CN117203231B

CN117203231B - Anti-IL-8 antibody

Info

Publication number: CN117203231B
Application number: CN202280008469.2A
Authority: CN
Inventors: 姚瑛英; 陈琳; 唐亮
Original assignee: Suzhou Kaijian Biotechnology Co ltd
Current assignee: Suzhou Kaijian Biotechnology Co ltd
Priority date: 2022-11-28
Filing date: 2022-11-28
Publication date: 2025-09-16
Anticipated expiration: 2042-11-28
Also published as: CN117203231A; WO2024113122A1

Abstract

The present invention relates to an anti-IL-8 antibody. Specifically, the present invention provides a heavy chain variable region of an antibody comprising three Complementarity Determining Regions (CDRs) of 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 antibodies of the invention are capable of binding IL-8 with high affinity and blocking or inhibiting 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

Anti-IL-8 antibody

Technical Field

The invention relates to the field of biotechnology, in particular to an anti-IL-8 antibody.

Background

Interleukin 8 (IL-8, also known as CXCL 8), previously known as monocyte-derived neutrophil chemokine (MDNCF) or neutrophil-inducible/activatable protein-1 (NAP-1), is an ELR ⁺ chemokine that exhibits chemotactic activity for a specific type of leukocyte in inflammatory diseases.

IL-8 is a polypeptide, and IL-8 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 to participate in migration of neutrophils to sites of inflammation through binding to high affinity receptors (CXCR 1 and CXCR 2) on the surface of neutrophils. IL-8 activates neutrophils by accelerating degranulation, reactive Oxygen Species (ROS) production, and destruction of infiltrating tissue.

Although neutrophil inflammatory responses are critical for destroying pathogens that invade the body, inappropriate neutrophil activation and infiltration can lead to a number of diseases. It is speculated 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 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) into the Tumor Microenvironment (TME) and facilitate tumor immune evasion.

Human tumor cells such as melanoma, breast cancer, hepatocellular carcinoma, castration-resistant prostate cancer, colorectal cancer, glioma express IL-8, which plays a role in tumor invasion and metastasis. Inhibition of IL-8 prevents inflammatory cells from infiltrating tissue, reduces angiogenesis, reduces trafficking of MDSCs, and ameliorates disease.

Accordingly, there is a need in the art to develop an antibody capable of binding IL-8 for the diagnosis and treatment of IL-8 mediated diseases.

Disclosure of Invention

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.

In a first aspect the invention provides a heavy chain variable region of an antibody comprising 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.

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

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

In another preferred embodiment, any of the above amino acid sequences further comprises a derivative sequence of at least 1 (e.g., 1-3, preferably 1-2, more preferably 1) amino acid, optionally with additions, deletions, modifications and/or substitutions.

In another preferred embodiment, the derivatized sequence retains the binding affinity of IL-8.

In another preferred embodiment, the derivative is anti-IL-8.

In another preferred embodiment, the heavy chain variable region further comprises an FR region of human origin or an FR region of murine origin.

In another preferred embodiment, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO. 7.

In a second aspect the invention provides a heavy chain of an antibody, said heavy chain comprising a heavy chain variable region according to the first aspect of the invention.

In another preferred embodiment, the heavy chain of the antibody further comprises a heavy chain constant region.

In another preferred embodiment, the heavy chain constant region is of human, murine or rabbit origin.

In a third aspect the invention provides a light chain variable region of an antibody, said light chain variable region comprising 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.

In another preferred embodiment, the derivative is anti-IL-8.

In another preferred embodiment, the light chain variable region further comprises an FR region of human origin or an FR region of murine origin.

In another preferred embodiment, the light chain variable region has the amino acid sequence shown in SEQ ID NO. 8.

In a fourth aspect the invention provides a light chain of an antibody, said light chain comprising a light chain variable region according to the third aspect of the invention.

In another preferred embodiment, the light chain of the antibody further comprises a light chain constant region.

In another preferred embodiment, the light chain constant region is of human, murine or rabbit origin.

In a fifth aspect, the invention provides an antibody comprising:

(1) The heavy chain variable region according to the first aspect of the invention, and/or

(2) The light chain variable region according to the third aspect of the invention.

In another preferred embodiment, the antibody comprises a heavy chain as described in the second aspect of the invention and/or a light chain as described in the fourth aspect of the invention.

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

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

In another preferred embodiment, the antibody is selected from the group consisting of an animal-derived antibody, a chimeric antibody, a humanized antibody, or a combination thereof.

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

In another preferred embodiment, the antibody is a monoclonal antibody.

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

In another preferred embodiment, the heavy chain variable region sequence of the antibody is shown in SEQ ID NO. 7, and/or

The light chain variable region sequence of the antibody is shown as SEQ ID NO. 8.

