Detection method of AXL-GAS6 compound and application of AXL-GAS6 compound in myocardial infarction patients

Abstract

The invention relates to a method for detecting an AXL-GAS6 compound and application of the AXL-GAS6 compound in a myocardial infarction patient, in particular to a method for detecting the level of the AXL-GAS6 compound in a sample, which comprises the following steps: (a) contacting the sample with an anti-AXL-GAS6 antibody or an antigen binding fragment thereof; and (b) detecting the binding of the AXL-GAS6 antibody or antigen-binding fragment thereof to the AXL-GAS complex and determining the level of the AXL-GAS6 complex in the sample; the invention also relates to application of the anti-AXL-GAS6 antibody or the antigen binding fragment thereof in preparation of a medicine or a kit for diagnosing myocardial infarction or evaluating the risk of myocardial infarction.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to antibodies that bind to the AXL-GAS6 complex, as well as methods for detecting the AXL-GAS6 complex and its application in patients with myocardial infarction. Background Technology

[0002] AXL is a member of the TAM receptor tyrosine kinase family, and its full-length cDNA was first cloned in 1991. AXL comes from the Greek word *anexelekto*, meaning "uncontrolled." AXL contains two immunoglobulin-like (IgL) domains, two fibronectin III (FNIII) domains, and one kinase domain, and is primarily expressed as a dimer on the cell surface. AXL mainly transduces signals into the cytoplasm by binding to its ligand GAS6 (Growth Arrest-Specific Protein 6), which links to phosphatidylserine (PtdSer) expressed on the surface of apoptotic cells. Under physiological conditions, AXL / GAS6 primarily functions to phagocytose and eliminate apoptotic cells, inhibiting inflammatory responses.

[0003] Over the past three decades, hundreds of articles have reported the association between AXL and its ligand expression and various cancers. These studies have found that overexpression or upregulation of AXL in tumor cells is often accompanied by poor prognosis in tumors such as acute myeloid leukemia (AML), glioblastoma multiforme, melanoma, pancreatic cancer, and esophageal cancer.

[0004] Studies have found that the interaction between GAS6 and AXL can activate the PI3K-Akt pathway to help tumor cells survive. Activated Akt leads to the inactivation of the pro-apoptotic mediator Bad, while increasing the expression of the anti-apoptotic protein Bcl-2. GAS6 / AXL binding can also directly bind to Grb2, thereby activating the Ras / ERK signaling pathway and stimulating tumor cell proliferation. Furthermore, it can upregulate Slug phosphorylation through extracellular signal-regulated kinase ERK, stimulating tumor cell migration. Thus, GAS6, by binding to AXL, activates different downstream signaling pathways, promoting tumor survival, proliferation, and migration, directly affecting tumor prognosis. Currently, based on these mechanisms, a series of anti-tumor drugs targeting AXL have been developed clinically, including small molecule inhibitors, monoclonal antibodies, antibody-drug conjugates, and soluble receptors.

[0005] Antibody-targeted therapy offers better specificity than small-molecule inhibitors and can serve as a carrier to deliver cytotoxic substances such as chemical drugs, toxins, and radionuclides to the tumor lesion site, specifically killing tumor cells. Currently, large-molecule drug candidates targeting AXL mainly focus on blocking the binding of AXL to the ligand GAS6 on the tumor cell surface. However, the affinity of GAS6 for AXL is actually very high, and the applicant's previous experiments have demonstrated that most tumor cells highly expressing AXL have already pre-bound GAS6, making it difficult to competitively inhibit the binding of GAS6 to AXL with antibodies. Currently, among the candidate drugs that competitively inhibit the binding of AXL to GAS6 on the tumor cell surface, only modified AXL fusion proteins have shown relatively smooth progress, while competitive AXL antibody drugs have not demonstrated promising therapeutic effects. The antibody of this invention does not require competition for the binding of GAS6 to AXL; it directly binds to the AXL-GAS6 complex, thereby inhibiting downstream signaling pathways involved in the binding of GAS6 to AXL, thus inhibiting the survival, proliferation, and migration of tumor cells.

[0006] GAS6 is a vitamin K-dependent protein, first discovered in mouse embryonic fibroblasts in 1988. Its biological function is mediated through interaction with the TAM receptor. The TAM receptor belongs to the receptor tyrosine kinase family. Currently identified GAS6 receptors include Tyro3, ​​AXL, and Mer, with AXL showing the highest affinity for GAS6. GAS6 consists of 678 amino acids with a molecular weight of approximately 75 kDa. Its structure comprises a γ-carboxyglutamate domain (49-90 aa), four EGF-like domains (118-278 aa), and two laminin G (LG)-like domains (279-678 aa). The LG region is the receptor-binding region.

[0007] GAS6 / AXL is highly expressed in various tumor tissues. The activation of AXL in the GAS6 / AXL signaling pathway involves GAS6-induced AXL dimerization and autophosphorylation. Current research suggests that the mechanism by which GAS6 regulates AXL dimerization is as follows: the LG region of one GAS6 molecule binds to the IG region of one AXL molecule, forming a GAS6-LG / AXL-IG complex. This complex then dimers with another GAS6-LG / AXL-IG complex via lateral diffusion. Following GAS6-induced AXL dimerization, autophosphorylation occurs at the tyrosine residue of the AXL receptor's intracellular kinase region, activating the AXL receptor's own tyrosine protease activity. This catalyzes downstream signal transduction, including JAK-STAT, PI3K-AKT, and RAS-RAF-MEK-ERK, promoting tumor cell survival, proliferation, migration, invasion, angiogenesis, and immune evasion.

