral delivery system, exemplary delivery means is liposome. Consider using lipid preparation, to introduce nucleic acid into host cell (in vitro, in vitro or in vivo). On the other hand, this nucleic acid can be associated with lipid. The nucleic acid associated with lipid can be encapsulated in the aqueous interior of liposome, be dispersed in the lipid bilayer of liposome, be attached to liposome through the connecting molecule that is associated with liposome and oligonucleotide, be trapped in liposome, be compounded with liposome, be dispersed in the solution that comprises lipid, mix with lipid, be united with lipid, be included in lipid as suspension, be included in micelle or be compounded with micelle, or be associated with lipid in other ways. The lipid, lipid / DNA or lipid / expression vector associated with composition are not limited to any concrete structure in solution. For example, they can be present in bilayer structure, as micelle or have " collapsed " structure. They also can be simply dispersed in solution, may form aggregates of size or shape inhomogeneity. Lipid is fatty substance, and it can be natural generation or synthetic lipid. For example, lipids include fat droplets that occur naturally in the cytoplasm as well as compounds that contain long-chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0189] In a preferred embodiment of the present invention, the vector is a lentiviral vector.

[0190] preparation

[0191] The present invention provides a formulation comprising the heavy chain variable region of the first aspect of the present invention, the heavy chain of the second aspect of the present invention, the light chain variable region of the third aspect of the present invention, the light chain of the fourth aspect of the present invention, the antibody of the fifth aspect of the present invention, the recombinant protein of the sixth aspect of the present invention, the antibody-drug conjugate of the seventh aspect of the present invention, the polynucleotide of the eighth aspect of the present invention, the vector of the ninth aspect of the present invention, and / or the genetically engineered host cell of the tenth aspect of the present invention, and a pharmaceutically acceptable carrier, diluent, or excipient. In one embodiment, the formulation is a liquid formulation. Preferably, the formulation is an injectable formulation.

[0192] In one embodiment, the formulation may include a buffer such as neutral buffered saline, sulfate buffered saline, or the like; a carbohydrate such as glucose, mannose, sucrose, or dextran, mannitol; a protein; a polypeptide or amino acid such as glycine; an antioxidant; a chelating agent such as EDTA or glutathione; an adjuvant (e.g., aluminum hydroxide); and a preservative. The formulation of the present invention is preferably formulated for intravenous or intraperitoneal administration.

[0193] Detection Uses and Kits

[0194] The antibodies of the present invention can be used in detection applications, for example, for detecting a sample to provide diagnostic information.

[0195] In the present invention, the samples used include cells, tissue samples and biopsy specimens. The term "biopsy" as used in the present invention should include all types of biopsies known to those skilled in the art. Therefore, the biopsy used in the present invention can include, for example, tissue samples prepared by endoscopic methods or puncture or needle biopsy of an organ.

[0196] Samples used in the present invention include fixed or preserved cell or tissue samples.

[0197] The present invention also provides a kit comprising the antibody (or fragment thereof) and scFv of the present invention. In a preferred embodiment of the present invention, the kit further comprises a container, instructions for use, a buffer, etc. In a preferred embodiment, the antibody of the present invention can be fixed to a detection plate.

[0198] The main advantages of the present invention include:

[0199] The present invention provides an antibody that binds to IL-8 with high affinity, and its use in blocking or inhibiting IL-8-induced activities, such as pro-inflammatory activity, chemotactic activity and angiogenesis, to treat IL-8-related immune, autoimmune, inflammatory or infectious diseases and cancers.

[0200] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally carried out under conventional conditions or under conditions recommended by the manufacturer.

[0201] Example 1

[0202] 1.1 Screening of phage antibody Fab library

[0203] Specific phage-activated human anti-IL-8 Fab antibodies were obtained from a combinatorial phage Fab display library created by Sanyou Biopharmaceutical Co., Ltd. Human IL-8 was screened by phage display [Science. 1985 Jun 14; 228(4705): 1315-7; Nature. 1991 Aug 15; 352(6336): 624-8] using magnetic particles, KingFisher instruments, and immunotube-based screening methods.

[0204] In the bioscreening process, the antigen (biotinylated IL-8) was immobilized on the surface of streptavidin magnetic particles by incubating the protein with the particles at room temperature on a rotator for 1 hour. The particles were then washed with PBS (pH 7.4) and blocked with 2.5% BSA in PBS (pH 7.4) for 1 hour. A phage solution containing 2.5% BSA in PBS (pH 7.4) was then added to the antigen-bound magnetic particles. The mixture was incubated with stirring at room temperature for 40 minutes. The magnetic particles were washed several times with PBST (PBS containing 0.05% Tween-20) to remove unbound phage. Phage that remained bound to the antigen on the magnetic particle surface were eluted with trypsin solution over 15 minutes with stirring. The resulting phage were then infected with Escherichia coli (SS320); phage were produced in the infected E. coli and isolated for the next round of screening. After three rounds of selection, DNAs (phagemids) were isolated from the phages and the antibody variable domain genes were cloned into expression vectors for Fab production in E. coli cells. Unless otherwise stated, phage display-derived anti-IL-8 antibodies were used in the following examples.