In another preferred embodiment, the antibody is IgG, igM, igA, igD or IgE type.

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

In a sixth aspect, the present invention provides a recombinant protein comprising:

(i) A heavy chain variable region according to the first aspect of the invention, a heavy chain according to the second aspect of the invention, a light chain variable region according to the third aspect of the invention, a light chain according to the fourth aspect of the invention, or an antibody according to the fifth aspect of the invention, and

(Ii) Optionally a tag sequence to assist expression and/or purification.

In another preferred embodiment, the tag sequence comprises a 6His tag.

In another preferred embodiment, the recombinant protein comprises a fusion protein.

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

In a seventh aspect, the present invention provides an antibody drug conjugate comprising:

(a) An antibody moiety selected from the group consisting of a heavy chain variable region according to the first aspect of the invention, a heavy chain according to the second aspect of the invention, a light chain variable region according to the third aspect of the invention, a light chain according to the fourth aspect of the invention, or an antibody according to the fifth aspect of the invention, or a combination thereof, and

(B) A coupling moiety coupled to the antibody moiety, the coupling moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.

In another preferred embodiment, the antibody moiety is coupled to the coupling moiety via a chemical bond or linker.

In an eighth aspect, the invention provides a polynucleotide encoding a polypeptide selected from the group consisting of:

(1) A heavy chain variable region according to the first aspect of the invention, a heavy chain according to the second aspect of the invention, a light chain variable region according to the third aspect of the invention, a light chain according to the fourth aspect of the invention, or an antibody according to the fifth aspect of the invention, or

(3) The recombinant protein according to the sixth aspect of the invention.

According to a ninth aspect of the present invention there is provided a vector comprising a polynucleotide according to the eighth aspect of the present invention.

In another preferred embodiment, the vector comprises a bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, or other vector.

In a tenth aspect the invention provides a genetically engineered host cell comprising a vector according to the ninth aspect of the invention or having integrated into its genome a polynucleotide according to the eighth aspect of the invention.

In an eleventh aspect, the present invention provides a pharmaceutical composition comprising:

The heavy chain variable region according to the first aspect of the invention, the heavy chain variable region according to the second aspect of the invention, the light chain variable region according to the third aspect of the invention, the light chain according to the fourth aspect of the invention, or the antibody according to the fifth aspect of the invention, the recombinant protein according to the sixth aspect of the invention, the antibody drug conjugate according to the seventh aspect of the invention, the polynucleotide according to the eighth aspect of the invention, the vector according to the ninth aspect of the invention and/or the genetically engineered host cell according to the tenth aspect of the invention.

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

In another preferred embodiment, the pharmaceutical composition is a liquid formulation.

In another preferred embodiment, the pharmaceutical composition is in the form of an injection.

In another preferred embodiment, the injection is an intravenous injection.

The twelfth aspect of the invention provides the use of a heavy chain variable region according to the first aspect of the invention, a heavy chain variable region according to the second aspect of the invention, a light chain variable region according to the third aspect of the invention, an antibody according to the fourth aspect of the invention, and/or a recombinant protein according to the sixth aspect of the invention for (i) preparing a medicament or formulation for blocking or inhibiting IL-8 induced activity, (ii) preparing a medicament or formulation for preventing and/or treating a disease associated with IL-8, and/or (iii) preparing a detection reagent or kit.

In another preferred embodiment, the IL-8-induced activity comprises pro-inflammatory activity, chemotactic activity and/or angiogenesis.

In another preferred embodiment, the IL-8-associated disorder includes autoimmune, inflammatory, infectious, and/or neoplastic disorders.

In another preferred embodiment, the disorder associated with IL-8 is a disorder associated with elevated or unbalanced IL-8 levels.

In another preferred example, the IL-8-associated disease is an immune, autoimmune, inflammatory, infectious disease or a disease characterized by elevated or unbalanced levels of human IL-8, 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, acute pancreatitis.

In another preferred example, the IL-8-associated disorder is an inflammatory or dermatological disorder, such as psoriasis, palmoplantar impetigo (PPP), bullous pemphigoid, pemphigus, contact dermatitis, eczema, lupus erythematosus, and atopic dermatitis.

In another preferred embodiment, the IL-8-associated disorder is a human virus-associated disorder or infection, such as the common cold caused by human rhinovirus, coronavirus, other enteroviruses, herpes viruses, influenza viruses, parainfluenza viruses, respiratory syncytial virus or adenovirus infection, and hepatitis C.