[0008] Furthermore, recent studies have found that differences in plasma soluble AXL (sAXL) levels have certain diagnostic and prognostic value for various diseases. A 2019 study found significantly elevated plasma sAXL levels in patients with myocardial infarction, with even more pronounced elevations in patients with both myocardial infarction and heart failure. Further analysis revealed that sAXL is an independent risk factor for ventricular remodeling after myocardial infarction. Human plasma exists in two forms: free sAXL and the sAXL-GAS6 complex. The clinical relevance of AXL and the AXL-GAS6 complex is currently unclear, and there are no commercially available diagnostic kits that can directly detect the AXL-GAS6 complex. Therefore, there is an urgent need to develop novel AXL antibodies that can bind to the AXL-GAS6 complex. Summary of the Invention

[0009] The present invention aims to provide an anti-AXL-GAS6 antibody or a binding fragment thereof targeting the AXL-GAS6 complex. The antibody of the present invention can bind to cells expressing AXL in the presence of different concentrations of GAS6; it can also bind to cells expressing AXL in the presence of human GAS6 or mouse GAS6. The antibody of the present invention can bind to GAS6 in the presence of AXL and can also bind to cells expressing GAS6. The antibody of the present invention specifically binds to the complex formed by human AXL and mammalian-derived GAS6 (e.g., human GAS6 or mouse GAS6). The antibody of the present invention binds to the first domain of AXL in the presence of human or mouse GAS6. The antibody of the present invention can also bind to tumor cells expressing both AXL and GAS6, or it can specifically bind to AXL-positive and GAS6-positive tumor cells, thereby being used to treat AXL-positive and GAS6-positive tumor diseases. The antibody of the present invention specifically binds only to the AXL-GAS6 complex, and does not bind to the free form of AXL or GAS6. Therefore, the antibody of the present invention can be used to detect the presence of the AXL-GAS6 complex and has shown good anti-tumor efficacy in animal experiments.

[0010] The present invention provides a nucleic acid encoding an antibody of the present invention or an antigen-binding fragment thereof, a vector containing the nucleic acid, and a host cell containing the vector.

[0011] The present invention also provides a method for preparing the antibody of the present invention or its antigen-binding fragment.

[0012] The present invention also provides immunoconjugates, pharmaceutical compositions, and combination products comprising the antibodies of the present invention.

[0013] On the one hand, the present invention provides a method and kit for detecting AXL-GAS6 complexes in samples using the anti-human AXL-GAS6 antibody of the present invention or its antigen-binding fragment, including, for example, for detecting AXL-GAS6 complexes in blood or plasma.

[0014] Furthermore, this invention also provides methods and uses for treating AXL-expressing tumors using the antibodies of this invention or their antigen-binding fragments. The antibodies of this invention can be used as the sole active agent or in combination with other therapies or therapeutic agents.

[0015] Specifically, the present invention provides the following technical solutions:

[0016] 1. A method for detecting the level of the AXL-GAS6 complex in a sample, including

[0017] (a) Contacting the sample with anti-AXL-GAS6 antibody or its antigen-binding fragment; and

[0018] (b) Detect the binding of the AXL-GAS6 antibody or its antigen-binding fragment to the AXL-GAS complex and determine the level of the AXL-GAS6 complex in the sample;

[0019] The anti-AXL-GAS6 antibody comprises three complementarity-determining regions (HCDRs) in the heavy chain variable region and three complementarity-determining regions (LCDRs) in the light chain variable region, wherein:

[0020] The amino acid sequence of HCDR1 is shown in SEQ ID NO:1;

[0021] The amino acid sequence of HCDR2 is shown in SEQ ID NO:2;

[0022] The amino acid sequence of HCDR3 is shown in SEQ ID NO:3;

[0023] The amino acid sequence of LCDR1 is shown in SEQ ID NO:4;

[0024] The amino acid sequence of LCDR2 is shown in SEQ ID NO:5; and

[0025] The amino acid sequence of LCDR3 is shown in SEQ ID NO:6.

[0026] 2. The method described in Project 1, wherein the detection of the binding level of the antibody or its antigen-binding fragment to the AXL-GAS complex in step (b) is performed by enzyme-linked immunosorbent assay (ELISA).

[0027] 3. The method according to Project 2, wherein the ELISA is a capture sandwich ELISA.

[0028] 4. The method described in Project 1, wherein the sample is plasma.

[0029] 5. Use of anti-AXL-GAS6 antibody or its antigen-binding fragment in the preparation of medicines or kits for the diagnosis of myocardial infarction or the assessment of myocardial infarction risk.

[0030] 6. According to the use described in Item 5, the anti-AXL-GAS6 antibody comprises three complementarity-determining regions (HCDRs) of the heavy chain variable region and three complementarity-determining regions (LCDRs) of the light chain variable region, wherein:

[0031] The amino acid sequence of HCDR1 is shown in SEQ ID NO:1;

[0032] The amino acid sequence of HCDR2 is shown in SEQ ID NO:2;

[0033] The amino acid sequence of HCDR3 is shown in SEQ ID NO:3;

[0034] The amino acid sequence of LCDR1 is shown in SEQ ID NO:4;

[0035] The amino acid sequence of LCDR2 is shown in SEQ ID NO:5; and

[0036] The amino acid sequence of LCDR3 is shown in SEQ ID NO:6.

[0037] 7. As described in Item 5, the myocardial infarction is ST-segment elevation myocardial infarction.

[0038] 8. As described in item 5, wherein the kit further includes ELISA detection reagents.

[0039] 9. According to the intended use described in Item 5, the kit further includes an instruction manual, wherein the instruction manual indicates:

[0040] When the AXL-GAS complex level is >1.8 ng / ml, it indicates a high risk of myocardial infarction or myocardial infarction.

[0041] When the AXL-GAS complex level is ≤1.8 ng / ml, it indicates no myocardial infarction or low risk of myocardial infarction.

[0042] The myocardial infarction mentioned above is ST-segment elevation myocardial infarction.

[0043] the term

[0044] All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0045] In this document, the term "antibody" refers to a polypeptide containing at least a light or heavy chain immunoglobulin variable region that specifically recognizes and binds to an antigen. This term encompasses a wide range of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, single-chain or multi-chain antibodies, monospecific or multispecific antibodies (e.g., bispecific antibodies), fully human antibodies, chimeric antibodies, or humanized antibodies, full-length antibodies, and antibody fragments, provided they exhibit the desired antigen-binding activity.

