CN118440205B - An anti-ITGAX antibody and its application - Google Patents

Abstract

The present invention belongs to the field of biomedicine technology, and specifically, relates to an anti-ITGAX antibody and its application. The present invention discloses a monoclonal antibody AH64D and/or a humanized chimeric antibody hAH64D against ITGAX. The antibody provided by the present invention has the characteristics of high affinity and specificity, and also provides a method for detecting ITGAX on the surface of human peripheral blood lymphocytes by flow cytometry based on the above-mentioned hAH64D antibody. The present invention solves the problem of relative shortage of antibody raw materials for ITGAX detection projects on the surface of human peripheral blood lymphocytes in the current market. The antibody provided by the present invention can be used as an auxiliary diagnosis of neurodegenerative diseases such as Alzheimer's disease, Nassau-Hakora disease, Parkinson's disease, amyotrophic lateral sclerosis and frontotemporal dementia through the detection of ITGAX on the surface of human peripheral blood lymphocytes.

Description

An anti-ITGAX antibody and its application

Technical Field

The present invention belongs to the field of biomedicine technology, and specifically relates to an anti-ITGAX antibody and an application thereof.

Background Art

ITGAX (Integrin alpha-X), also known as CD11c, is a receptor for fibrinogen. It recognizes the sequence G-P-R in fibrinogen. It mediates cell-cell interactions during inflammatory responses. It is particularly important in monocyte adhesion and chemotaxis. It is a specific protein expressed by dendritic cells (DCs), which binds to β2-integrin to mediate the phagocytosis of DCs.

ITGAX is a type I transmembrane glycoprotein of approximately 150 kDa that is part of the complement receptor 4 (CR4). Together with CR3, also known as Mac-1 or CD11b/CD18, CR4 belongs to the β2 integrin family of adhesion molecules. CR3 and CR4 constitute heterodimeric integral membrane proteins, consisting of a CD18 β chain (β2) and either an integrin αM chain (ITGAM, CD11b) or an integrin αX chain (ITGAX, CD11c), respectively. CR3 and CR4 have overlapping ligand binding specificities, and their extracellular domains share 87% sequence homology. Complement receptors provide an important link between cellular functions and soluble components, factors, and other associated proteins. Thus, CR3 and CR4 are able to bind a variety of ligands, such as fibrinogen or heparin. In addition, both CR3 and CR4 are involved in cell adhesion, migration, and phagocytosis. However, while CR3 is involved in phagocytosis of iC3b-opsonized bacteria, CR4 directs cell adhesion to fibrinogen in human monocyte-derived macrophages and dendritic cells. Our data suggest that ITGAX plays an important role in the regulation of hematopoietic stem and progenitor cells (HSPSs) under stress conditions.

Inflammatory clusters are frequently found in the liver under inflammatory conditions. These clusters include T cells, CD11b ⁺ neutrophils, cells of the monocyte/macrophage lineage, and ITGAX ⁺ dendritic cells. The expression of ITGAX is a marker of microglial activation, and the presence of ITGAX-positive microglia around Aβ plaques in Alzheimer's disease (AD) has been demonstrated in multiple studies.

Currently, there are no diagnostic reagent raw materials with high specificity for ITGAX in the industry. Since ITGAX is a single transmembrane protein, it only exists on the surface of the cell membrane and its content in peripheral blood and body fluids is extremely low. Traditional immunodiagnostic methods, such as chemiluminescence assay (CLIA), radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), latex enhanced turbidimetry, etc., are suitable for the detection of secretory proteins in peripheral blood and other body fluids, but not for the detection of transmembrane proteins. Immunohistochemistry methods have the disadvantages of long detection time, complex operation, poor repeatability, and low sensitivity. Flow cytometry can detect natural cell membrane proteins, so it is more suitable for ITGAX analysis and detection.

Currently, there is an urgent need to develop an ITGAX antibody with strong specificity and high sensitivity for the development of ITGAX kits.

Summary of the invention

The purpose of the present invention is to overcome the problems existing in the prior art and provide an anti-ITGAX antibody and its application. The present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to ITGAX, which can be in the form of a monoclonal antibody or a human chimeric antibody. The antibody provided by the present invention has the characteristics of high affinity and specificity.

The purpose of the present invention and the solution to the technical problem are achieved by adopting the following technical solutions.

The first aspect of the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to ITGAX, comprising a light chain variable region VL and a heavy chain variable region VH, wherein the light chain variable region VL comprises LCDR1-3 having an amino acid sequence as shown in SEQ ID NO: 3-5, or having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or at least 100% identity thereto, and the heavy chain variable region VH comprises HCDR1-3 having an amino acid sequence as shown in SEQ ID NO: 6-8, or having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or at least 100% identity thereto.

In some embodiments of the invention, the amino acid sequence of the VL is as shown in SEQ ID NO:1 or has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity thereto, and the amino acid sequence of the VH is as shown in SEQ ID NO:2 or has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity thereto.

In some embodiments of the present invention, the amino acid sequence of VL comprises LCDR1, LCDR2 and LCDR3 sequences having a light chain variable region as shown in SEQ ID NO: 1, or having one or more conservative amino acid substitutions, deletions or insertions or any combination thereof; the amino acid sequence of VH comprises HCDR1, HCDR2 and HCDR3 sequences having a heavy chain variable region as shown in SEQ ID NO: 2, or having one or more conservative amino acid substitutions, deletions or insertions or any combination thereof.

In some embodiments of the present invention, it further comprises a light chain constant region CL and/or a heavy chain constant region CH, wherein the light chain constant region CL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO:13, and the heavy chain constant region CH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO:14.

In some embodiments of the invention, the antibody is of an isotype selected from IgG.

In some embodiments of the present invention, the antibody is of the IgG1 subtype.

In some embodiments of the present invention, the antigen-binding fragment is selected from a diabody.

In some embodiments of the present invention, the antibody is a murine monoclonal antibody or a humanized chimeric antibody.

In some embodiments of the present invention, the antibody is a monoclonal antibody, wherein the antibody is a monoclonal antibody, clone number is AH64D, which further comprises a light chain and a heavy chain, the light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 15, and the heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 16.

In some embodiments of the present invention, the antibody is a humanized chimeric antibody, wherein the antibody is a humanized chimeric antibody, clone number is hAH64D, which further comprises a light chain and a heavy chain, the light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 17, and the heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 18.

The second aspect of the present invention provides a nucleic acid comprising a nucleic acid encoding any of the above-mentioned antibodies or antigen-binding fragments thereof that specifically bind to ITGAX.

In some embodiments of the invention, the nucleic acid is DNA, such as cDNA or recombinant DNA.

The third aspect of the present invention provides a vector comprising the above-mentioned nucleic acid.

The fourth aspect of the present invention provides a host cell comprising the above-mentioned nucleic acid or the above-mentioned vector.

In some embodiments of the present invention, the host cell is a eukaryotic cell.

In some embodiments of the present invention, the host cell is a mammalian cell, including but not limited to 293F cells and CHO cells.

The fifth aspect of the present invention provides a hybridoma cell, which can produce any of the above-mentioned antibodies or antigen-binding fragments that specifically bind to ITGAX.

The sixth aspect of the present invention provides a method for preparing the antibody or antigen-binding fragment thereof described in any one of the above items, comprising: culturing the host cell or hybridoma cell described in any one of the above items under conditions that allow the antibody or antigen-binding fragment thereof to be expressed.

A seventh aspect of the present invention provides a composition comprising:

(i) an antibody or antigen-binding fragment thereof, nucleic acid, vector, host cell or hybridoma cell that specifically binds to ITGAX as described in any of the above items;

(ii) a pharmaceutically acceptable carrier or adjuvant.

The eighth aspect of the present invention provides an antibody-conjugate, which comprises any of the above antibodies or antigen-binding fragments thereof that specifically bind to ITGAX.

The ninth aspect of the present invention provides a detection reagent, which comprises any of the above-mentioned antibodies or antigen-binding fragments thereof that specifically bind to ITGAX.

In some embodiments of the present invention, the antibody or antigen-binding fragment thereof that specifically binds to ITGAX is fluorescently labeled and used as a component of a flow cytometry fluorescence detection reagent that specifically recognizes ITGAX.

In some embodiments of the present invention, substances used for fluorescent labeling include but are not limited to PE, PerCP, FITC, and APC.

The tenth aspect of the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to ITGAX, or a detection reagent as described in any of the above items, for use in preparing a reagent or product for detecting the ITGAX content in a sample, a reagent or product for diagnosing ITGAX-related diseases, or a drug for preventing and/or treating ITGAX-related diseases.

In some embodiments of the present invention, the ITGAX-related disease is selected from the group consisting of leukemia, liver inflammation, glioma, Alzheimer's disease, Nassau-Hakora disease, Parkinson's disease, amyotrophic lateral sclerosis and frontotemporal dementia, Huntington's disease, amyotrophic lateral sclerosis, and neuroinflammation.

The eleventh aspect of the present invention provides an antibody or antigen-binding fragment thereof, nucleic acid, vector, host cell, hybridoma cell, composition and/or antibody-conjugate specifically binding to ITGAX as described in any one of the above items for use in preparing any of the following products:

(1) Test ITGAX products;

(2) Products that stimulate or enhance the immune response;

(3) Products for the prevention and/or treatment of ITGAX-related diseases.