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

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

In a thirteenth aspect, the invention provides a method for in vitro detection of IL-8 in a sample, said method comprising the steps of:

(1) Contacting the sample with an antibody according to the fifth aspect of the invention in vitro;

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

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

In a fourteenth aspect the invention provides a test plate comprising a substrate (support plate) and a test strip comprising an antibody according to the fifth aspect of the invention or an antibody drug conjugate according to the seventh aspect of the invention.

The fifteenth aspect of the present invention provides a method of blocking or inhibiting IL-8-induced activity, and/or preventing and/or treating a disease associated with IL-8, comprising administering to the subject an antibody according to the fifth aspect of the present invention, a recombinant protein according to the sixth aspect of the present invention, an antibody drug conjugate according to the seventh aspect of the present invention, a polynucleotide according to the eighth aspect of the present invention, a vector according to the ninth aspect of the present invention, a genetically engineered host cell according to the tenth aspect of the present invention, and/or a 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 a disease associated with IL-8.

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

In another preferred embodiment, the non-human mammal comprises a cow, horse, sheep, dog, cat or mouse.

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

FIG. 1 shows the results of ELISA assays for binding of anti-IL-8 antibodies of the invention to human IL-8.

FIG. 2 shows the results of a Biological Layer Interferometry (BLI) binding assay performed using GATOR instruments. Dissociation (KD or KD) and binding (ka) rate constants were obtained using GATOR software. The equilibrium dissociation constant (K _D) was calculated from the ratio of kd to ka.

FIG. 3 shows the results of Differential Scanning Fluorescence (DSF) analysis performed in an ABI7500 rapid real-time PCR instrument.

FIG. 4 is a graph showing the results of an assay for IL-8 mediated inhibition of CXCR1+ cell chemotaxis by an anti-IL-8 antibody using a Boyden chamber migration assay, wherein "B30" refers to antibody LJH001-B30 and "positive" refers to monoclonal mouse IgG1 clone #6217.

Detailed Description

The present invention provides an antibody that binds to IL-8 with high affinity, and its use for blocking or inhibiting 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.

Terminology

As used herein, the terms "comprising," "including," and "containing" are used interchangeably, and include not only open-ended definitions, but also semi-closed, and closed-ended definitions. In other words, the terms include "consisting of" and "consisting essentially of.

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

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

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 an antigen. Examples of ScFv include antibody polypeptides formed by recombinant DNA techniques, and wherein Fv regions of immunoglobulin heavy (H chain) and light (L chain) chain fragments are linked via a spacer sequence. Various methods of improving ScFv are known to those skilled in the art.

As used herein, the term "administering" refers to the application of an exogenous drug, therapeutic, diagnostic, or composition to an animal, human, subject, cell, tissue, organ, or biological fluid. "administration" may refer to therapeutic, pharmacokinetic, diagnostic, research and experimental methods. Treatment of a cell includes contacting a reagent with the cell, contacting a reagent with a fluid, and contacting a fluid with the cell. "administration" also means in vitro and ex vivo treatment by an agent, diagnosis, binding composition, or by another cell. "administration" when applied to a human, animal or study subject refers to therapeutic, prophylactic or preventative measures, studies and diagnostics, including the contact of an antibody against human IL-8 with a human or animal, subject, cell, tissue, physiological compartment or physiological fluid.

As used herein, the term "treatment" refers to the administration of an internal or external therapeutic agent comprising any of the anti-human IL-8 antibodies of the invention, and compositions thereof, to a patient having one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. Typically, the patient is administered an amount of the therapeutic agent (therapeutically effective amount) effective to alleviate one or more symptoms of the disease.

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

"Sequence identity" as used herein refers to the degree of identity between two nucleic acid or two amino acid sequences when optimally aligned and compared with appropriate substitutions, insertions, or deletions of mutations. The sequence identity between the sequences described in the present invention and sequences with which it has identity 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%, 100%.

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

IL-8

IL-8 is a polypeptide, and IL-8 is secreted by fibroblasts, vascular endothelial cells, macrophages, dendritic Cells (DCs), lymphocytes, keratinocytes, melanocytes, hepatocytes, and various tumor cells.

Antibodies to

As used herein, the term "antibody" refers to an immunoglobulin that is a tetrapeptide chain structure formed from two identical heavy chains and two identical light chains joined by an interchain disulfide bond. The immunoglobulin heavy chain constant region differs in amino acid composition and sequence, and thus, in antigenicity. Accordingly, immunoglobulins can be categorized into five classes, or isotypes, igM, igD, igG, igA and IgE, with their respective heavy chains being the μ, δ, γ, α, and epsilon chains, respectively. The Ig of the same class can be further classified into different subclasses according to the amino acid composition of the heavier chain region and the number and position of disulfide bonds of the heavy chain, such as IgG can be classified into IgG1, igG2, igG3 and IgG4. Light chains are classified as either kappa chains or lambda chains depending on the constant region. Each of the five classes of Ig may have either a kappa chain or a lambda chain. Subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those skilled in the art.