[0046] The antibody of this invention comprises at least two heavy chains (H) and two light chains (L). Each heavy chain consists of a heavy chain variable region (abbreviated VH herein) and a heavy chain constant region. The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated VL herein) and a light chain constant region. The light chain constant region consists of one domain: CL. The variable region is a domain in the heavy or light chain of the antibody that participates in the binding of the antibody to its antigen. The constant region does not directly participate in the binding of the antibody to the antigen but exhibits various effector functions. The light chain of the antibody can be classified into one of two types (called kappa (κ) and lambda (λ)) based on the amino acid sequence of its constant domain. Antibody heavy chains can be classified into five main types based on the amino acid sequence of their heavy chain constant regions: IgA, IgD, IgE, IgG, and IgM. Several of these types can be further subdivided into subclasses, such as IgG1, IgG2, IgG3 and IgG4, IgA1, and IgA2. The heavy chain constant regions corresponding to different antibody types are called α, δ, ε, γ, and μ, respectively. The term "isotype" refers to the antibody type determined by the antibody heavy chain constant region. See, for example, Fundamental Immunology, Ch.7 (edited by Paul, W., 2nd ed., Raven Press, NY (1989)).

[0047] "Anti-angiogenic agents" refer to compounds that block or interfere with blood vessel development to some extent. Anti-angiogenic agents can be, for example, small molecules or antibodies that bind to growth factors or growth factor receptors involved in promoting angiogenesis. In one embodiment, the anti-angiogenic agent is an antibody that binds to vascular endothelial growth factor (VEGF), such as bevacizumab (AVASTIN).

[0048] The term "PD-1 axis binding antagonist" refers to a molecule that inhibits the interaction of a PD-1 axis binding partner with one or more of its binding partners, thereby removing T cell dysfunction originating from signal transduction along the PD-1 signaling axis, resulting in the restoration or enhancement of T cell function (e.g., proliferation, cytokine production, target cell killing). As used herein, PD-1 axis binding antagonists include PD-1 binding antagonists (e.g., anti-PD-1 antibodies), PD-L1 binding antagonists (e.g., anti-PDL1 antibodies), and PD-L2 binding antagonists (e.g., anti-PD-L2 antibodies).

[0049] In this invention, the myocardial infarction includes ST-segment elevation myocardial infarction and non-ST-segment elevation myocardial infarction. ST-segment elevation myocardial infarction refers to an upward-convex elevation of the ST segment on the electrocardiogram, affecting the entire thickness of the ventricular wall, resulting in acute coronary artery occlusion and an extremely high sudden death rate. Non-ST-segment elevation myocardial infarction, on the other hand, lacks characteristic changes in the ST segment on the electrocardiogram, sometimes presenting as ST-segment depression. In terms of ventricular involvement, it is a non-transmural myocardial infarction (not involving the epicardium), with incomplete coronary artery occlusion, often characterized by 90-99% stenosis or plaque rupture, and a relatively high sudden death rate.

[0050] In this invention, sAXL and AXL can be used interchangeably.

[0051] In this invention, the AXL-4# antibody obtained by this invention is used to detect the level of the AXL-GAS6 complex via a capture sandwich ELISA method. This capture sandwich ELISA method is a conventional method in the art, also known as sandwich ELISA. It involves first coating a 96-well plate with the capture antibody AXL-4#, then adding the target antigen (i.e., the AXL-GAS6 complex). The antigen specifically binds to the capture antibody. After washing to remove unbound proteins, a biotin-labeled detection antibody (i.e., biotin-labeled AXL-4# antibody or a commercially available AXL detection antibody) is added. Both antibodies bind to the antigen, forming a double-antibody sandwich structure. HRP-labeled avidin is then added, and subsequent color development is performed using an avidin-biotin reaction system. The TMB substrate produces a color reaction under the action of HRP. Finally, a stop solution is added to complete the experiment. The completed 96-well plate is placed in a microplate reader to read the values, and the content information of the target antigen is obtained according to the standard curve technique.

[0052] The present invention will now be described in detail.

[0053] I. The anti-AXL-GAS6 antibody of the present invention

[0054] The antibodies of this invention specifically bind to AXL-GAS6, preferably in the presence of human or mouse GAS6, to antibodies or antigen-binding fragments thereof that bind to human AXL protein (e.g., the human AXL sequence of Genbank accession number NM_021913.5). The antigen-binding fragments of the antibodies of this invention are selected from antibody fragments such as Fab, Fab', Fab'-SH, Fv, single-chain antibodies such as scFv, (Fab')2 fragments, single-domain antibodies, bispecific antibodies (dAbs), or linear antibodies. The antibodies of this invention or their antigen-binding fragments bind to the first domain of AXL in the presence of human or mouse GAS6. The antibodies of this invention can bind to tumor cells expressing AXL that express GAS6.

[0055] This invention provides an anti-human AXL-GAS6 antibody targeting the human AXL-GAS6 complex. The antibody of this invention can bind to cells expressing AXL in the presence of different concentrations of GAS6; it can also bind to cells expressing AXL in the presence of human or mouse GAS6. The antibody of this invention can bind to GAS6 in the presence of AXL and can also bind to cells expressing GAS6. The antibody of this invention binds to the first domain of AXL in the presence of human or mouse GAS6. The antibody of this invention can also bind to tumor cells expressing both AXL and GAS6. The antibody of this invention can be used to detect the presence of the AXL-GAS6 complex and has demonstrated good antitumor efficacy in animal experiments.

[0056] Antibody CDR region

[0057] The "complementarity-determining region" or "CDR region" is the amino acid region in the antibody variable region that is primarily responsible for binding to the antigen epitope. The CDRs of the heavy and light chains are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus. CDRs located within the variable domain of the antibody heavy chain are called HCDR1, HCDR2, and HCDR3, while those located within the variable domain of the antibody light chain are called LCDR1, LCDR2, and LCDR3.