By means of the above technical solution, the present invention has at least the following advantages:

The present invention provides an anti-ITGAX monoclonal antibody and a modified human chimeric antibody hITGAX, which avoids the HAMA reaction in natural samples. The antibody or antigen-binding fragment thereof provided by the present invention has the characteristics of high affinity and specificity. The product of the present invention solves the problem of poor specificity and low sensitivity of antibody raw materials for ITGAX detection projects on the market. The antibody disclosed in the present invention has high sensitivity and specificity and can be used as an auxiliary diagnosis for neurodegenerative diseases such as Alzheimer's disease, Nassau-Hakora disease, Parkinson's disease, amyotrophic lateral sclerosis and frontotemporal dementia.

When screening antibodies, the present invention selects a variety of natural cell antigens and compares antibody specificity detection. After multiple rounds of screening, AH64D is obtained as an antibody with good specificity for ITGAX. Multiple subcutaneous and multi-point injections are used in the animal immunization process to improve the titer of the final animal serum and the positive clone rate. The addition of HAT screening reagents is postponed from the day of fusion to the second day, which significantly improves the cell positive rate.

The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the preferred embodiments of the present invention are described in detail as follows.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a diagram of the construction of the pCDNA3.1-ITGAX plasmid;

FIG2 is a graph showing the purity of the ITGAX target protein detected by SDS-PAGE experiment;

FIG3 is a gel image showing the subtype identification of the antibody expressed by the hybridoma cell line AH64D and the cloned variable regions of the antibody according to an embodiment of the present invention, and the lane sequence is shown in Table 7;

FIG4 is a plasmid map of a humanized chimeric antibody expression plasmid pGmab-K-hAH64D constructed using antibody AH64D according to an embodiment of the present invention;

FIG5 is a plasmid map of a humanized chimeric antibody expression plasmid pGmab-H-hAH64D constructed using antibody AH64D according to an embodiment of the present invention;

FIG6 shows the application of antibody hAH64D in detecting ITGAX on the surface of human peripheral lymphocytes by flow cytometry according to an embodiment of the present invention.

DETAILED DESCRIPTION

In order to make the technical means, creative features, objectives and effects of the present invention easy to understand, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

In order to make the present invention more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present invention belongs.

As used herein, the terms "first" and "second" are used for descriptive purposes only and are not to be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of the features. In the description of the present disclosure, the meaning of "plurality" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined.

As used herein, the terms “comprise” or “include” are open expressions, namely including the contents specified in the present invention but not excluding other contents.

As used herein, the terms "optionally," "optional," or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

In one aspect, the present disclosure provides an antibody or an antigen-binding fragment thereof that specifically binds to ITGAX, comprising a light chain variable region VL and a heavy chain variable region VH, wherein the light chain variable region VL comprises LCDR1-3 having an amino acid sequence as shown in SEQ ID NO: 3-5, or at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or at least 100% identical thereto, respectively, and the heavy chain variable region VH comprises HCDR1-3 having an amino acid sequence as shown in SEQ ID NO: 6-8, or at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or at least 100% identical thereto, respectively.

In some embodiments of the antibodies or antigen-binding fragments thereof that specifically bind to ITGAX disclosed herein, the amino acid sequence of the VL is as shown in SEQ ID NO: 1, or has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity thereto, and the amino acid sequence of the VH is as shown in SEQ ID NO: 2, or has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity thereto.

In some embodiments of the antibodies or antigen-binding fragments thereof that specifically bind to ITGAX disclosed herein, the amino acid sequence of the VL comprises LCDR1, LCDR2 and LCDR3 sequences having a light chain variable region as shown in SEQ ID NO: 1, or having one or more conservative amino acid substitutions, deletions or insertions, or any combination thereof; the amino acid sequence of the VH comprises HCDR1, HCDR2 and HCDR3 sequences having a heavy chain variable region as shown in SEQ ID NO: 2, or having one or more conservative amino acid substitutions, deletions or insertions, or any combination thereof.

As used herein, the term "antibody" refers to an immunoglobulin molecule that has the ability to specifically bind to a specific antigen. Antibodies typically contain a variable region and a constant region in each heavy chain and light chain. The variable regions of the antibody heavy and light chains contain a binding domain that interacts with the antigen. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system such as C1q (the first component in the classical pathway of complement activation). Therefore, most antibodies have a heavy chain variable region (VH) and a light chain variable region (VL) that together form the antibody portion that binds to the antigen.

As used herein, the term "antibody analogs" refers to derivatives produced by biological or chemical methods, based on the antibody structure, by deletion, addition, or modification of chemical groups (such as amino acids). These derivatives still contain structures similar to the antibody variable region (or the CDR region in the antibody variable region), and can undergo reactions similar to antigen-antibody binding through these structures.

As used herein, the term "binding" or "specific binding" refers to a non-random binding reaction between two molecules, such as an antibody and its target antigen. In certain embodiments, an antibody that specifically binds to an antigen refers to an antibody that binds to the antigen with an affinity corresponding to a KD of less than about 10 5M, such as less than about 10 6M, 10 7M, 10 8M, 10 9M or 10 10M or less. As used herein, "KD" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the KD, the higher the binding affinity between the antibody and the antigen.

As used herein, the term "antibody variable region" refers to a domain in the heavy and light chains of an antibody. It includes a light chain variable region (VL) and a heavy chain variable region (VH). In nature, the antibody variable region is encoded by V, D (only applicable to heavy chains), and J fragments in immunoglobulin (heavy and light chain) genes through gene recombination splicing. The amino acid sequences of the variable regions between different antibodies have a high degree of diversity (the amino acid sequences of other regions of the antibody are relatively highly identical) and are responsible for recognizing and binding to specific antigenic determinants. In the antibody variable region, the VL domain and the VH domain both contain a framework region (FR) and a CDR region (comlementarity determining region) from the amino terminus to the carboxyl terminus. A typical antibody variable region has 3 framework regions and 3 CDR regions, which are interspersed with each other: FR1, CDR1, FR2, CDR2, FR3 and CDR3. The framework region FR mainly plays a role in the formation of the protein domain framework, while the CDR region mainly plays a role in the specific recognition and binding of antigen-antibody. The CDR1, CDR2 and CDR3 of the VL domain are also referred to herein as LCDR1, LCDR2 and LCDR3, respectively; the CDR1, CDR2 and CDR3 of the VH domain are also referred to herein as HCDR1, HCDR2 and HCDR3, respectively.

The amino acid arrangement of each VL domain and VH domain is consistent with any conventional definition of CDR. Conventional definitions include Kabat definition (Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991)), Chothia definition (Chothia and Lesk, J. Mol. Biol. 196: 901 917, 1987; Chothia et al., Nature 342: 878 883, 1989); a composite of Chothia Kabat CDRs, wherein CDR H1 is a composite of Chothia CDRs and Kabat CDRs; the AbM definition used by Oxford Molecular's antibody modeling software; and the CONTACT definition of Martin et al. (World Wide Web bioinfo.org.uk/abs). Kabat provides a widely used numbering convention (Kabat numbering system), in which corresponding residues between different heavy chains or between different light chains are given the same number. The present disclosure may use CDRs defined according to either of these numbering systems, but preferred embodiments use CDRs defined according to Kabat or Chothia.

As used herein, the term "anti-ITGAX antibody (or antibody analog)" exists in a single (or monoclonal) form, and a single antibody (or antibody analog) contains a single structure (such as a single amino acid sequence).

As used herein, the term "identity" is used to describe an amino acid sequence or a nucleic acid sequence relative to a reference sequence, and the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences is determined by conventional methods, for example, see Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN program (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research There are many algorithms for aligning sequences and determining sequence identity, including the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48:443; the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85:2444; the Smith-Waterman algorithm (Meth. Mol. Biol. 48:443); the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85:24 ... l.70:173-187 (1997); and BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J. Mol. Biol. 215:403-410). Computer programs that utilize these algorithms are also available, and include, but are not limited to: ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al., Meth. Enzym., 266:460-480 (1996)); or GAP, BESTFIT, BLAST Altschul et al., supra, FASTA, and TFASTA, available in the Genetics Computing Group (GCG) package, Version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC/Gene program provided by Intelligenetics, Mountain View, California.

Under the premise of not substantially affecting the activity of the antibody (retaining at least 95% of the activity), those skilled in the art can replace, add and/or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more) amino acids in the sequence of the present invention to obtain variants of the sequence of the antibody or its functional fragment. They are all considered to be included in the scope of protection of the present invention. For example, amino acids with similar properties are replaced in the variable region. The variant sequence of the present invention may have at least 80% identity (or homology) with the reference sequence. The sequence identity of the present invention can be measured using sequence analysis software. For example, the computer program BLAST with default parameters is used, especially BLASTP or TBLASTN. The amino acid sequences described in the present invention are all shown in a manner from N-terminus to C-terminus.

As used herein, the term "conservative amino acid substitution" refers to the replacement of an amino acid with another amino acid residue that is biologically, chemically or structurally similar. Biologically similar means that the replacement does not destroy the biological activity of the ITGAX antibody or the ITGAX antigen. Structurally similar means that the amino acids have side chains of similar length, such as alanine, glycine or serine, or have side chains of similar size. Chemical similarity means that the amino acids have the same charge or are hydrophilic or hydrophobic. For example, the hydrophobic residues isoleucine, valine, leucine or methionine replace each other. Or with polar amino acids, such as arginine replacing lysine, glutamic acid replacing aspartic acid, glutamine replacing asparagine, serine replacing threonine, etc.

Therefore, the antibodies of the present invention can also be directly synthesized according to the antibody variable region amino acid sequence provided by the present invention, and can be derived into different antibody analogs through further chemical modification.

In some embodiments of the antibodies or antigen-binding fragments thereof that specifically bind to ITGAX disclosed herein, they further comprise a light chain constant region CL and/or a heavy chain constant region CH, wherein the light chain constant region CL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 13, and the heavy chain constant region CH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 14.