The antibody light chains of the present invention may further comprise a light chain constant region comprising a kappa, lambda chain of human or murine origin or variants thereof.

In the present invention, the antibody heavy chain of the present invention may further comprise a heavy chain constant region comprising IgG1, igG2, igG3, igG4 or variants thereof of human or murine origin. The sequences of the heavy and light chains of the antibody near the N-terminus vary widely, being the variable region (Fv region) and the remaining amino acid sequences near the C-terminus are relatively stable, being the constant region. The variable region includes 3 hypervariable regions (HVRs) and 4 Framework Regions (FR) that are relatively conserved in sequence. The 3 hypervariable regions determine the specificity of the antibody, also known as Complementarity Determining Regions (CDRs). Each of the Light Chain Variable Region (LCVR) and Heavy Chain Variable Region (HCVR) consists of 3 CDR regions and 4 FR regions, arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 from amino-terminus to carboxy-terminus. 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.

Antibodies of the invention include murine antibodies, chimeric antibodies, humanized antibodies, preferably humanized antibodies. The term "murine antibody" is herein a monoclonal antibody against human IL-8 prepared according to the knowledge and skill in the art. The preparation is performed by injecting the test subjects with IL-8 antigen and then isolating hybridomas expressing antibodies having the desired sequence or functional properties. In a preferred embodiment of the invention, the murine IL-8 antibody or antigen binding fragment thereof may further comprise a murine kappa, lambda chain or variant light chain constant region or further comprise a murine IgG1, igG2, igG3 or variant heavy chain constant region.

The term "chimeric antibody" refers to an antibody in which a variable region of a murine antibody is fused to a constant region of a human antibody, and which can reduce the immune response induced by the murine antibody.

The term "humanized antibody", also known as CDR-grafted antibody, refers to an antibody produced by grafting murine CDR sequences into the framework of human antibody variable regions, i.e., the framework sequences of different types of human germline antibodies. Humanized antibodies can overcome the heterologous response induced by chimeric antibodies that carry large amounts of murine protein components. Such framework sequences may be obtained from public DNA databases including germline antibody gene sequences or published references. To avoid a decrease in immunogenicity while at the same time causing a decrease in activity, the human antibody variable region framework sequences may be subjected to minimal reverse or back-mutations to maintain activity.

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). Fragments of full length antibodies have been shown to be useful for performing the antigen binding function of antibodies. Examples of binding fragments encompassed within the term "antigen-binding fragment of an antibody" include

(I) A Fab fragment, a monovalent fragment consisting of V _L、V_H, CL and CH1 domains;

(ii) A F (ab') ₂ fragment comprising a bivalent fragment of two Fab fragments linked by a disulfide bridge over the longer chain region;

(iii) An Fd fragment consisting of V _H and a CH1 domain;

(iv) Fv fragments consisting of the V _H and V _L domains of the single arm of the antibody.

Fv antibodies contain antibody heavy chain variable regions, light chain variable regions, but no constant regions, and have a minimal antibody fragment of the entire antigen binding site. Generally, fv antibodies also comprise a polypeptide linker between the V _H and V _L domains and are capable of forming the structures required for antigen binding.

The term "CDR" refers to one of the 6 hypervariable regions within the variable domain of an antibody that contribute primarily to antigen binding.

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). Epitopes generally comprise at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 contiguous or non-contiguous amino acids in a unique spatial conformation.

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

The term "competitive binding" refers to an antibody that recognizes the same epitope (also referred to as an epitope) or a portion of the same epitope on the extracellular region of human IL-8 as a monoclonal antibody of the invention and binds to the antigen. An antibody that binds to the same epitope as the monoclonal antibody of the 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 invention.

The term "KD" or "KD" refers to the dissociation equilibrium constant of a particular antibody-antigen interaction.

As used herein, the term "epitope" refers to a discrete, three-dimensional spatial site on an antigen that is recognized by an antibody or antigen-binding fragment of the invention.

The invention includes not only whole antibodies but also fragments of antibodies having immunological activity or fusion proteins of antibodies with other sequences. Thus, the invention also includes fragments, derivatives and analogues of said antibodies.

In the present invention, antibodies include murine, chimeric, humanized or fully human antibodies prepared by 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 DNA recombination techniques well known in the art.

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

In the present invention, antibodies may be monospecific, bispecific, trispecific, or more multispecific.