[0058] Table 1 below shows some exemplary antibody VH and VL sequence combinations of the present invention:

[0059]

[0060] Various schemes for determining the CDR sequence in a given VH or VL amino acid sequence are well known in the art. For example, the Kabat complementarity-determining region (CDR) is determined based on sequence variability and is the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Chothia refers to the location of the structural loop (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). AbM HVR is a compromise between Kabat HVR and Chothia structural loop and is used by the AbM antibody modeling software from Oxford Molecular. “Contact” HVR is based on the analysis of available complex crystal structures. IMGT defines a unique scheme for variable domains in antibodies and T cell receptors (Lefranc, Leukemmia, 17:260-206 (2003)). Based on different CDR determination schemes, these CDR residues are shown in Table 2 below.

[0061]

[0062]

[0063] The positions of residues in the variable region of the antibody of the present invention (including heavy chain variable region residues and light chain variable region residues) are determined according to the IMGT numbering system. Table 3 below provides exemplary CDR sequences of the present invention:

[0064] Table 3

[0065]

[0066] In one embodiment, the antibody or its binding fragment of the present invention comprises three complementarity-determining regions (HCDRs) of the heavy chain variable region and three complementarity-determining regions (LCDRs) of the light chain variable region, wherein

[0067] HCDR1 contains or is composed of the amino acid sequence of SEQ ID NO:1, HCDR2 contains or is composed of the amino acid sequence of SEQ ID NO:2, HCDR3 contains or is composed of the amino acid sequence of SEQ ID NO:3, LCDR1 contains or is composed of the amino acid sequence of SEQ ID NO:4, LCDR2 contains or is composed of the amino acid sequence of SEQ ID NO:5, and LCDR3 contains or is composed of the amino acid sequence of SEQ ID NO:6.

[0068] Antibody variable region

[0069] In one embodiment, the antibody of the present invention comprises the heavy chain variable region VH sequence of the antibodies listed in Table 2, or is composed of the amino acid sequence described therein. In another embodiment, the antibody of the present invention comprises a variant of the VH sequence.

[0070] In another embodiment, the antibody of the present invention comprises the light chain variable region (VL) sequence of the antibodies listed in Table 2, or is composed of the amino acid sequence described therein. In yet another embodiment, the antibody of the present invention comprises a variant of the VL sequence.

[0071] In one embodiment, the antibody or antigen-binding fragment of the present invention comprises a heavy chain variable region and a light chain variable region, wherein:

[0072] The heavy chain variable region comprises: the amino acid sequence shown in SEQ ID NO:7, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with it. Variant versions of the VH sequence, compared to the reference VH sequence, (preferably, in the full length or in the CDR1, 2, and 3 regions) contain at least one and no more than 30, 10, or 5, 4, 3, 2, 1, or 0 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions). Preferably, the sequence differences do not occur in the CDR regions.

[0073] In one embodiment, the antibody or antigen-binding fragment of the present invention comprises a heavy chain variable region and a light chain variable region, wherein the light chain variable region comprises: the amino acid sequence shown in SEQ ID NO:8, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with it. Variant sequences of the VL sequence, compared to a reference VL sequence, (preferably, in the full length or in the CDR1, 2, and 3 regions), contain at least one and no more than 30, 10, or 5, 4, 3, 2, 1, or 0 amino acid changes (preferably amino acid substitutions, preferably conserved substitutions). Preferably, the sequence differences do not occur in the CDR regions.

[0074] In one embodiment, the antibody or antigen-binding fragment of the present invention comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:7 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:8.

[0075] Antibody heavy chain and light chain

[0076] The antibodies of the present invention comprise heavy chain constant regions and / or light chain constant regions. In some embodiments, the antibodies of the present invention comprise human heavy chain Fc regions, such as the Fc regions of human IgG1, IgG2, IgG3, or IgG4 isotypes, preferably human IgG1 and IgG4. In other embodiments, the antibodies of the present invention comprise mouse heavy chain Fc regions, such as the Fc regions of mouse IgG1, IgG2a, IgG2b, or IgG3 isotypes, preferably mouse IgG1 or IgG2a. In still other embodiments, the antibodies of the present invention comprise κ light chain constant regions, such as human κ light chain constant regions or mouse κ light chain constant regions. The sequences of the heavy chain constant regions and light chain constant regions are known, for example, available from GenBank.

[0077] "IgG form antibody" refers to an antibody whose heavy chain constant region belongs to the IgG form. All antibodies of the same type have the same heavy chain constant region, while antibodies of different types have different heavy chain constant regions. For example, an IgG1 form antibody means that its heavy chain constant region Ig domain is the Ig domain of IgG1.

[0078] II. Polynucleotides, Vectors, and Hosts

[0079] This invention provides nucleic acids encoding any of the above-mentioned anti-human AXL-GAS6 antibodies or fragments thereof. The invention also provides vectors comprising said nucleic acids. In one embodiment, the vector is an expression vector. A host cell comprising said nucleic acid or said vector is also provided. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells (e.g., CHO cells or 293 cells). In yet another embodiment, the host cell is prokaryotic.

[0080] The nucleic acid may be a nucleic acid comprising an amino acid sequence encoding the light chain variable region and / or the heavy chain variable region of an antibody, or a nucleic acid comprising an amino acid sequence encoding the light chain and / or the heavy chain of an antibody. An exemplary nucleic acid sequence encoding the antibody heavy chain variable region comprises a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with a nucleic acid sequence selected from SEQ ID NO:9. An exemplary nucleic acid sequence encoding the antibody light chain variable region comprises a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with a nucleic acid sequence selected from SEQ ID NO:10.

[0081] This invention also provides polynucleotides encoding typically all three CDR regions of the heavy chain VH or light chain VL sequence of the aforementioned antibody-binding human AXL-GAS6. The polynucleotides encode the complete or substantially complete variable region sequence of the heavy chain and / or light chain of the aforementioned antibody-binding human AXL-GAS6. Those skilled in the art will understand that, due to codon degeneracy, each antibody or polypeptide amino acid sequence can be encoded by multiple nucleic acid sequences.

[0082] Nucleic acid sequences encoding antibodies or antigen-binding fragments of AXL-GAS6 can be synthesized from de novo solid-phase DNA or by PCR mutagenesis using methods well-known in the art.