As used herein, the term "heavy chain constant region" includes an amino acid sequence derived from an immunoglobulin heavy chain. The polypeptide comprising the heavy chain constant region includes at least one of the following: a CH1 domain, a hinge (e.g., an upper hinge region, a middle hinge region, and/or a lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. For example, the antigen-binding polypeptide for use in the present disclosure may include: a polypeptide chain comprising a CH1 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH2 domain; a polypeptide chain comprising a CH1 domain and a CH3 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH3 domain; or a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, a CH2 domain, and a CH3 domain. In another embodiment, the polypeptide of the present disclosure includes a polypeptide chain containing a CH3 domain. In addition, the antibody for use in the present disclosure may lack at least a portion of a CH2 domain (e.g., all or part of a CH2 domain). As described above, those of ordinary skill in the art will appreciate that the heavy chain constant region can be modified so that it differs in amino acid sequence from a naturally occurring immunoglobulin molecule.

Based on the amino acid sequence of the constant region of the heavy chain of the antibody, immunoglobulin molecules can be divided into five classes (isotypes): IgA, IgD, IgE, IgG and IgM, and can be further divided into different subtypes, such as IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, etc. Based on the amino acid sequence of the light chain, the light chain of the antibody can be divided into lambda (λ) chain and kappa (κ) chain. The antibodies disclosed herein can be any of the above classes or subtypes.

In some embodiments of the antibodies or antigen-binding fragments thereof disclosed herein that specifically bind to ITGAX, the antibody has an isotype selected from IgG. In some embodiments, the antibody has a subtype selected from IgG1.

As used herein, the term "antigen-binding fragment" is also referred to as "antibody fragment". Antibody fragments generally refer to antigen-binding antibody fragments, which may include a portion of a complete antibody, generally an antigen-binding region or a variable region. Examples of antibody fragments include Fab, Fab', F(ab') ₂ , Fd, Fv, scFv, dAb, Fab/c, complementary determining region, single-chain antibody, double-chain antibody, or single-domain antibody molecule, etc. Among them, the "Fab fragment" is composed of a light chain and the CH1 and variable region of a heavy chain. The heavy chain of the Fab molecule cannot form a disulfide bond with another heavy chain molecule. Fab' fragment, which has one or more cysteine residues at the C-terminus of the CH1 domain of the Fab fragment; F(ab') ₂ fragment, which is a bivalent fragment comprising two Fab' fragments linked by a disulfide bond at the hinge region; Fd fragment, which has VH and CH1 domains; Fv fragment, which has VL and VH domains in a single arm of the antibody; dAb fragment, which consists of a VH domain or a VL domain; isolated CDR regions; and modified forms of any of the above fragments that retain antigen-binding activity.

In some embodiments of the antibodies or antigen-binding fragments thereof disclosed herein that specifically bind to ITGAX, the antigen-binding fragment is selected from a diabody.

In some embodiments of the antibody or antigen-binding fragment thereof disclosed herein that specifically binds to ITGAX, the antibody is a murine monoclonal antibody or a humanized chimeric antibody.

In some embodiments of the antibodies or antigen-binding fragments thereof that specifically bind to ITGAX disclosed herein, the antibody is a monoclonal antibody further comprising a light chain and a heavy chain, the light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 15, and the heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 16.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population. That is, except for a small amount of mutations that may occur naturally, each antibody constituting the population is identical. Monoclonal antibodies are highly specific and are directed against a single antigen. The term "monoclonal antibody" herein is not limited to antibodies produced by hybridoma technology, nor should it be construed as requiring antibodies produced by any particular method.

In some embodiments of the antibodies or antigen-binding fragments thereof that specifically bind to ITGAX disclosed herein, the antibody is a humanized chimeric antibody further comprising a light chain and a heavy chain, the light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 17, and the heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 18.

As used herein, the term "chimeric antibody" refers to an antibody in which a portion of the heavy chain and/or light chain is derived from one source or species, while the rest of the heavy chain and/or light chain is derived from a different source or species. "Humanized chimeric antibody" refers to an antibody obtained by inserting the nucleic acid sequence of the light chain V region and the nucleic acid sequence of the heavy chain V region of a monoclonal antibody into the constructed plasmids of pGmab-K (containing the constant region of the human antibody kappa chain) and pGmab-H (containing the constant region of the human antibody IgG1 chain) expression plasmids by molecular cloning methods, and transfecting host cells to express the obtained antibody. The term "humanized chimeric antibody" herein is not limited to antibodies produced by the techniques described herein, nor should it be interpreted as requiring antibodies produced by any particular method.

In yet another aspect, the present invention provides a nucleic acid comprising a nucleotide sequence encoding an antibody or antigen-binding fragment thereof that specifically binds to ITGAX disclosed herein.

In yet another aspect, the present disclosure provides a vector comprising a nucleic acid disclosed herein.

As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is connected. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop to which additional DNA fragments can be connected. Another type of vector is a viral vector, in which additional DNA fragments can be connected to the viral genome. Some vectors can replicate autonomously in the host cell in which they are introduced (e.g., bacterial vectors and free mammalian vectors with bacterial replication origins). Other vectors (e.g., non-free mammalian vectors) can be integrated into the genome of the host cell after being introduced into the host cell, thereby replicating together with the host genome. In addition, some vectors can guide the expression of the gene operably connected thereto. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In some embodiments, the vector includes but is not limited to: (1) plasmid; (2) phagemid; (3) cosmid; (4) artificial chromosome, such as yeast artificial chromosome, bacterial artificial chromosome or artificial chromosome derived from P1; (5) bacteriophage, such as λ phage or M13 phage; (6) animal virus, such as retrovirus, adenovirus, adeno-associated virus, sporovirus, poxvirus, baculovirus.

When the nucleic acid disclosed in the present invention is connected to a vector, the nucleic acid can be directly or indirectly connected to the control elements on the vector, as long as these control elements can control the translation and expression of the nucleic acid. Of course, these control elements can come directly from the vector itself, or they can be exogenous, that is, they are not from the vector itself. Of course, the polynucleotide can be operably connected to the control elements. In this article, "operably connected" means connecting the exogenous gene to the vector so that the control elements in the vector, such as transcription control sequences and translation control sequences, etc., can play their intended function of regulating the transcription and translation of the exogenous gene. Of course, the polynucleotides used to encode the heavy and light chains of the antibody can be independently inserted into different vectors, and it is common to insert them into the same vector.

In another aspect, the present disclosure provides a host cell comprising a nucleic acid disclosed herein or a vector disclosed herein.

As used herein, the term "host cell" refers to a cell into which an expression vector has been introduced. The expression vector can be introduced into a host cell to construct a recombinant cell, and then the recombinant cells are used to express the antibody or antigen-binding fragment provided by the present invention. The corresponding antibody can be obtained by culturing the recombinant cell. In some embodiments, the host cell includes, for example, a CHO cell, such as a CHOS cell and a CHO K1 cell, or a HEK293 cell, such as HEK293A, HEK293T, and HEK293F.

In some embodiments of the host cells disclosed herein, the host cell is a eukaryotic cell.

In some embodiments of the host cells disclosed herein, the host cells are mammalian cells, including but not limited to 293F cells and CHO cells.

In another aspect, the present disclosure provides hybridoma cells that can produce antibodies or antigen-binding fragments that specifically bind to ITGAX disclosed in the present disclosure.

As used herein, the term "hybridoma cell" refers to a cell formed by the fusion of myeloma cells and B lymphocytes in the process of preparing monoclonal antibodies. In the classic monoclonal antibody preparation method, a suitable antigen is first required and used to immunize animals. In order to prepare anti-ITGAX antibodies (or antibody analogs), the appropriate antigen must contain ITGAX. This antigen can be isolated and purified from natural human tissues or blood, or expressed in prokaryotic or eukaryotic cells and purified by artificial recombinant protein expression. Antigens are used for animal immunization and antibody screening detection.

In the classic monoclonal antibody preparation method, animals are first immunized with ITGAX antigens, and blood is collected at intervals to verify whether the animals have produced antibody responses to the ITGAX antigens. B cells are then isolated from the spleen of animals with antibody responses and fused with immortal myeloma cells in vitro to obtain hybridoma cells. These hybridoma cells are then diluted to the limit in culture plates and regrown (monoclonal hybridoma cell lines), and the culture supernatants of these hybridoma cell lines are taken to detect whether they contain specific antibodies against the antigen. Based on the antibody yield, quality and cell line growth characteristics, the best monoclonal antibody production cell line can be selected for subsequent monoclonal antibody production.

Other methods can also be used to obtain monoclonal antibodies. For example, spleen cells from the above animals can be isolated and then incubated with labeled antigens (such as fluorescein-labeled ITGAX). Since antibody-producing B cells usually have antibody molecules presented on the cell membrane surface, these cells will be bound (stained) by the labeled antigen and can be sorted by a fluorescent flow cytometer. The mRNA of these sorted B cells can be isolated, and a library of antibody variable region cDNA can be obtained by in vitro reverse transcription and specific PCR reactions. This cDNA library can be inserted into an expression plasmid (such as an antibody expression plasmid suitable for expression in mammalian cells, or a phage expression plasmid suitable for expression in bacterial cells) and expressed in a host cell suitable for this expression plasmid. These host cells (or phages) can be isolated and purified (cloned) by different methods (such as the cell limiting dilution culture method described above, or the phage plaque plating method). These cell lines or phage clones can be used to produce antibodies and analyze and identify the antibodies.

In another aspect, the present disclosure provides a method for preparing the antibody or antigen-binding fragment thereof disclosed in the present invention, comprising: culturing the host cell or hybridoma cell disclosed in the present invention under conditions that allow the antibody or antigen-binding fragment thereof to be expressed.