In the present invention, the antibodies of the invention also include conservative variants thereof, meaning that up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids are replaced by amino acids of similar or similar nature to the amino acid sequence of the antibodies of the invention to form a polypeptide. These conservatively variant polypeptides are preferably generated by amino acid substitutions according to Table A.

Table A

IL-8 specific antibodies

The present invention provides an anti-human IL-8 antibody (hereinafter referred to as IL-8 antibody). In particular, the invention provides a high specificity and high affinity antibody to IL-8 comprising a heavy chain variable region (V _H) amino acid sequence and a light chain comprising a light chain variable region (V _L) amino acid sequence.

In a preferred embodiment of the invention, the heavy chain variable region comprises 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.

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

In a preferred embodiment of the invention, the light chain variable region comprises 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.

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

Any of the amino acid sequences described above includes sequences having IL-8 binding affinity that have been added, deleted, modified and/or substituted with at least 1 (e.g., 1-5, 1-3, preferably 1-2, more preferably 1) amino acids.

In another preferred embodiment, the sequence formed by adding, deleting, modifying and/or substituting at least 1 (e.g. 1-5, 1-3, preferably 1-2, more preferably 1) amino acid sequences is preferably an amino acid sequence having a homology of at least 80%, preferably at least 85%, more preferably at least 90%, most preferably at least 95%.

The antibody of the present invention may be a double-or single-chain antibody, and may be selected from animal-derived antibodies, chimeric antibodies, humanized antibodies, more preferably humanized antibodies, human-animal chimeric antibodies, and even 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 known antibody derivatives in the art, etc., as well as IgA, igD, igE, igG and any one or more of IgM antibodies or other subclasses of antibodies.

Wherein the animal is preferably a mammal, such as a mouse.

The antibodies of the invention may be murine antibodies, chimeric antibodies, humanized antibodies, CDR-grafted and/or modified antibodies that target human IL-8.

In a preferred embodiment of the invention, any one or several of the sequences of SEQ ID NOS 1, 2 and 3 as described above, or a sequence having IL-8 binding affinity, in which at least one amino acid has been added, deleted, modified and/or substituted, is located in the CDR region of the heavy chain variable region (V _H).

In a preferred embodiment of the invention, any one or several of the sequences of SEQ ID NOS 4, 5 and 6 as described above, or a sequence having IL-8 binding affinity, in which at least one amino acid has been added, deleted, modified and/or substituted, is located in the CDR region of the light chain variable region (V _L).

In a preferred embodiment of the invention, V _H CDR1, CDR2, CDR3 are each independently selected from any one or several sequences of SEQ ID NO 1,2 and 3 or sequences thereof with IL-8 binding affinity, which are added, deleted, modified and/or substituted for at least one amino acid, and V _L CDR1', CDR2', CDR3' are each independently selected from any one or several sequences of SEQ ID NO 4, 5 and 6 or sequences thereof with IL-8 binding affinity, which are added, deleted, modified and/or substituted for at least one amino acid.

In the above-described aspect of the present invention, the number of amino acids added, deleted, modified and/or substituted is preferably not more than 40%, more preferably not more than 35%, more preferably 1 to 33%, more preferably 5 to 30%, more preferably 10 to 25%, more preferably 15 to 20% of the total amino acids in the original amino acid sequence.

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

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 the group consisting of 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.

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 the group consisting of 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.

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.

Preparation of antibodies

Any method suitable for producing monoclonal antibodies can be used to produce the IL-8 antibodies of the invention. For example, animals may be immunized with a linked or naturally occurring IL-8 protein or fragment thereof. Suitable immunization methods may be used, including adjuvants, immunostimulants, repeated booster immunizations, and one or more routes may be used.

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

The humanized antibody may 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 an IgG1 or IgG4 subtype is used.

Also, any type of light chain may be used in the compounds and methods herein. In particular, kappa, lambda chains or variants thereof are useful in the compounds and methods of the present invention.

The sequence of the DNA molecule of the antibody or fragment thereof of the present invention can be obtained by a conventional technique such as amplification by PCR or screening of a genomic library. In addition, the coding sequences for the different light and heavy chains may be fused together in different combinations to form single chain antibodies. And the optimized single-chain antibody is obtained by detecting and analyzing the functions of the single-chain antibodies modified by different combinations or different links.

Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.

Furthermore, the sequences concerned, in particular fragments of short length, can also be synthesized by artificial synthesis. In general, fragments of very long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. The DNA sequence can then be introduced into a variety of existing DNA molecules (or vectors, for example) and cells known in the art.

The term "nucleic acid molecule" refers to both DNA molecules and RNA molecules. The nucleic acid molecule may be single-stranded or double-stranded, but is 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.

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 may be ligated.