[0083] In one embodiment, the present invention provides one or more vectors comprising the nucleic acids of the present invention. The vector is an expression vector, such as a eukaryotic expression vector. Vectors include, but are not limited to, viruses, plasmids, granules, λ phages, or yeast artificial chromosomes (YAC).

[0084] In one embodiment, a host cell comprising the vector is provided. Suitable host cells for cloning or expressing the vector encoding an antibody include prokaryotic or eukaryotic cells. For example, antibodies can be produced in bacteria, such as expression in *Escherichia coli*. After expression, the antibody can be separated from the bacterial cell paste in a soluble fraction and can be further purified.

[0085] In one implementation, the host cell is eukaryotic. The host cell is selected from yeast cells or mammalian cells. Used mammalian host cell lines include 293 and CHO.

[0086] III. Antibody Preparation

[0087] The antibody against the human AXL-GAS6 complex of this invention is prepared using authoritative monoclonal antibody preparation methods (e.g., Paterson, HMVaY (Jone Wiley and Sons, Inc., New York, 1995). Production of Antibodies. Current Protocols in Immunology). The AXL antigen used to prepare the monoclonal antibody can be a synthetic AXL gene ligated into an expression vector, or RNA can be extracted from cells expressing AXL, obtained through AXL-specific primer reverse transcription PCR, and then ligated into an expression vector. The expression vector can be a eukaryotic expression vector or a prokaryotic expression vector. The AXL antigen used to immunize the animal can be a purified protein, transiently transfected cells expressing AXL (such as 293 cells or L929 cells), or cells stably expressing AXL (such as cells infected with lentiviruses).

[0088] Mice are the preferred animal for preparing hybridomas. The procedure for generating hybridomas using mice is well-established and well-developed. Immunization procedures and techniques for isolating immune spleen cells used for fusion are well known in the art. Fusion partners (e.g., mouse myeloma cells) and fusion methods are also well known.

[0089] In addition to obtaining anti-antibodies by amplifying hybridoma cells in vivo or in vitro, the encoding gene of the antibody against human AXL-GAS6 of the present invention can also be cloned into a eukaryotic expression vector using conventional molecular cloning methods, and the antibody can be obtained through eukaryotic expression and purification.

[0090] IV Immunoconjugates

[0091] This invention provides immunoconjugates generated by conjugating the antibody of the present invention to a heterologous molecule. In one embodiment, the antibody of the present invention (or its antigen-binding fragment) is conjugated to a therapeutic or diagnostic agent in the immunoconjugate. In some embodiments, the antibody of the present invention may be conjugated to the heterologous molecule in the form of a full-length antibody or an antibody fragment. For example, conjugation may be in the form of Fab fragments, Fab' fragments, F(ab)'2 fragments, single-chain scFab antibodies, single-chain scFv, etc.

[0092] The antibodies of this invention can be conjugated to diagnostic or detectable agents. Such conjugates can be used as part of clinical testing methods (e.g., to determine the efficacy of a specific therapy) to monitor or predict the onset, development, progression, and / or severity of a disease or condition. Such diagnostics and detections can be achieved by conjugating antibodies to detectable agents, including but not limited to various enzymes such as, but not limited to, horseradish peroxidase; prosthetic groups such as, but not limited to, streptavidin / biotin and avidin / biotin; fluorescent substances; luminescent substances; radioactive substances; and positron-emitting metal and non-radioactive paramagnetic metal ions used in various positron emission tomography (PET) imaging techniques.

[0093] V. Pharmaceutical compositions and pharmaceutical preparations

[0094] The present invention also includes compositions comprising anti-AXL-GAS6 antibodies or their immunoconjugates (including pharmaceutical compositions or pharmaceutical formulations) and compositions comprising polynucleotides encoding anti-AXL-GAS6 antibodies or their immunoconjugates. These compositions may optionally also contain suitable pharmaceutical excipients, such as pharmaceutical carriers, pharmaceutical excipients, including buffers, known in the art.

[0095] VI. Methods and Applications

[0096] This invention provides methods and uses for applying the anti-AXL-GAS6 antibody of the present invention or its antigen-binding fragment. For example, the methods and uses of the present invention relate to the treatment of a disease in an individual subject. The methods and uses of the present invention also relate to detecting the presence of the AXL-GAS6 complex in a sample, for example, from a subject. The present invention also provides the use of the AXL-GAS6 antibody of the present invention or its antigen-binding fragment in the preparation of products (e.g., pharmaceutical compositions, pharmaceutical products, or detection products) for the above-described uses.

[0097] The anti-human AXL-GAS6 antibody of the present invention can be used to prepare drugs for treating cancer, including hematologic cancers and solid tumors expressing GAS6 and AXL. Hematologic cancers include leukemia, such as chronic lymphocytic leukemia, myeloid leukemia, acute myeloid leukemia, and chronic myeloid leukemia; and lymphomas, such as non-Hodgkin's lymphoma and multiple myeloma. Solid tumors expressing GAS6 and AXL include lung cancer, epidermoid carcinoma, colorectal cancer such as colorectal cancer and colorectal adenoma, bladder cancer, bone cancer such as chondrosarcoma, breast cancer such as triple-negative breast cancer, central nervous system-eluting cancers such as glioblastoma, astrocytoma, neuroblastoma, cervical cancer, connective tissue cancer, endometrial cancer, fibroblastic cancer, gastric cancer, head and neck cancer, kidney cancer, liver cancer, muscle cancer, nerve tissue cancer, ovarian cancer, pancreatic cancer, and skin cancer such as malignant melanoma and soft tissue sarcoma. Attached Figure Description

[0098] Figure 1 The binding of the anti-AXL-GAS6 antibody was shown; among them, Figure 1 A shows that the anti-AXL-GAS6 antibody binds to AXL expressed on the cell membrane surface at the flow cytometry level in the presence of GAS6; Figure 1 B shows the binding of anti-AXL-GAS6 antibody to hGAS6 at flow cytometry levels in the presence of different concentrations of human GAS6.

[0099] Figure 2 ELISA results showing the interaction between anti-AXL-GAS6 antibody and different concentrations of hGAS6.

[0100] Figure 3 The flow cytometry results show that the anti-AXL-GAS6 antibody binds to membrane-expressed GAS6 in the presence of secreted AXL.