In another aspect, the present disclosure provides a composition comprising: (i) an antibody or antigen-binding fragment thereof, nucleic acid, vector, host cell or hybridoma cell disclosed in the present invention that specifically binds to ITGAX; and (ii) a pharmaceutically acceptable carrier or adjuvant.

As used herein, the term "pharmaceutically acceptable" means that the carrier or adjuvant is compatible with the other ingredients of the composition and not substantially toxic to the recipient thereof, and/or such carrier or adjuvant is approved or available for inclusion in pharmaceutical compositions for parenteral administration to humans.

In some embodiments, the carrier or adjuvant used with the compositions disclosed herein include, but are not limited to, sterile liquids, such as water and oils, including oils of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. In some embodiments, when the pharmaceutical composition is administered intravenously, the carrier can be water. Saline solutions and aqueous glucose solutions and glycerol solutions can also be used as liquid carriers, particularly for injection solutions. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences by E.W.Martin, which is incorporated herein by reference. Such compositions will contain a clinically effective dose of an antibody or antibody fragment, together with a suitable carrier, to provide a dosage form suitable for the patient. The preparation should be suitable for the mode of administration. The preparation can be packaged in an ampoule, a disposable syringe or a multi-dose vial made of glass or plastic.

In yet another aspect, the present disclosure provides an antibody-conjugate comprising the antibody or antigen-binding fragment thereof that specifically binds to ITGAX disclosed in the present invention.

In another aspect, the present invention provides a detection reagent comprising the antibody or antigen-binding fragment thereof that specifically binds to ITGAX disclosed in the present invention.

In some embodiments of the detection reagent disclosed herein, the antibody or antigen-binding fragment thereof that specifically binds to ITGAX is fluorescently labeled and used as a component of a flow cytometry fluorescence detection reagent that specifically recognizes ITGAX.

In the present disclosure, suitable substances for fluorescent labeling include but are not limited to PE (phycoerythrin), PerCP (peridinocyanin-chlorophyll-protein complex), FITC (fluorescein isothiocyanate), and APC (allophycocyanin).

On the other hand, the present disclosure provides the use of the antibody or antigen-binding fragment thereof that specifically binds to ITGAX, or the detection reagent disclosed in the present invention, in the preparation of a reagent or product for detecting the content of ITGAX in a sample, a reagent or product for diagnosing ITGAX-related diseases, or a drug for preventing and/or treating ITGAX-related diseases.

In some embodiments of the uses disclosed herein, the ITGAX-related disease is selected from the group consisting of leukemia, liver inflammation, glioma, Alzheimer's disease, Nassau-Hakora disease, Parkinson's disease, amyotrophic lateral sclerosis and frontotemporal dementia, Huntington's disease, amyotrophic lateral sclerosis, neuroinflammation.

In another aspect, the present disclosure provides the use of the antibody or antigen-binding fragment thereof, nucleic acid, vector, host cell, hybridoma cell, composition and/or antibody-conjugate disclosed in the present invention that specifically binds to ITGAX in the preparation of any of the following products:

(1) Test ITGAX products;

(2) Products that stimulate or enhance the immune response;

(3) Products for the prevention and/or treatment of ITGAX-related diseases.

The anti-ITGAX antibody of the present invention can be used to detect ITGAX on the surface of human peripheral blood lymphocytes, or substances containing ITGAX, and substances that can specifically bind to ITGAX (such as anti-ITGAX antibodies). The principle of detection is mainly based on the specific recognition and binding of the ITGAX antibody of the present invention to ITGAX, and detection is performed using chemical labeling technology (such as labeled antibodies, or labeled specific secondary antibodies) or physical detection technology (such as light scattering technology, plasma resonance technology, etc.).

The present invention provides anti-ITGAX monoclonal antibody AH64D and/or humanized chimeric antibody hAH64D, which has the characteristics of high affinity and specificity, and also provides a flow detection method based on the above antibodies. The present invention solves the problem of relative shortage of antibody raw materials for ITGAX detection projects in the current market, can be used as an auxiliary diagnosis for related neurological diseases, and fills the gap in the domestic related market.

The nucleic acid encoding the heavy chain and/or light chain of the antibody of the present invention is within the scope of the present invention. According to the amino acid sequence of the heavy chain and/or light chain, those skilled in the art can easily obtain the corresponding nucleic acid sequence, as shown in Table 1. It should be noted that the CDR sequences listed in Table 1 below are obtained based on the IMGT database. Those skilled in the art should know that the CDR sequences analyzed from different databases may be different, but these changes should be included in the scope of protection of the present invention.

Table 1 Sequence information corresponding to different numbers

The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.

Example 1: Construction of ITGAX recombinant protein expression plasmid

The nucleic acid sequence of ITGAX was based on the NCBI Gene ID: NM_000887.5 (ITGAX) sequence. When synthesizing the DNA sequence, the extracellular region sequence of ITGAX was connected to a protein purification tag sequence (6×His) at the 3' end, and the upstream and downstream of the DNA sequence were designed with EcoRI and XbaI sites, respectively. The above designed DNA sequence was synthesized by a third-party gene synthesis company and constructed into the pUC57 plasmid. After construction, the plasmid was named pUC57-ITGAX. After the NEB Cutsmart nuclease system, 2μg of pUC57-ITGAX and pCDNA3.1 plasmids were added with 2UEcoRI and 2U XbaI endonucleases, respectively. After 2 hours of enzyme digestion at 37℃, the positive bands were separated and recovered by 1% agarose gel, and the concentration of the recovered DNA fragments was detected by A260. 2nmol of the double-digested fragments of pUC57-ITGAX and pCDNA3.1 plasmids were taken, 1U T4 ligase was diluted to 20 μL with T4 ligase buffer and ultrapure water, and transformed into DH5α competent cells after overnight ligation at 16°C, and spread on LB plates. Single colonies were picked and sequenced with FCMV and SV40 universal primers. The gene sequence was correct after sequencing. The constructed plasmid was named pCDNA3.1-ITGAX, and its structure is shown in Figure 1.

Example 2: Expression and identification of ITGAX recombinant protein

First, the expression plasmid pCDNA3.1-ITGAX was transfected into CHO-S cells. The transfection reagent used for transfection of suspension cultured CHO-S cells was Free Style MAX (Invitrogen, 16447100), and the transfection procedure was performed as per the manufacturer's instructions.

Subsequently, the transfected CHO-S cells were transferred to a 125 mL triangular shake flask in 30 mL FreeStyleCHO medium at a cell density of 1×10 ⁶ cells/mL. The cells were cultured in a CO ₂ incubator (37°C, 5% CO ₂ ) with a shaker speed of 130 rpm. The culture supernatant was collected after 7 days of cell culture, and the concentration of ITGAX recombinant protein (expressed and secreted by CHO-S cells) in the supernatant was detected by sandwich ELISA.

Specifically, in the sandwich ELISA, purified antibodies (abcam, #ab254183, #ab195534) were used as coating antibodies and detection antibodies, respectively. In order to determine the active concentration of ITGAX, a known concentration (detected by A280 method) of ITGAX control antigen (Sino Biological, #CT017-H2508H) was used to make a concentration standard curve. 1 μg/mL mouse anti-ITGAX antibody (abcam, #ab254183) diluted with pH 9.6 carbonate buffer, 100 μL/well coated 96-well ELISA plate, overnight at 4°C; washed twice with PBST; added 200 μL of 1% BSA in PBS to each well, blocked at room temperature for 2 hours, and then placed on trifold paper to pat dry; sample addition: PTB diluted ITGAX transfection supernatant and control antigen in different proportions or concentrations, 0.1 mL of diluted sample was added to the reaction well, incubated at 37°C for 1 hour, and then washed, and blank wells (no sample added), negative control wells and positive control wells were made at the same time. 0.1 mL of newly diluted antibody was added to each reaction well, incubated at 37°C for 1 hour, and then washed 3 times. Add enzyme-labeled secondary antibody: 0.1 mL of newly diluted enzyme-labeled antibody was added to each reaction well. Incubated at 37°C for 1 hour, and washed 3 times. Add substrate solution for color development: Add 0.1 mL of TMB substrate solution to each reaction well and let stand at room temperature for 10 minutes. Add 0.1 mL of 1M H ₂ SO ₄ to each reaction well. Measure the OD value to determine the result. Use a microplate reader to measure the absorbance value (A450) at 450 nm. The results are shown in Table 2.

Finally, ELISA CALC was used to calculate the 4-parameter logistic curve of the control protein as the standard curve, where R ² = 0.997. The ITGAX protein expression concentration in the supernatant of the cells to be tested was calculated to be about 35.6 ng/mL, and the results showed that the target protein was significantly expressed in the supernatant of CHO-S cells transfected with pCDNA3.1-ITGAX.

Table 2 Absorbance values corresponding to different protein concentrations

Example 3: Screening and identification of ITGAX CHO-S stable cell lines

In order to screen and obtain cell lines stably expressing ITGAX, CHO-S cells transfected with pCDNA3.1-ITGAX were diluted to a density of 100 cells/well and added to the microwells of a 96-well plate, 200 μL CD-CHO medium/well, and the medium contained 12.5 μM MSX reagent (glutamine synthetase GS inhibitor, used to screen GS resistance gene). The microwell culture plate was cultured in a CO ₂ incubator (37°C, 5% CO ₂ ) for about 20 days to allow resistant cells to grow. The cells in the microwells with resistant cell growth were transferred to a 24-well cell culture plate for expansion culture (and quantitative determination of the expression level of ITGAX protein in different cell lines), with about 1000 cells/well at the time of transfer. After 7 days of culture, the cell supernatant in the plate was taken out for ITGAX protein concentration detection.