The invention also relates to vectors comprising the above-described suitable DNA sequences and suitable promoter or control sequences. These vectors may be used to transform an appropriate host cell to enable expression of the protein.

The term "host cell" refers to a cell into which an expression vector has been introduced. The host cell may be a prokaryotic cell, such as a bacterial cell, or 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).

The steps described herein for transforming a host cell with recombinant DNA may be performed using techniques well known in the art. The transformant obtained can be cultured by a conventional method, and the transformant expresses the polypeptide encoded by the gene of the present invention. Depending on the host cell used, it is cultivated in conventional medium under suitable conditions.

Typically, the transformed host cell is cultured under conditions suitable for expression of the antibodies of the invention. The antibodies of the invention are then purified by conventional immunoglobulin purification procedures, such as protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography, using conventional separation and purification means well known to those skilled in the art.

The resulting monoclonal antibodies can be identified by conventional means. For example, the binding specificity of a monoclonal antibody can be determined using immunoprecipitation or in vitro binding assays, such as Radioimmunoassays (RIA) or enzyme-linked immunosorbent assays (ELISA).

Carrier body

Nucleic acid sequences encoding a 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 vectors known to include the gene, or by direct isolation from cells and tissues containing the gene using standard techniques. Alternatively, the gene of interest may be produced synthetically.

The invention also provides vectors into which the expression cassettes of the invention are inserted. Vectors derived from retroviruses such as lentiviruses are suitable tools for achieving long-term gene transfer, as they allow 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 transduce non-proliferating cells, such as hepatocytes. They also have the advantage of low immunogenicity.

In brief summary, the expression cassette or nucleic acid sequence of the invention is typically operably linked to a promoter and incorporated into an expression vector. The vector is suitable for replication and integration of eukaryotic cells. Typical cloning vectors contain transcriptional and translational terminators, initiation sequences, and promoters useful for regulating expression of the desired nucleic acid sequence.

The expression constructs of the invention may also be used in nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods of gene delivery are known in the art. In another embodiment, the invention provides a gene therapy vector.

The nucleic acid may be cloned into many types of vectors. For example, the nucleic acid may be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses and cosmids. Specific vectors of interest include expression vectors, replication vectors, probe-generating vectors, and sequencing vectors.

Further, the expression vector may be provided to the cell in the form of a viral vector. Viruses that may be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. In general, suitable vectors comprise an origin of replication functional in at least one organism, a promoter sequence, a convenient restriction enzyme site and one or more selectable markers.

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

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

One example of a suitable promoter is the immediate early Cytomegalovirus (CMV) promoter sequence. The promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is extended growth factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including but not limited to the simian virus 40 (SV 40) early promoter, the mouse mammary carcinoma virus (MMTV), the Human Immunodeficiency Virus (HIV) Long Terminal Repeat (LTR) promoter, the MoMuLV promoter, the avian leukemia virus promoter, the ebustan-balr (Epstein-Barr) virus immediate early promoter, the ruses sarcoma virus promoter, and human gene promoters such as but not limited to the actin promoter, myosin promoter, heme promoter, and creatine kinase promoter. Further, the invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present invention. The use of an inducible promoter provides a molecular switch that is capable of switching on expression of a polynucleotide sequence operably linked to the inducible promoter when such expression is desired, or switching off expression when expression is undesired. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

Methods for introducing genes into cells and expressing genes into cells are known in the art. In the context of expression vectors, the vector may be readily introduced into a host cell, e.g., a mammalian, bacterial, yeast or insect cell, by any method known in the art. For example, the expression vector may be transferred into the host cell by physical, chemical or biological means.

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

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

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. An exemplary colloidal system for use as an in vitro and in vivo delivery tool is a liposome (e.g., an artificial membrane vesicle).

In the case of non-viral delivery systems, an exemplary delivery means is a liposome. Lipid formulations are contemplated for introducing nucleic acids into host cells (in vitro, ex vivo, or in vivo). In another aspect, the nucleic acid can be associated with a lipid. The nucleic acid associated with the lipid may be encapsulated into the aqueous interior of the liposome, dispersed within the lipid bilayer of the liposome, attached to the liposome via a linking molecule associated with both the liposome and the oligonucleotide, entrapped in the liposome, complexed with the liposome, dispersed in a solution comprising the lipid, mixed with the lipid, associated with the lipid, contained in the lipid as a suspension, contained in or complexed with the micelle, or otherwise associated with the lipid. The lipid, lipid/DNA or lipid/expression vector associated with the composition is not limited to any particular structure in solution. For example, they may be present in a bilayer structure, as micelles or have a "collapsed" structure. They may also simply be dispersed in solution, possibly forming aggregates of non-uniform size or shape. Lipids are fatty substances, which may be naturally occurring or synthetic lipids. For example, lipids include fat droplets, which naturally occur in the cytoplasm as well as in such compounds comprising long chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

In a preferred embodiment of the invention, the vector is a lentiviral vector.