[0205] 1.2. Screening of Fabs that specifically bind to human IL-8

[0206] ELISA was used to screen for Fab binding to human IL-8. All subsequent steps were performed according to the standard ELISA protocol. The ELISA plate wells were coated with 30 μl of human IL-8 (2 μg / ml), sealed and incubated overnight at 4°C. The plate wells were washed three times with PBST (PBS containing 0.05% Tween-20). To block nonspecific binding, 20 μL of PMSM blocking buffer (PBS containing 5% skim milk) was added. The plate was incubated on a shaker at room temperature for 1 hour. After washing three times with PBST (PBS containing 0.05% Tween-20), 30 μl of test cell supernatant containing the Fab to be tested mixed with an equal volume of PMSM (PBS containing 5% skim milk) was added to each well. The well plate was incubated again, shaken at room temperature for 1 hour, and then each well was washed 6 times with PBST (PBS containing 0.05% Tween-20) buffer. Then in PBST (PBS containing 0.05% Tween-20), add the secondary antibody of anti-M13-HRP coupling (30 μ L / well) with the ratio of 1:8000. The orifice plate is shaken on a rotary shaker (50 min, room temperature) and washed 9 times with PBST buffer (PBS containing 0.05% Tween-20). By adding TMB (30 μ L / well) until saturation (average 5-10 min) to show colorimetric signal, then by adding stop solution (30 μ L / well, 2M sulfuric acid) stop color development. Use plate reader (Molecular Device) to measure colorimetric signal at a wavelength of 450 nm.

[0207] 1.3. Affinity Analysis of Anti-IL-8 Antibodies

[0208] The V clone of the Fab sequence from "1.1 Screening of phage antibody Fab library" L and V H The antibody of the present invention is expressed in the form of 100 μg / mL of 100 μg / mL of 100 μg / mL of 100 μg / mL of 100 μg / mL.The antibody of the present invention is expressed in the form of 100 μg / mL ...

[0209] ELISA is used to screen for anti-IL-8 antibodies that bind to human IL-8. ELISA plate wells are coated with 30 μl of human IL-8 (2 μg / ml), sealed, and incubated overnight at 4°C. The wells are washed three times with PBST. 20 μl of PMSM blocking buffer is added, and the plate is incubated on a shaker at room temperature for 1 hour. After washing three times with PBST, 30 μl of the anti-IL-8 monoclonal antibody to be tested mixed with an equal volume of PMSM is added to each well. The plate is incubated again, shaken at room temperature for 1 hour, and then each well is washed three times with PBST buffer to remove unbound antibody. Anti-human Kappa + Lambda HRP is then added (30 μl / well) at a ratio of 1:5000 in PBST and incubated for 1 hour. The wells are washed six times with PBST buffer. Color development is performed by adding TMB (30 μl / well), followed by stopping the color development by adding stop solution (30 μl / well, 2 M sulfuric acid). Optical density was measured at a wavelength of 450 nm using a suitable plate reader (Molecular Devices). Figure 1 As shown, the antibody binds to human IL-8 with high affinity.

[0210] BLI is a label-free biomolecular detection method that detects biomolecular interactions by measuring the interference pattern of white light reflected from the biosensor surface. Dissociation (kd) and association (ka) rate constant data were obtained using GATOR software. The equilibrium dissociation constant (K D ) is calculated from the ratio of kd to ka. Figure 2 As shown in Table 1 , all anti-IL-8 monoclonal antibodies bound to IL-8 with high affinity.

[0211] Table 1 Kinetic data of screened anti-IL-8 monoclonal antibodies

[0212]

[0213] Note: “E” is the scientific notation. For example, “1.93E-09” means 1.93×10 -9 , “2.41E-10” means 2.41×10 -10 ; "1.70E+06" means 1.70×10 6 , “8.67E+05” means 8.67×10 5 , and so on.