The specific operation of ITGAX protein concentration detection is as follows: 1 μg/mL mouse anti-ITGAX antibody (abcam, #ab254183) diluted with pH 9.6 carbonate buffer solution, 100 μL/well coated 96-well ELISA plate, 4°C overnight; PBST washed twice; 200 μL of 1% BSA in PBS was added to each well, blocked at room temperature for 2 hours, and then placed on trifold paper and patted dry; sample addition: PTB diluted the culture supernatant of different ITGAX transfected CHO-S stable cell lines and control antigen (Sino Biological, #CT017-H2508H) in different proportions or concentrations, 0.1 mL of the diluted sample was placed in the reaction well, incubated at 37°C for 1 hour, and then washed, and blank wells (no sample added), negative control wells and positive control wells were made at the same time. 0.1 mL of the newly diluted antibody was added to each reaction well, incubated at 37°C for 1 hour, and then washed 3 times. Add enzyme-labeled secondary antibody: Add 0.1 mL of freshly diluted enzyme-labeled antibody to each reaction well. Incubate at 37°C for 1 hour and wash 3 times. Add substrate solution for color development: Add 0.1 mL of TMB substrate solution to each reaction well and let stand at room temperature for 10 minutes. Add 0.1 mL of 1M H ₂ SO ₄ to each reaction well. Measure the OD value to determine the result. Use a microplate reader to measure the absorbance value (A450) at 450 nm. The results are shown in Table 3.

The 4-parameter logistic curve of the control protein was calculated by ELISA CALC as the standard curve, where R ² =0.998. The ITGAX protein expression concentration in the supernatant of the cells to be tested was calculated, and the results showed that the supernatant of the 3A4 cell line of CHO-S transfected with pCDNA3.1-ITGAX had obvious expression of the target protein of 442.1 ng/mL. Combined with the ITGAX expression level and cell growth characteristics, the 3A4 cell line was finally selected for the later ITGAX protein production.

Table 3 Absorbance values corresponding to different protein concentrations

Example 4: Production and purification of ITGAX recombinant protein

Cell culture stage: The ITGAX stable cell line 3A4 was cultured at a concentration of 2×10 ⁵ cells/ml in a 1L shake flask containing 400 mL of LCD-CHO medium containing 12.5 μM MSX reagent. The shake flask was placed in a shaker (130 rpm) in a CO ₂ incubator (37°C, 5% CO ₂ ) and cultured for 10 days. The cell culture medium after 10 days of culture was taken out, centrifuged at 200 g for 5 minutes (to separate cells and coarse cell debris), and the supernatant was transferred to a new bottle and centrifuged at 3000 g for 15 minutes (to separate fine cell debris). The separated supernatant was sterilized by filtration using a 0.22 μM filter cup and stored at 4°C.

Protein purification stage: The sterilized cell supernatant was balanced to room temperature and then purified using a Ni column. Ni column filler (medium), company GE Healthcare, catalog number: #17-3712-02. 30mL of the chromatography column was loaded, and the final volume of the column medium was 10mL. The cell supernatant was loaded in an amount of 400mL, the flow rate was 1.0mL/min, and then eluted with different concentrations of imidazole eluent (20mMTris-HCl, pH 8.5, 50-1000mM imidazole concentration gradient). ITGAX recombinant protein was eluted in 100mM imidazole eluent. Samples from each elution step of the purification experiment were taken, and the purity of the ITGAX target protein was detected by SDS-PAGE experiment. The results are shown in Figure 2, where Lanes 1-9 were loaded with samples: supernatant, flow-through, Marker, washing solution, 10mM imidazole eluent, 50mM imidazole eluent, 100mM imidazole eluent, 200mM imidazole eluent, and 500mM imidazole eluent; the ITGAX purified protein was mainly present in Lane 7 (i.e., in the 100mM imidazole eluent). As shown in the results of Figure 2, the purified ITGAX recombinant protein was mainly present in Lane 7 (Lane 7), and the protein molecular weight was 127.8KDa, which was consistent with the theoretical value; the visual purity was 95%.

The eluate (Lane 7) containing ITGAX recombinant protein was concentrated using a 10KDa ultrafiltration tube (Millipore, UFC900396) and dialyzed overnight using a 10KDa dialysis bag with a 10mM pH7.4 PBS buffer. The recombinant protein after dialysis was tested for protein concentration using the A280 method. The typical total protein concentration was 1.12mg/mL. The purified ITGAX was stored at 4°C.

Example 5: Immunization of BALB/c mice with ITGAX recombinant protein

BALB/c mice aged 6 to 8 weeks were selected for the following immunization procedure: for the first immunization, 25 μg of ITGAX protein was mixed with an equal amount of Freund's complete adjuvant and injected subcutaneously at multiple points after emulsification; 14 days after the first immunization, 12.5 μg of ITGAX protein was mixed with Freund's incomplete adjuvant and emulsified for booster immunization. 14 days after the second immunization, 12.5 μg of ITGAX protein was mixed with Freund's incomplete adjuvant and emulsified for booster immunization. 14 days after the third immunization, blood was collected and serum was separated. ELISA plates were coated with ITGAX protein for indirect ELISA test to determine the serum titer. The results showed that the titer of the prepared mouse antiserum was 1:72900. The typical test results of immunized mouse serum are shown in Table 4.

Table 4 Mouse antiserum titer detection

Serum dilution multiple OD450 100 2.875 300 2.425 900 2.104 2700 1.865 8100 1.245 24300 0.952 72900 0.568 Background 0.042

Example 6: Cell fusion

In Example 5, after the third immunization, the mice were boosted with the following specific steps: 12.5 μg of ITGAX protein was injected and mixed with PBS to a volume of 200 μL for intraperitoneal injection. Cell fusion was performed 3 days after the boosted immunization of mice. After removing the mouse eyeballs to collect blood, the mice were killed by dislocation, placed in a 70% alcohol bottle for 2 minutes, and then fixed on a foam board in a biosafety cabinet, the abdominal skin was untied to find the spleen, which was removed with tweezers, placed in a 200-mesh stainless steel filter and gently crushed, and the cells were gently rinsed with DMEM culture medium (Thermo, 11965092), and then centrifuged at 200g for 10 minutes in a centrifuge at room temperature, and the supernatant was discarded for later use.

When preparing feeder cells, kill the mouse by dislocation, place it in a 70% alcohol bottle for 2 minutes, and then fix the mouse on a foam board in a biosafety cabinet. Untie the abdominal skin, draw PBS with a syringe and gently inject it under the peritoneum. Wash out the liquid containing the feeder cells from the other side, then centrifuge at 200g in a centrifuge at room temperature for 10 minutes, discard the supernatant and set aside. 2.0×10 ⁷ FO myeloma cells and 2.0×10 ⁸ spleen cells were mixed, centrifuged at 200 g for 10 minutes, the supernatant was discarded, and the mixture was gently shaken to mix. In a 37°C water bath, 1 mL of a 50% PEG-1450 (Merk, P1458) aqueous solution was added dropwise within 90 seconds, followed by 20 mL of DMEM culture medium. The mixture was centrifuged at 200 g for 10 minutes, the supernatant was discarded, and the washing was repeated. The mixture was centrifuged at 200 g for 10 minutes, the supernatant was discarded, and the hybridoma cells were obtained. The cells were plated into 10 96-well culture plates with 150 μL per well. 10,000 cells/well of feeder layer cells were added to the above 10 96-well cell culture plates, 100 μL per well, and the culture plates were marked and placed in a cell culture incubator at 37°C containing 5% CO _2. HAT selection medium (Merk, H0262) was added on the second day, and a large number of tumor cells died within 1-2 days of HAT selection culture. After 3-4 days, the tumor cells disappeared and hybrid cells formed small colonies. After 7-10 days of HAT selection culture medium, HT culture medium (Merk, H0137) should be used, and then maintained for another 2 weeks, and then 20% FBS (ExCell, FSP500) DMEM culture medium was used to continue culture. During the above selection culture period, when the hybridoma cells covered 1/10 of the bottom area of the well, specific antibodies could be detected to screen out the desired hybridoma cell lines. During the selection culture period, half of the culture medium was generally replaced every 2-3 days.

Example 7: Screening and subcloning of positive hybridoma cell lines

First, the optimal coating amount of ITGAX protein as an antigen was determined by the founder experiment. 0.5, 1.0, 2.0, and 4.0 μg of ITGAX protein were coated on a 96-well plate, and 6 wells of each concentration were set as 3 positive and 3 negative. Founder titration was performed with positive serum from mice immunized with ITGAX protein at different dilutions, and negative serum from unimmunized mice was used as a negative control. 96-well ELISA plates were coated with 0.5 μg of purified ITGAX protein per well and incubated at 4°C overnight; washed twice with PBST; 200 μL of 1% BSA in PBS was added to each well, blocked at room temperature for 2 hours, and then placed on trifold paper and patted dry; sample addition: 0.1 mL of the sample to be tested was added to the reaction well, incubated at 37°C for 1 hour, and then washed, and blank wells (no sample added), negative control wells, and positive control wells were made at the same time. 0.1 mL of newly diluted antibody was added to each reaction well, incubated at 37°C for 1 hour, and then washed 3 times. Add enzyme-labeled secondary antibody: Add 0.1 mL of freshly diluted enzyme-labeled antibody (Solebol, #SE131) to each reaction well. Incubate at 37°C for 1 hour and wash 3 times. Add substrate solution for color development: Add 0.1 mL of TMB substrate solution to each reaction well and let stand at room temperature for 10 minutes. Add 0.1 mL of 1M H ₂ SO ₄ to each reaction well. Measure the OD value to determine the result, and use an enzyme reader to measure the absorbance value (A450) at 450 nm. A value that is 2.1 times or more of the negative control OD value is considered positive (calculated after adjusting the blank control well to zero). Select the hybridoma cell monoclonal clone against ITGAX.