Formulations

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

In one embodiment, the formulation may include buffers such as neutral buffered saline, sulfate buffered saline, and the like, carbohydrates such as glucose, mannose, sucrose or dextran, mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), and preservatives. The formulations of the present invention are preferably formulated for intravenous or intraperitoneal administration.

Detection application and kit

The antibodies of the invention may be used in detection applications, for example for detecting samples, thereby providing diagnostic information.

In the present invention, the samples (specimens) used include cells, tissue samples and biopsy specimens. The term "biopsy" as used herein shall include all kinds of biopsies known to a person skilled in the art. Thus biopsies used in the present invention may include tissue samples prepared, for example, by endoscopic methods or by puncture or needle biopsy of an organ.

Samples for use in the present invention include fixed or preserved cell or tissue samples.

The invention also provides a kit comprising the antibody (or fragment thereof), scFv and the like, and in a preferred embodiment of the invention, the kit further comprises a container, instructions for use, a buffer and the like. In a preferred embodiment, the antibody of the present invention may be immobilized on a detection plate.

The main advantages of the invention include:

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental methods, in which specific conditions are not noted in the following examples, are generally conducted under conventional conditions or under conditions recommended by the manufacturer.

Example 1

1.1 Screening of phage antibody Fab library

Antibodies to human anti-IL-8 Fab were obtained from a combinatorial phage Fab display library created by Sanyou biomedical Co. Human IL-8 was screened by phage display [ science.1985, month 14;228 (4705): 1315-7; nature.1991, month 15;352 (6336): 624-8], using magnetic particles, kingFisher apparatus and immune tube based screening methods.

In biological screening, the antigen (biotinylated IL-8) was immobilized on the surface of streptavidin magnetic particles by incubating the protein with the particles at room temperature and on a rotator for 1h. The particles were then washed with PBS (pH 7.4) and then blocked with 2.5% BSA PBS (pH 7.4) solution for 1h. Phage PBS (pH 7.4) solution containing 2.5% BSA was then added to the antigen-binding magnetic particles. The mixture was incubated at room temperature with stirring for 40min. 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 surface of the magnetic particles were eluted with trypsin solution in 15min under stirring. Coli was infected with the obtained phage (SS 320), and phage were produced in the infected escherichia coli and isolated for the next round of screening. After three rounds of screening, DNAs (phagemids) were isolated from phages and antibody variable domain genes were cloned into expression vectors for Fab production in E.coli cells. Phage display derived anti-IL-8 antibodies were used in the following examples unless otherwise indicated.

1.2. Fab screening for specific binding to human IL-8

ELISA was used to screen Fab binding to human IL-8. All subsequent steps were performed according to standard ELISA protocols. ELISA plate wells were coated with 30. Mu.l human IL-8 (2. Mu.g/ml), sealed and incubated overnight at 4 ℃. Plate wells were washed three times with PBST (PBS containing 0.05% tween-20). To block non-specific binding, 20 μl of PMSM blocking buffer (PBS with 5% skim milk) was added. Plates were incubated on a shaker for 1h at room temperature. After three washes with PBST (PBS containing 0.05% Tween-20), 30. Mu.l of test cell supernatant containing the Fab to be tested mixed with an equal volume of PMSM (PBS containing 5% skimmed milk) was added to each well. The wells were again incubated, shaken at room temperature for 1h, and each well was washed 6 times with PBST (PBS containing 0.05% Tween-20) buffer. anti-M13-HRP conjugated secondary antibody (30. Mu.L/well) was then added to PBST (PBS containing 0.05% Tween-20) at a ratio of 1:8000. The well plate was shaken on a rotary shaker (50 min, room temperature) and washed 9 times with PBST buffer (PBS containing 0.05% tween-20). The color signal was visualized by adding TMB (30. Mu.L/well) until saturated (average 5-10 min), then the color development was stopped by adding stop solution (30. Mu.L/well, 2M sulfuric acid). The colorimetric signals were measured at a wavelength of 450nm using a plate reader (Molecular Device).

1.3. Affinity analysis of anti-IL-8 antibodies

The cloned V _L and V _H regions from Fab sequences in "screening of 1.1 phage antibody Fab library" were subcloned into the expression cassette of the immunoglobulin expression vector (pcdna3.4). The expression vector was transfected into chinese hamster ovary (CHO-s) cells. Cultures were incubated on an orbital shaker platform rotating at 135 rpm. The medium was collected after 4-7 days. After purification by protein-a affinity chromatography, the affinity of recombinant antibodies produced from CHO cells was assessed by ELISA and kinetic analysis using the Biological Layer Interferometry (BLI) technique in GATOR instruments.