[0101] Figure 4 This diagram shows the constructs that express the full-length AXL and express domain 1 and domain 234.

[0102] Figure 5 The anti-AXL-GAS6 antibody AXL-4# binds to domain 1 of AXL.

[0103] Figure 6 The results show the binding of the anti-AXL-GAS6 antibody to cells expressing human AXL or mouse AXL in the presence of human or mouse GAS6. 6A represents the flow cytometry results, indicating that the anti-AXL-GAS6 antibody AXL-4# binds to the complexes of human AXL and human GAS6, or human AXL and mouse GAS6, but not to the complexes of mouse AXL and human GAS6, or mouse AXL and mouse GAS6. Figure 6 B Display Figure 6 A. Statistical data of flow cytometry results.

[0104] Figure 7 The antibody against AXL-GAS6 was shown to bind to cancer cells expressing AXL.

[0105] Figure 8 The figure shows the nucleotide and amino acid sequences of the variable region of the AXL-4# heavy chain of the AXL-GAS6 antibody. The black shaded areas in the figure indicate the signal peptide region, CDR1 region (SEQ ID NO:1), CDR2 region (SEQ ID NO:2), and CDR3 region (SEQ ID NO:3).

[0106] Figure 9 The figure shows the nucleotide and amino acid sequences of the variable region of the light chain of the anti-AXL-GAS6 antibody AXL-4#. The black shaded areas in the figure indicate the signal peptide region, CDR1 region (SEQ ID NO:4), CDR2 region (SEQ ID NO:5), and CDR3 region (SEQ ID NO:6).

[0107] Figure 10 The comparison results of the amino acid sequence (SEQ ID NO:7) of the heavy chain variable region of the anti-AXL-GAS6 antibody AXL-4# with the mouse amino acid sequence are shown. The heavy chain variable region composition of the AXL-GAS6 monoclonal antibody is as follows: V gene is IGHV5-17, D gene is IGHD1-2, and J gene is IGHJ4.

[0108] Figure 11 The comparison results of the amino acid sequence (SEQ ID NO:8) of the variable region of the light chain of the anti-AXL-GAS6 antibody AXL-4# with the mouse amino acid sequence are shown. The variable region of the light chain of the anti-AXL-GAS6 monoclonal antibody is composed of: V gene IGKV8-30 and J gene IGKJ2.

[0109] Figure 12A and Figure 12B The results of a capture sandwich ELISA assay using an anti-human AXL-GAS6 antibody-coated complex are shown.

[0110] Figure 13 An exemplary ESLIA standard curve is shown.

[0111] Figure 14 This shows the level of the AXL-GAS6 complex in myocardial infarction patients as detected using anti-human AXL-GAS6 antibody.

[0112] Figure 15 The ROC curve is displayed.

[0113] Figure 16 The correlation between AXL-Gas6, AXL and clinical indicators (A: AXL-Cr; B: PLT; C: WBC; D: HDL; E: CMKB; F: So-FMC) was shown in STEMI patients. Detailed Implementation

[0114] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0115] Unless otherwise specified, the methods used in the following examples are conventional methods, and the reagents used are commercially available reagents.

[0116] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0117] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0118] In the quantitative experiments described below, three replicate experiments were conducted, and the average value of the results was taken.

[0119] Example 1: Preparation of cells expressing human AXL, cells expressing mouse AXL, and cells expressing membrane-bound human GAS6.

[0120] 1. Preparation of full-length human AXL cells

[0121] Cells that stably express full-length human AXL, cells that express AXL domain 1, or cells that express AXL domain 234 (i.e., domains 2, 3, and 4, referred to herein as domain 234) were constructed respectively.

[0122] Design primers based on Genbank accession number NM_021913.5.

[0123] hAXL-F:5'-ccgaattcgccaccatggcgtggcggtgccccaggatgggc-3'(SEQ ID NO:11)

[0124] hAXL-R:5'-ccagcagccccagggcaggaggatggtgcctgaggatccgcc-3'(SEQ ID NO:12)

[0125] From isolated human CD5+ dendritic cells (human CD5+) + For the isolation of dendritic cells, see Yin Xiangyun, Yu Haisheng, Jin Xiaoyang, et al., Human Blood CD1c+Dendritic Cells Encompass CD5high and CD5low Subsets That Differ Significantly in Phenotype, Gene Expression, and Functions, J Immunol (2017) 198(4): 1553-1564). Total RNA was extracted using TRIzol (Invitrogen). According to the manufacturer's instructions, cDNA was synthesized using Oligo (dT) and MMLV reverse transcriptase (Promega, M1705). Using cDNA as a template, PCR amplification was performed using primers hAXL-F and hAXL-R. The obtained PCR amplification product was the full-length human AXL nucleotide sequence.

[0126] The PCR amplification product was digested with restriction endonucleases EcoRI and BamHI and ligated into the similarly digested pEGFP-N1 (Addgene) vector to obtain the pEGFP-N1-hAXL plasmid. In this plasmid, the EGFP gene is located downstream of the AXL sequence; when AXL is expressed, a GFP signal can be observed. The pEGFP-N1-hAXL plasmid was transfected into 293T cells (purchased from ATCC) using lipofectamine transfection reagent (ThermoFisher) to obtain the 293T-hAXL cell line expressing hAXL.

[0127] 2. Preparation of cells expressing human AXL domain 1 or domain 234

[0128] Using a plasmid containing full-length hAXL (e.g., the pEGFP-N1-hAXL plasmid prepared above in this embodiment) as a template, the following primers were used respectively.

[0129] P1u-EcoRI:5'-gcGAATTCGCCACCatggcgtggcggtgcccca-3'(SEQ ID NO:13)

[0130] P2d6s,5'- CCCTGGGCGCCAcaagccctccagcccaacatagcc-3'(SEQ ID NO:14)

[0131] Fragment 1 was obtained through amplification;

[0132] Use the following primers

[0133] P6u-5'-TGGCGCCCAGGGCAAGCAC-3'(SEQ ID NO:15)

[0134] p6d-BamHI:5'-cgGGATCCTCAGGCACCATCCTCCTGCCCT-3'(SEQ ID NO:16)

[0135] Fragment 2 was obtained from the amplification.