According to the above method, the positive hybridoma cells screened were subcloned, and the original wells were diluted with HAT selection medium by limiting dilution method and redistributed into 96-well culture plates, and then the morphology and number of cells were observed. The cells were adjusted to 3-10 cells/mL. Take the cell culture plate with feeder cell layer prepared the day before, and add 100 μL of diluted cells to each well. Cultured statically in a 37°C, 5% CO ₂ incubator. Change the medium on the 7th day, and change the medium once every 2-3 days thereafter. Cell clones can be seen on the 8th to 9th day, and the antibody activity is detected in time. The cells in the positive wells were transferred to a 24-well plate for expansion and culture. The binding activity of antibodies and ITGAX proteins in the culture supernatants of different cell lines was compared by indirect ELISA detection, and the cell line AH64D with the highest OD value (OD ₄₅₀ = 2.324) was selected for later experiments. The cells should be frozen as soon as possible, and the hybridoma cell line with clone number AH64D was finally selected for antibody production.

Example 8: Mass preparation of monoclonal antibodies and determination of antibody titer

(1) Large-scale preparation of monoclonal antibodies

8-week-old BALB/C mice were injected intraperitoneally with 0.5 mL of Freund's incomplete adjuvant. Two weeks later, 1×10 ⁶ hybridoma cells were injected intraperitoneally. Ascites can be produced 7 to 10 days after the cells were inoculated. The health status of the animals and the signs of ascites were closely observed. When the ascites was as much as possible and the mice were on the verge of death, the mice were killed and the ascites was sucked into a test tube with a dropper. 5 to 10 mL of ascites can be obtained from one mouse. Ascites can also be extracted with a syringe and can be collected repeatedly several times. The ascites obtained was centrifuged at 3000g for 10 minutes, the upper layer of fat and the bottom sediment were discarded, the supernatant was collected and aliquoted at -20℃, and after the ascites supernatant was thawed and equilibrated to room temperature, 1/10 volume of 1M Tris-HCl pH8.0 was added to adjust the sample pH to 8.0. The protein G affinity column was equilibrated with 20 column volumes of 100mM Tris-HCl pH 8.0, and the ascites supernatant after adjusting the pH to 8.0 was applied to the column, followed by washing with 20 column volumes of 100mM Tris-HCl pH 8.0, and finally the antibody was eluted with 100mM Glycine-HCL pH 2.5. The antibody eluate was added to a concentrator tube (Millipore, UFC801008, 10K) and centrifuged at 3000×g for 20 minutes at room temperature in a centrifuge (Xiangyi, L550). Batch centrifugation was performed until the solution volume reached 1mL/concentrator tube (2 tubes), 4mL of 10mM PBS pH 7.4 buffer was added, and centrifugation at 3000×g for 20 minutes at room temperature was continued. The centrifugation was repeated 3 times to make the antibody buffer 10mM PBS pH 7.4, and 10mM PBS pH 7.4 was added to a total volume of 10mL. Finally, 2 mL/tube of the concentrated antibody solution was dispensed into centrifuge tubes and stored at -80° C. The antibody concentration was determined using a BCA kit (Solabo, PC0020), and the concentration of the purified monoclonal antibody AH64D was 1.8 mg/mL.

(2) Antibody titer determination

The titer of anti-ITGAX antibody AH64D was detected by indirect ELISA. ITGAX protein was diluted with PBS and coated in a 96-well ELISA plate at 0.2 μg/mL and 100 μL/well. After overnight at 4°C, the ELISA plate was washed twice with PBST solution, 300 μL/well each time, and patted dry after washing. The ELISA plate was blocked with 1% BSA PBS solution, 200 μL/well, at room temperature for 2 hours, and patted dry after blocking; anti-ITGAX antibody AH64D diluted to 20 ng/mL with PTB was added, reacted in a 37°C incubator for 1 hour, and the ELISA plate was washed twice with PBST solution, 300 μL/well each time. Patted dry after washing. HRP-labeled goat anti-mouse antibody diluted 5000 times with PTB was added, reacted in a 37°C incubator for 1 hour, and the ELISA plate was washed twice with PBST solution, 300 μL/well each time. Patted dry after washing. TMB substrate solution was added, 100 μL/well, reacted at room temperature for 10 minutes, and 100 μL/well of 1M H ₂ SO ₄ was added to terminate the reaction. The absorbance (A450) was measured at 450 nm using an ELISA reader. The results are shown in Table 5, and it can be seen that the titer of the purified antibody is 1:729,000.

Table 5 Antibody titer determination

Antibody dilution factor OD450 1000 2.452 3000 2.124 9000 1.869 27000 1.539 81000 1.104 243000 0.875 729000 0.362 Background 0.048

Example 9: Sequence analysis of monoclonal antibodies

(1) Monoclonal antibody subtype identification

The hybridoma cell line AH64D was cultured in a 10 cm diameter cell culture dish (37°C, 5% CO ₂ ) using DMEM medium (GIBCO, #C11995500BT) supplemented with 10% serum. After 7 days of culture, the cells were transferred to a 15 mL centrifuge tube, and 4×10 ⁶ cells were taken out after counting with a hemocytometer. After centrifugation at 200 g for 5 minutes, the supernatant was discarded, and the centrifuge tube was inverted to drain the liquid in the tube. The cells in the tube were synthesized into cDNA using the reverse transcription kit (Qiagen, 74134) of QIAGEN.

The antibody subtype was determined by PCR using antibody subtype-specific primers. The above-synthesized cDNA was used as a PCR reaction template. The primer sequence information used in PCR is shown in Table 6.

Table 6 PCR primer sequence information

Note: In the table, S=C or G, M=A or C, R=A or G, and W=A or T.

PCR reaction solution system: TAKARA Ex Taq (5U/μL, TAKARA, RR001B), 0.25μL; 10×Ex TaqBuffer, 5μL; dNTP mixture (2.5mM each), 4μL; template cDNA, 1μL; upstream primer (100μM), 1μL; downstream primer (100μM), 1μL; add double distilled water to a total volume of 50μL.

PCR reaction temperature program: 94°C for 5 minutes of pre-denaturation, 30 temperature cycles (94°C for 1 minute, 57°C for 1 minute, 72°C for 1 minute), and extension at 72°C for 10 minutes.

After the reaction, 10 μL of each PCR product was loaded on a 1% agarose gel for electrophoresis. The electrophoresis diagram is shown in FIG3 , and the order of samples added to each lane is shown in Table 7. As shown in FIG3 , through electrophoresis diagram analysis, lane 2 (Lane2) and lane 6 (Lane6) amplified IgG1 heavy chain and kappa light chain 650 bp positive bands, respectively, thereby determining that the monoclonal antibody AH64D obtained in the present invention is of IgG1 subtype heavy chain and kappa subtype light chain. The subtype of the antibody can be inferred from the PCR product results, and the results are shown in Table 7. As can be seen from Table 7, the monoclonal antibody AH64D obtained in the present invention is of IgG1 heavy chain and kappa light chain.

Table 7 Sample information corresponding to the lane

(2) Sequencing of the variable region (V region) of the hybridoma cell line AH64D antibody

The fragment obtained after PCR amplification of the V region of the antibody of the cell line AH64D (see the above bar) was cut from the agarose gel and extracted using a DNA extraction kit (Qiagen, 74134). The extracted DNA fragment was linked to the pEASY-T1 cloning vector and transformed into Trans1-T1 competent cells (Transgen, CT101-1). The transformed bacterial colonies were picked into LB culture medium and DNA sequencing was performed after overnight culture. The nucleic acid sequence of the light chain V region of the anti-ITGAX antibody (AH64D) provided by the present invention is shown in SEQ ID NO.9, and the nucleic acid sequence of the heavy chain V region is shown in SEQ ID NO.10.

Based on the above, the amino acid sequences of the light chain variable region (VL) and heavy chain variable region (VH) of the monoclonal antibody AH64D obtained are shown in Table 8. The CDR sequence of the antibody according to the IMGT database is shown in Table 9. The light chain constant region and heavy chain constant region sequences of the antibody are shown in Table 10. The complete heavy chain and light chain sequences of the antibody are shown in Table 11.