ELISA was used to screen for anti-IL-8 antibodies that bind to human IL-8. ELISA plate wells were coated with 30. Mu.l human IL-8 (2. Mu.g/ml), sealed and incubated overnight at 4 ℃. Wells were washed three times with PBST. Add 20. Mu.L of PMSM blocking buffer and incubate the plates on a shaker for 1h at room temperature. After three washes with PBST, 30. Mu.l of the monoclonal antibody containing the anti-IL-8 to be tested mixed with an equal volume of PMSM was added to each well. The wells were again incubated, shaken at room temperature for 1h, and each well was then washed 3 times with PBST buffer to remove unbound antibody. Anti-human kappa+lambda HRP (30. Mu.L/well) was then added to PBST at a ratio of 1:5000 and incubated for 1h. Wash 6 times with PBST buffer. The color development was stopped by adding TMB (30. Mu.L/well) for color development, and then by adding a stop solution (30. Mu.L/well, 2M sulfuric acid). Optical density was measured at a wavelength of 450nm using a suitable plate reader (Molecular Device). As shown in FIG. 1, the antibodies bind to human IL-8 with high affinity.

BLI is a label-free biomolecule detection method that detects a biomolecule interaction by measuring an interference pattern of white light reflected from a biosensor surface. Data for dissociation (kd) and association (ka) rate constants were obtained using GATOR software. The equilibrium dissociation constant (K _D) was calculated from the ratio of kd to ka. As shown in FIG. 2 and Table 1, all anti-IL-8 monoclonal antibodies bind IL-8 with high affinity.

TABLE 1 kinetic data of anti-IL-8 monoclonal antibodies screened

Note that "E" is an expression of 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.

Wherein the heavy chain variable region of antibody LJH001-B30 has the following three Complementarity Determining Regions (CDRs):

CDR1 (GGTFSSYAIS) as shown in SEQ ID NO. 1,

CDR2 (GIIPIFGTANYAQKFQG) as shown in SEQ ID NO. 2, and

CDR3 as shown in SEQ ID NO. 3 (PRYFDWSEDHYDAFDI);

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

The light chain variable region of antibody LJH001-B30 has the following three Complementarity Determining Regions (CDRs):

CDR1' (SGSSSNIGSYTVN) as shown in SEQ ID NO. 4,

CDR2' (SNTQRPS) as shown in SEQ ID NO 5, and

CDR3' (AAWDDSLHGAV) as shown in SEQ ID NO. 6;

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

1.4 Determination of anti-IL-8 antibody stability

The thermal stability of anti-IL-8 antibodies was determined by Differential Scanning Fluorescence (DSF) techniques in an ABI7500 rapid real-time PCR instrument. DSF can be conveniently used to determine the melting temperature (Tm) of a protein.

DSF (also known as protein thermal shift assay) determines thermally induced protein denaturation by measuring the change in fluorescence of a dye that preferentially binds to an unfolded protein (e.g., sypro orange stain, which binds to the hydrophobic region of the protein exposed by unfolding), typically by using a real-time PCR instrument. The stability of a protein is related to its unfolded gibbs free energy, which is temperature dependent. Briefly, in ABI7500 rapid real-time PCR, 20. Mu.l of 0.2mg/ml anti-IL-8 antibody sample PBS (pH 7.4) (containing 100 XSYPRO orange stain) was heated in 1℃increments from 25℃to 99℃at an excitation wavelength of 492nm and an emission wavelength of 580nm. The inflection point (Tm) of the transition curve is calculated using a simple equation such as boltzmann's equation, and the result is shown in fig. 3.

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

Inhibition of IL-8 antibody LJH001-B30 migration ability of IL-8 induced Jurkat-CXCR1 (Jurkat cells overexpressing human CXCR 1) was determined by a 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 (4X 10 ⁵ cells) was incubated in the upper compartment and assayed at 37℃for 4-6 h. The data shown in FIG. 4 shows that anti-IL-8 antibodies inhibited the chemotaxis of Jurkat-CXCR1 in a dose dependent manner.

All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.

Claims

1. An anti-IL-8 antibody, characterized in that the antibody comprises:

(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 vector, characterized in that the vector contains the polynucleotide according to claim 4.

6. The vector according to claim 5, wherein the vector comprises: a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus, a mammalian cell virus, a retrovirus, 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 comprises:

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 plate, characterized in that the detection plate comprises: a substrate and a test strip, and the test strip comprises the antibody according to claim 1.