[0136] Using a mixture of fragments 1 and 2 as a template, overlap PCR was performed using primers P1u-EcoRI (SEQ ID NO:13) and p6d-BamHI (SEQ ID NO:16) to amplify fragment 3, which is the AXL fragment containing domain 1 but lacking domain 234. This fragment was then digested with EcoRI and BamHI and ligated into the pEGFP-N1 vector, which had undergone the same digestion process, to obtain the pEGFP-N1-AXL-D1 plasmid.

[0137] Using a plasmid containing full-length hAXL (e.g., the pEGFP-N1-hAXL plasmid prepared above in this embodiment) as a template, the following primers were used respectively.

[0138] P1u:5'-gcGAATTCGCCACCatggcgtggcggtgcccca-3'(SEQ ID NO:17)

[0139] P1d-3s:5'-CAGCCCAACATAGCCcgtgcccctgggggccatgc-3'(SEQ ID NO:18)

[0140] Fragment 4 was obtained from the amplification;

[0141] Use the following primers

[0142] P3u:5'-GGCTATGTTGGGCTGGAGGGCTTG-3'(SEQ ID NO:19)

[0143] p6d-BamHI:5'-cgGGATCCTCAGGCACCATCCTCCTGCCCT-3'(SEQ ID NO:20)

[0144] Fragment 5 was obtained from the amplification.

[0145] Using a mixture of fragments 4 and 5 as a template, overlap PCR was performed using primers P1u-EcoRI (SEQ ID NO:13) and p6d-BamHI (SEQ ID NO:20) to amplify fragment 6, which is the AXL fragment lacking domain 1 and containing domain 234. This fragment was then digested with EcoRI and BamHI and ligated to the similarly digested vector pEGFP-N1 to obtain the pEGFP-N1-AXL-D234 plasmid. Schematic diagrams of the constructs expressing full-length AXL and those expressing domain 1 and domain 234 are shown below. Figure 4 As shown.

[0146] Using lipofectamine transfection reagent (ThermoFisher), pEGFP-N1-AXL-D1 plasmid or pEGFP-N1-AXL-D234 plasmid was transfected into 293T cells (purchased from ATCC) to obtain cells expressing 293T-hAXL-domain 1 or 293T-hAXL-domain 234, respectively.

[0147] 3. Preparation of cells expressing mouse AXL (mAXL)

[0148] In this step, cells expressing full-length mAXL, used in this invention, were constructed.

[0149] According to GenBank accession number NM_009465.4, full-length mAXL was synthesized and ligated to plasmid pEGFP-N1 (synthesized by Shanghai Jierui Biotechnology Co., Ltd.). In this plasmid, the EGFP gene is located downstream of the mAXL sequence; when mAXL is expressed, a GFP signal can be observed. Following the method in Example 1.1, plasmid pEGFP-N1-mAXL was prepared. Using Lipofectamine transfection reagent (Thermo Fisher), the pEGFP-N1-mAXL plasmid was transfected into 293T cells (purchased from ATCC) to obtain the mAXL-expressing cell line 293T-mAXL.

[0150] 4. Preparation of cells expressing membrane-bound GAS6 (MemGAS6)

[0151] In this step, cells stably expressing membrane-bound GAS6 (MemGAS6) used in this invention were constructed.

[0152] The hGAS6 gene was synthesized according to GenBank accession number NM_000820.4. An EcoRI restriction site and a signal peptide sequence (SEQ ID NO:21) were added upstream of the 5' end of the synthesized hGAS6 gene, and the platelet-derived growth factor receptor (PDGFR) transmembrane region (SEQ ID NO:22) and an AvaI restriction site were added downstream of the 3' end of the hGAS6 gene. The synthesized gene was digested with EcoRI and AvaI, and then ligated to the similarly digested vector pDisplay (purchased from Thermo) to construct the plasmid pDisplay-hGAS6. This plasmid was transfected into 293T cells to obtain cell line 293T-memGAS6 expressing membrane-bound GAS6.

[0153] The sequence of the signal peptide sequence SEQ ID NO:21 is as follows:

[0154] ATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGTGAC(SEQID NO:21)

[0155] The sequence of the transmembrane region of the platelet-derived growth factor receptor (PDGFR) SEQ ID NO:22 is as follows:

[0156] AATGCTGTGGGCCAGGACACGCAGGAGGTCATCGTGGTGCCACACTCCTTGCCCTTTAAGGTGGTGGTGATCTCAGCCATCCTGGCCCTGGTGGTGCTCACCATCATCTCCCTTATCATCCTCATCATGCTTTGGCAGAAGAAGCCACGTTAG (SEQ ID NO: 22)

[0157] Example 2: Preparation of secretory AXL

[0158] Using the plasmid containing full-length human AXL from Example 1 (i.e., pEGFP-N1-hAXL plasmid) as a template, the following primers were used:

[0159] Upstream primer hAXL-F-EcoRI:

[0160] 5'-GCGAATTCGCCACCATGGCGTGGCGGTGCCCCAGGATGGGCAGGGTC-3' (SEQ ID NO: 23), and

[0161] Downstream primer hAXL-R-his:

[0162] 5'-CGGGATCCTTAGTGATGGTGGTGGTGATGTGGGCCAGGCCTCCAGG-3' (SEQ ID NO: 24),

[0163] PCR amplification was performed using the high-fidelity enzyme PrimStar (purchased from Takara Bio). The PCR product was then double-digested with EcoRI and BamHI and ligated with the similarly digested vector pTT3 (purchased from Thermo) to construct the expression plasmid pTT3-hAXL-his, which fused with the extracellular domain of human AXL.

[0164] Plasmids were extracted using an endotoxin-free plasmid extraction kit (purchased from Tiangen Biotech). The plasmids were mixed thoroughly with the PEI transfection reagent and allowed to stand. The mixture was then added to 293T cells (purchased from ATCC) and cultured at 37°C for 4 days in a 5% CO2 shaker. The supernatant was collected by centrifugation and purified using a nickel column followed by elution with 10 mM imidazole. The eluted protein was transferred to a 30 kDa ultrafiltration tube, PBS was added, and the mixture was centrifuged at 4000 rpm to obtain the purified AXL-his antigen.