Table 8 VL and VH sequences of monoclonal antibody AH64D

Table 9 CDR sequences of monoclonal antibody AH64D

LCDR1 Q S L V H S N G N S Y SEQ ID NO:3 LCDR2 E V S SEQ ID NO:4 LCDR3 F Q G T H L P G S T SEQ ID NO:5 HCDR1 G Y S F T G Y Y SEQ ID NO:6 HCDR2 V N P Y N G G T SEQ ID NO:7 HCDR3 A P D V R A N D F A R W Y F D V SEQ ID NO:8

Table 10 Light chain constant region and heavy chain constant region of monoclonal antibody AH64D

Table 11 Light and heavy chains of monoclonal antibody AH64D

Example 10: Transformation, expression and purification of human chimeric antibody AH64D

(1) Construction of pGmab-K-hAH64D/pGmab-H-hAH64D plasmid

The nucleic acid sequence of the light chain V region of the antibody AH64D obtained in the early stage is shown as SEQ ID NO.9, and the nucleic acid sequence of the heavy chain V region is shown as SEQ ID NO.10. The above designed DNA sequences were synthesized by a third-party gene synthesis company and constructed into the pUC57 plasmid. After construction, the plasmids were named pUC57-AH64D-VL and pUC57-AH64D-VH. 2 μg of pUC57-AH64D-VL or pUC57-AH64D-VH and pGmab-K (containing the human antibody kappa chain constant region) or pGmab-H (containing the human antibody IgG1 chain constant region) plasmids were added to 2U XbaI and 2U BamHI endonuclease, 37 ℃ digestion for 2 hours, 1% agarose gel separation and recovery of positive bands and use A260 to detect the concentration of recovered DNA fragments, respectively take the insert fragment (AH64D-VL or AH64D-VH) and the expression plasmid double digestion fragment (pGmab-K or pGmab-H) 2nmol, add 1U T4 ligase, use T4 ligase buffer and ultrapure water to make up to 20μL, 16 ℃ overnight connection, transform into DH5α competent cells, and spread on LB plates, pick single colonies and sequence with FCMV and SV40 universal primers, and the gene sequence is correct after sequencing. After construction, the expression plasmids are named pGmab-K-hAH64D/pGmab-H-hAH64D, and the plasmid maps are shown in Figures 4 and 5.

The light chain variable region (VL) and heavy chain variable region (VH) amino acid sequences of the hAH64D antibody are shown in Table 12. The CDR sequences of the antibody according to the Kabat system are shown in Table 13. The heavy chain and light chain sequences of the antibody are shown in Table 14.

Table 12 Amino acid sequences of the light chain variable region (VL) and heavy chain variable region (VH) of hAH64D antibody

Table 13 CDR sequences of hAH64D antibody

LCDR1 Q S L V H S N G N S Y SEQ ID NO:3 LCDR2 E V S SEQ ID NO:4 LCDR3 F Q G T H L P G S T SEQ ID NO:5 HCDR1 G Y S F T G Y Y SEQ ID NO:6 HCDR2 V N P Y N G G T SEQ ID NO:7 HCDR3 A P D V R A N D F A R W Y F D V SEQ ID NO:8

Table 14 Light and heavy chains of hAH64D antibody

(2) Expression and purification of human chimeric antibody hAH64D

First, the two plasmids pGmab-K-hAH64D/pGmab-H-hAH64D constructed in the above process were mixed at a ratio of 1:1 and transfected into CHO-S cells. The transfection reagent used for transfection of suspension cultured CHO-S cells was Free StyleMAX (Invitrogen, 16447100), and the transfection operation steps were carried out as described in the manufacturer's product manual. Subsequently, the transfected CHO-S cells were transferred to a 125 mL triangular shake flask, and the cell density was 1×10 ⁶ cells/mL in 30 mL FreeStyle CHO medium. The cells were cultured in a CO ₂ incubator (37°C, 5% CO ₂ ) with a shaker speed of 130 rpm. Finally, the culture supernatant was collected after 7 days of cell culture, and the activity concentration of the anti-ITGAX humanized antibody (named hAH64D) in the supernatant (expressed and secreted by CHO-S cells) was detected by indirect ELISA. In this experiment, ITGAX protein was diluted with PBS as an antigen and coated in a 96-well ELISA plate at a coating concentration of 1 μg/mL and a volume of 100 μL/well. After overnight coating at 4°C, the ELISA plate was washed twice with PBST solution, 300 μL/well each time. After washing, pat dry, the ELISA plate was blocked with PBS solution containing 1% BSA, 200 μL/well, at room temperature for 2 hours. Pat dry, add the supernatant of cells expressing hAH64D diluted with PTB in different ratios, add to the blocked ELISA plate, 100 μL/well, and react in a 37°C incubator for 1 hour. Pat dry, wash the plate 3 times with PBST, 300 μL/well each time. Pat dry, add HRP-labeled goat anti-human antibody diluted 5000 times with PTB to the ELISA plate, and react in a 37°C incubator for 1 hour. Wash the plate 3 times with PBST, 300 μL/well. Pat dry, add TMB substrate solution to the ELISA plate, 100 μL/well, at room temperature for 10 minutes, then add 100 μL/well 1M H ₂ SO ₄ to terminate the reaction. Use an ELISA reader to measure the absorbance (A450) at 450 nm, and the results are shown in Table 15. As shown in Table 15, the activity titer of the anti-ITGAX humanized antibody hAH64D in the cell supernatant was detected, and the titer of hAH64D in the cell supernatant was 24300.

Table 15 Detection of the titer of anti-ITGAX humanized antibody hAH64D in cell supernatant

Cell supernatant dilution factor HkDJ 10 2.435 30 2.124 90 1.862 2700 1.425 8100 0.582 24300 0.325 72900 0.118 Background 0.087

Subsequently, the cell supernatant was centrifuged at 3000×g for 10 minutes, and 1/10 volume of 1M Tris–HCl pH8.0 was added to adjust the sample pH to 8.0. The protein G affinity column was equilibrated with 20 column volumes of 100mM Tris–HCl pH8.0, and the ascites supernatant after adjusting the pH to 8.0 was loaded onto the column, followed by washing with 20 column volumes of 100mM Tris–HCl pH 8.0, and finally the antibody was eluted with 100mM Glycine–HCL pH 2.5. The antibody eluate was added to a concentrator tube (Millipore, UFC801008, 10K) and centrifuged at 3000×g for 20 minutes at room temperature in a centrifuge (Xiangyi, L550). The solution was centrifuged in batches until the volume of the solution reached 1mL/concentrator tube (2 tubes), 4mL of 10mM PBS pH 7.4 buffer was added, and centrifugation at 3000×g for 20 minutes at room temperature was continued. Repeat centrifugation 3 times to make the antibody buffer 10mM PBS pH 7.4, add 10mM PBS pH 7.4 to a total volume of 10mL. Finally, the concentrated antibody solution 2mL/tube was dispensed into centrifuge tubes and stored at -80°C. The antibody concentration was determined using a BCA kit (Solebo, PC0020), and the concentration of the purified human chimeric antibody hAH64D was determined to be 4.1mg/mL.

Activity titer test of purified anti-ITGAX humanized antibody hAH64D. In this experiment, ITGAX protein was diluted with PBS as antigen and coated in 96-well ELISA plate at a coating concentration of 1μg/mL and a volume of 100μL/well. After overnight coating at 4°C, the ELISA plate was washed twice with PBST solution, 300μL/well each time. After washing, pat dry, and the ELISA plate was blocked with PBS solution containing 1% BSA, 200μL/well, at room temperature for 2 hours. Pat dry, add different concentrations of antibody hAH64D diluted with PTB, add to the above blocked ELISA plate, 100μL/well, and react in a 37°C incubator for 1 hour. Pat dry, wash the plate 3 times with PBST, 300μL/well each time. Pat dry, add HRP-labeled goat anti-human antibody diluted 5000 times with PTB to the ELISA plate, and react in a 37°C incubator for 1 hour. The plate was washed 3 times with PBST, 300 μL/well. Patted dry, TMB substrate solution was added to the ELISA plate, 100 μL/well, at room temperature for 10 minutes, and then 100 μL/well of 1M H ₂ SO ₄ was added to terminate the reaction. The absorbance (A450) was measured at 450 nm using an ELISA reader, and the results are shown in Table 16. As shown in Table 16, the titer of hAH64D in the cell supernatant was 2430000.

Table 16: Titer detection of humanized antibody hAH64D after affinity purification

Antibody dilution factor HkDJ 10000 1.985 30000 1.235 90000 0.895 270000 0.652 810000 0.324 2430000 0.154 7290000 0.102 Background 0.068

Example 11: Identification of the sensitivity and specificity of antibody hAH64D in detecting ITGAX

(1) Identification of the sensitivity of antibodies hAH64D and AH64D in detecting ITGAX

The sensitivity of antibody hAH64D to detect ITGAX was evaluated by indirect ELISA. The sensitivity was compared with that of antibody AH64D and control antibody (mouse anti-ITGAX monoclonal antibody, abcam, #ab11029). In this experiment, ITGAX protein was diluted with PBS as antigen and coated in 96-well ELISA plate at a coating concentration of 1μg/mL and a volume of 100μL/well. After overnight coating at 4℃, the ELISA plate was washed twice with PBST solution, 300μL/well each time. After washing, pat dry, the ELISA plate was blocked with PBS solution containing 1% BSA, 200μL/well, at room temperature for 2 hours. Pat dry, add different concentrations of antibody AH64D, hAH64D, and control antibody diluted with PTB, add to the above blocked ELISA plate, 100μL/well, and react in a 37℃ incubator for 1 hour. Pat dry, wash the plate 3 times with PBST, 300 μL/well each time. Pat dry, add HRP-labeled goat anti-human antibody diluted 5000 times with PTB to the ELISA plate, and react in a 37°C incubator for 1 hour. Wash the plate 3 times with PBST, 300 μL/well. Pat dry, add TMB substrate solution to the ELISA plate, 100 μL/well, incubate at room temperature for 10 minutes, then add 100 μL/well of 1MH ₂ SO ₄ to terminate the reaction. Use an ELISA reader to measure the absorbance (A450) at 450 nm.

The experimental results are shown in Table 17. The sensitivity of antibodies AH64D and hAH64D in detecting ITGAX protein is better than that of control antibody, and hAH64D has a lower background value (0.063) than ITGAX protein. The results show that the standard curve of ITGAX protein has R ² =0.9981 and IC50=25ng/mL. The standard curve range of this method is 0-3000ng/mL, and the minimum detection limit of control antibody is 50ng/mL; AH64D antibody is 25ng/mL; and hAH64D antibody is 12.5ng/mL. The results are shown in Table 17.