[0214] Among them, the heavy chain variable region of antibody LJH001-B30 has the following three complementarity determining regions (CDRs):

[0215] CDR1 (GGTFSSYAIS) as shown in SEQ ID NO: 1,

[0216] CDR2 (GIIPIFGTANYAQKFQG) as shown in SEQ ID NO: 2, and

[0217] CDR3 (PRYFDWSEDHYDAFDI) as shown in SEQ ID NO: 3;

[0218] The heavy chain variable region of antibody LJH001-B30 has the amino acid sequence shown in SEQ ID NO: 7 (QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQ KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCASPRYFDWSEDHYDAFDIWGQGTMVTVSS);

[0219] The light chain variable region of antibody LJH001-B30 has the following three complementarity determining regions (CDRs):

[0220] CDR1' (SGSSSNIGSYTVN) as shown in SEQ ID NO: 4,

[0221] CDR2' (SNTQRPS) as shown in SEQ ID NO: 5, and

[0222] CDR3' (AAWDDSLHGAV) as shown in SEQ ID NO: 6;

[0223] The light chain variable region of antibody LJH001-B30 has the amino acid sequence shown in SEQ ID NO: 8 (QSALTQPPSASGTPGQRVTISCSGSSSNIGSYTVNWFQQLPGTAPKLLIYSNTQRPSGVPDRFSG SKSGTSASLAISGLQSEDEADYYCAAWDDSLHGAVFGGGTQLAVL).

[0224] 1.4 Anti-IL-8 Antibody Stability Determination

[0225] The thermal stability of anti-IL-8 antibodies was determined using differential scanning fluorimetry (DSF) on an ABI 7500 fast real-time PCR instrument. DSF can be conveniently used to determine the melting temperature (Tm) of proteins.

[0226] DSF (also known as protein thermal shift assay) measures heat-induced protein denaturation by measuring the fluorescence change of a dye that preferentially binds to unfolded proteins (such as Sypro Orange dye, which binds to hydrophobic regions of proteins exposed by unfolding), which is usually performed using a real-time PCR instrument. The stability of a protein is related to its Gibbs free energy of unfolding, which is temperature dependent. Briefly, in an ABI7500 fast real-time PCR, 20 μl of 0.2 mg / ml anti-IL-8 antibody sample in PBS (pH 7.4) (containing 100×SYPRO Orange dye) was heated from 25°C to 99°C in 1°C increments with an excitation wavelength of 492 nm and an emission wavelength of 580 nm. Simple equations such as the Boltzmann equation are used to calculate the inflection point (Tm) of the transition curve, and the results are shown in Figure 2. Figure 3 shown.

[0227] 1.5 Inhibition of IL-8-mediated CXCR1 / 2+ cell chemotaxis

[0228] The inhibition of IL-8 antibody LJH001-B30 on IL-8-induced migration of Jurkat-CXCR1 (Jurkat cells overexpressing human CXCR1) was determined by Boyden chamber migration assay. Human IL-8 (20 ng / ml) was incubated with different 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 chamber and incubated at 37°C for 4-6 h. Figure 4 The data shown show that anti-IL-8 antibodies inhibited the chemotaxis of Jurkat-CXCR1 in a dose-dependent manner.

[0229] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. An anti-IL-8 antibody, characterized in that The antibodies include: (1) heavy chain variable region; and (2) light chain variable region; The heavy chain variable region includes the following three complementarity determining regions (CDRs): CDR1 as shown in SEQ ID NO: 1, CDR2 as shown in SEQ ID NO: 2, and CDR3 as shown in SEQ ID NO: 3; The light chain variable region includes the following three complementarity determining regions (CDRs): CDR1' as shown in SEQ ID NO:4, CDR2' as shown in SEQ ID NO: 5, and CDR3' as shown in SEQ ID NO:

6.

2. The antibody according to claim 1, wherein The heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 7; and The light chain variable region has the amino acid sequence shown in SEQ ID NO:

8.

3. A recombinant protein, characterized in that The recombinant protein consists of the following components: (i) the antibody of claim 1; and (ii) an optional tag sequence to facilitate expression and / or purification.

4. A polynucleotide, characterized in that The polynucleotide encodes a polypeptide selected from the group consisting of: (1) the antibody according to claim 1; or (2) The recombinant protein according to claim 3.

5. A carrier, characterized in that The vector contains the polynucleotide according to claim 4.

6. The vector according to claim 5, wherein The vectors include bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses, retroviruses, or other vectors.

7. A genetically engineered host cell, characterized in that The host cell comprises the vector according to claim 6 or the polynucleotide according to claim 4 is integrated into the genome of the host cell.

8. A pharmaceutical composition, characterized in that The pharmaceutical composition include: The antibody according to claim 1, the recombinant protein according to claim 3, the polynucleotide according to claim 4, the vector according to claim 5 and / or the genetically engineered host cell according to claim 7.

9. A detection board, characterized in that: The detection plate comprises: a substrate and a test strip, and the test strip comprises the antibody as claimed in claim 1.

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