Table 17 Identification of the sensitivity of antibodies AH64D and hAH64D in determining ITGAX protein

ITGAX concentration (ng/mL) Control Antibodies AH64D HkDJ 3000 1.865 2.561 2.865 1000 1.354 2.235 2.674 300 0.985 2.124 2.145 100 0.625 1.825 1.824 50 0.325 0.825 0.985 25 0.21 0.535 0.548 12.5 0.145 0.325 0.268 0 0.105 0.321 0.098 Background 0.102 0.224 0.063

(2) Identification of the specificity of antibodies hAH64D and AH64D in detecting ITGAX

The specificity of antibodies hAH64D and AH64D for detecting ITGAX was evaluated using an indirect Cell based ELISA. In this experiment, cells (MCF7) and the corresponding MCF7 ITGAX knockout cell line (abcam, #ab287574, this cell line does not express ITGAX protein on the membrane surface due to ITGAX gene knockout) were cultured for 3 generations in DMEM medium with 10% serum. After culturing for 3 generations, the cells were transferred to 96-well cell culture plates at a cell density of 100,000 cells/well, cultured overnight, the supernatant was discarded, and 4% paraformaldehyde was added at a volume of 100μL/well to fix the cells for 30 minutes. The cells were washed twice with PBST solution, 300μL/well each time. After washing, the plates were patted dry and blocked with PBS solution containing 1% BSA, 200μL/well, at room temperature for 2 hours. Pat dry, add different concentrations of antibodies hAH64D and AH64D diluted with PTB, 100 μL/well, and react in a 37°C incubator for 1 hour. Pat dry, wash the plate 3 times with PBST, 300 μL/well each time. Pat dry, add HRP-labeled goat anti-mouse antibody diluted 5000 times with PTB to the ELISA plate, and react in a 37°C incubator for 1 hour. Wash the plate 3 times with PBST, 300 μL/well. Pat dry, add TMB substrate solution to the ELISA plate, 100 μL/well, room temperature for 10 minutes, and then add 100 μL/well of 1M H2SO4 to terminate the reaction. Use an ELISA reader to measure the absorbance value (A450) at 450nm. The experimental results are shown in Table 18, indicating that the antibody hAH64D has better specificity for detecting ITGAX protein on the surface of natural cell membranes than the antibody AH64D and the control antibody.

Table 18 Identification of the specificity of antibodies AH64D and hAH64D in detecting ITGAX

Example 12: Comparison of the use of antibodies hAH64D and AH64D in detecting ITGAX on the surface of human peripheral lymphocytes by flow cytometry

(1) Fluorescein isothiocyanate (FITC) labeling of antibodies hAH64D and AH64D and identification of labeled products

Antibodies hAH64D and AH64D (20 nmol, 3 mg) were taken to a volume of 0.5 mL and added to a dialysis bag (10 KDa, 1 cm width), and dialyzed in 2 L 10 mM PBS solution (pH 7.4) at 4 ° C overnight. The next day, the dialysis bag containing the antibody solution was placed in 1 L 10 mM carbonate buffer (pH 9.5) and dialyzed for 2 hours at room temperature, ready for coupling with activated FITC.

At the same time, use an analytical balance to accurately weigh 0.1 mg FITC, add it to the dialyzed antibody solution, add fluorescein while stirring to prevent the powder from adhering to the wall, and place it in a 4°C refrigerator or icehouse and continue stirring for 12 to 18 hours. After the binding is completed, first centrifuge the conjugate at 3000×g for 20 minutes, remove a small amount of precipitate, put it into a dialysis bag, and then dialyze it overnight in a beaker of 10mM PBS solution (pH 7.4). Take the marker that has been dialyzed overnight, pass it through a dextran gel G-25 column, separate the free FITC, collect the labeled fluorescent antibody and store it at 4°C.

(2) FTTC-labeled antibodies hAH64D and AH64D were used to detect ITGAX protein on the surface of human peripheral lymphocytes by flow cytometry.

In this experiment, human whole blood was processed using a modified protocol based on Chow et al, 2005 (PMID: 16080188). In brief, human whole blood was fixed in 4% paraformaldehyde at 22°C for 10 minutes. Red blood cells were then lysed by adding Triton X-100 (final concentration of 0.1%) at 37°C for 15 minutes. In the experiment, 100-fold diluted anti-FTTC labeled antibodies hAH64D and AH64D were used for staining at 4°C for 30 minutes. The isotype control antibody was FITC-labeled mouse monoclonal IgG1 (abcam, #ab170190) used under the same conditions. Using the CytoFLEX instrument, >30,000 total events were collected using a 50mW argon blue laser (488nm) and a 530/30 bandpass filter. Gating strategy events were collected by using the forward and side light scattering properties of living cells, and the antibody hAH64D was determined to recognize ITGAX protein on the surface of peripheral lymphocytes by comparing FITC values. The results are shown in Figure 6.

As shown in Figure 6, the peak value of the fluorescence value at 488nm of the negative antibody is (10×10 ⁴ ), the peak value of the fluorescence value at 488nm of the antibody (hAH64D) is (140×10 ⁴ ), and the peak value of the fluorescence value at 488nm of the antibody (AH64D) is (100×10 ⁴ and 140×10 ⁴ ). The results show that both antibodies (hAH64D and AH64D) can specifically recognize the ITGAX protein on the surface of human peripheral lymphocytes, but the hAH64D antibody shows a better single peak in the flow cytometry detection, while the AH64D flow cytometry detection has more background peaks, and the specificity is lower than that of the hAH64D antibody. It can be judged that the hAH64D antibody has a good advantage in the specific recognition of the ITGAX protein on the surface of human peripheral lymphocytes in the flow cytometry detection.

The present invention provides anti-ITGAX monoclonal antibodies AH64D and/or hAH64D, which have the characteristics of high affinity and specificity, and also provides a method for detecting ITGAX on the surface of human peripheral blood lymphocytes by flow cytometry based on the hAH64D antibody. The present invention solves the problem of relative shortage of antibody raw materials for ITGAX detection projects in the current market.

In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, unless they are contradictory.

The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technician familiar with this profession can make some changes or modifications to equivalent embodiments of equivalent changes by using the methods and technical contents disclosed above without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the technical solution of the present invention.

Claims (14)

1. An antibody or antigen-binding fragment thereof that specifically binds ITGAX, wherein the amino acid sequences of LCDR1-3 of the light chain variable region VL of said antibody or antigen-binding fragment thereof are shown in SEQ ID NOS 3, 4 and 5, respectively, and the amino acid sequences of HCDR1-3 of the heavy chain variable region VH of said antibody or antigen-binding fragment thereof are shown in SEQ ID NOS 6, 7 and 8, respectively.

2. The antibody or antigen-binding fragment thereof that specifically binds ITGAX of claim 1, wherein the VL comprises an amino acid sequence having at least 80% sequence identity to SEQ ID No. 1 and the VH comprises an amino acid sequence having at least 80% sequence identity to SEQ ID No. 2.

3. The antibody or antigen-binding fragment thereof that specifically binds ITGAX according to claim 1 or 2, wherein the light chain constant region CL comprises an amino acid sequence having at least 80% sequence identity to SEQ ID No. 13 and the heavy chain constant region CH comprises an amino acid sequence having at least 80% sequence identity to SEQ ID No. 14.

4. The antibody or antigen-binding fragment thereof that specifically binds ITGAX of claim 1 or 2, wherein the antigen-binding fragment is selected from the group consisting of diabodies; the antibody is a murine monoclonal antibody or a humanized chimeric antibody.

5. The antibody or antigen-binding fragment thereof that specifically binds ITGAX of claim 4, wherein the antibody is a monoclonal antibody whose light chain comprises an amino acid sequence having at least 80% sequence identity to SEQ id No. 15 and whose heavy chain comprises an amino acid sequence having at least 80% sequence identity to SEQ id No. 16.

6. The antibody or antigen-binding fragment thereof that specifically binds ITGAX of claim 4, wherein the antibody is a humanized chimeric antibody whose light chain comprises an amino acid sequence having at least 80% sequence identity to SEQ id No. 17 and heavy chain comprises an amino acid sequence having at least 80% sequence identity to SEQ id No. 18.

7. A nucleic acid comprising a nucleotide sequence encoding an antibody or antigen-binding fragment thereof that specifically binds ITGAX according to any one of claims 1-6.

8. A vector comprising the nucleic acid of claim 7.

9. A host cell comprising the nucleic acid of claim 7 or the vector of claim 8.

10. The host cell of claim 9, wherein the host cell is a mammalian cell including, but not limited to, 293F cells, CHO cells.

11. A method of making the antibody or antigen-binding fragment thereof of any one of claims 1-6, comprising: culturing the host cell according to claim 9 or 10 under conditions such that the antibody, antigen binding fragment thereof is expressed.

12. A detection reagent comprising the antibody or antigen-binding fragment thereof that specifically binds to ITGAX according to any one of claims 1 to 6, wherein the antibody or antigen-binding fragment thereof that specifically binds to ITGAX is fluorescently labeled as a component of a flow fluorescent detection reagent that specifically recognizes ITGAX.

13. Use of an antibody or antigen-binding fragment thereof that specifically binds ITGAX according to any one of claims 1-6, or of a detection reagent according to claim 12, in the manufacture of a product for detecting the content of ITGAX in a sample.

14. Use of an antibody or antigen-binding fragment thereof that specifically binds ITGAX according to any one of claims 1-6, a nucleic acid according to claim 7, a vector according to claim 8, a host cell according to claim 9 or 10 for the preparation of a product for detecting ITGAX.