[0165] Example 3: Production of anti-AXL-GAS6 antibody in mice

[0166] 1. Preparation of immunogens

[0167] The full-length human AXL fragment was ligated into the vector pEGFP-C1 (Clontech, Cat:#6084-1) to obtain the plasmid pEGFPAXL. Following the manufacturer's instructions, this plasmid was transfected into mouse L cells (ATCC, catalog number: ) using Lipofectamine 2000 (Invitrogen). CRL-2648 TM In this study, mouse L cells expressing the human AXL extracellular domain were obtained 48 hours after transfection. The transfection efficiency was determined by analyzing the expression of EGFP in the transfected mouse L cells and used in the following mouse immunization experiments. In this embodiment, mouse L cells expressing the human AXL extracellular domain were used as the immunogen. In reality, mouse L cells themselves can produce GAS6, and because AXL has a strong affinity for GAS6, the human AXL-GAS6 complex is present as an immunogen during the actual immunization process.

[0168] 2. Immunization of Balb / C mice

[0169] The mouse L cells expressing the human AXL extracellular domain obtained in step 1 were used as immunogens for human AXL. 5,000,000 mouse L cells expressing the human AXL extracellular domain were mixed with 20 μg CpG1826 (InvivoGen) to form a 0.5 ml suspension, which was then administered intraperitoneally to 6-week-old Balb / c mice. Immunization was repeated monthly for a total of four doses. Finally, a booster immunization was performed using the same method, and hybridoma fusion was performed four days later.

[0170] 3. Fusion of hybridomas

[0171] Mice awaiting fusion after immunization were sacrificed, and spleen cells were harvested. Cell counting was performed, and mouse spleen cells were mixed with mouse myeloma cell line SP2 / 0 (ATCC, CRL1581) at a ratio of 1:3, using 50% PEG (Sigma) for cell fusion. The fused cells were added to 60 ml of RPMI medium (Gibco, catalog number: C11875500BT) containing 1xHAT (10% fetal bovine serum), at a rate of 1–2 drops per well in a 96-well cell culture plate. The hybridoma cells were then cultured at 37°C and 5% CO2, with a half-volume medium change every 3–4 days. Antibody screening was performed approximately 10 days later.

[0172] Example 4: Screening of AXL and GAS6 antibodies

[0173] 1. Screening and subcloning of AXL and GAS6 antibodies

[0174] Aspirate approximately 100 μl of the supernatant from the 96-well cell culture plate into a 96-well U-shaped culture plate, and add 100 μl of fresh RPMI medium containing 1xHAT to the original wells to continue culturing the hybridoma cells.

[0175] Recombinant human GAS6 protein (R&D, NP_000811) was added to a suspension of 293T-hAXL cells (obtained in Example 1.1) digested with PBS containing 2 mM EDTA and incubated at room temperature for 30 minutes before being added to 96-well U-shaped bottom culture plates. 4Cells / 100 μL / well. Centrifuge at 2200 rpm for 3 minutes, discard the supernatant containing recombinant human GAS6 protein, add 100 μL / well of hybridoma cell culture supernatant, resuspend, and incubate at 4°C for 30 minutes. Wash cells twice with FACS buffer (PBS containing 2% FBS and 2 mMEDTA). Then incubate with 100 μL / well of PE-labeled goat anti-mouse IgG antibody (BioLegend, Poly4053, 405307) (1:500 dilution) at 4°C in the dark for 30 minutes. Wash cells twice with FACS buffer. After final washing, analyze GFP and PE signals using flow cytometry (Thermo Analyzer). GFP and PE double-positive wells are positive wells containing the AXL and GAS6 complex.

[0176] Cells from FACS-positive hybridoma wells were selected and limitedly diluted in 96-well plates. These cells were allowed to grow for 7 days. When sufficient cell volume was reached, the supernatant from each well was collected and re-screened using 293T-hAXL cells treated with recombinant GAS6 protein as described above (i.e., those producing the AXL-GAS6 complex) by binding to the cells. Figure 1 The binding status of the anti-AXL-GAS6 antibody was shown.

[0177] From each 96-well plate, monoclonal cells with cell-binding activity were expanded and subjected to a second round of limiting dilution. After 7 days, the supernatant from the cells in the 96-well plates was analyzed by FACS. Subcloning was performed three times. Stable monoclonal hybridoma cell lines expressing the AXL-4# antibody were obtained. Positive clones were further expanded and cultured to produce antibodies, and the antibody subtype was determined to be mIgG2a using a mouse monoclonal antibody typing kit (SinoScan SEK003). For detailed information on the AXL-4# antibody sequence, please refer to Example 9.

[0178] 2. Preparation of small-scale antibodies

[0179] The hybridoma cell lines were cultured in serum-free PFHMII (Thermo Fisher) medium. The hybridoma cell culture supernatant was collected, and protein A filler (Captiva) was added. TM PriMAB (cat: CA-PRI-1000) was packed into a column, and the antibody was adsorbed through the affinity chromatography column. The bound antibody AXL-4# was eluted from the affinity chromatography column with 0.1M glycine (pH 2.7) elution buffer. The antibody in the collection tube was used to determine the protein concentration. The liquids in the tubes containing protein were combined, and the antibody was diluted with PBS. The solution was added to a 30KD ultrafiltration centrifuge tube and concentrated by centrifugation at 3000 rpm and 4°C. The solution in which the antibody was dissolved was replaced with PBS.

[0180] Example 5: Interaction between AXL-4# antibody and human or mouse GAS6

[0181] 1. The interaction between enzyme-linked immunosorbent assay (ELISA) and human GAS6.

[0182] The interaction between antibody AXL-4# and human GAS6 was detected by ELISA. In short, hGAS at a maximum concentration of 2 μg / mL was serially diluted 2-fold with PBS, and 100 μL was coated per well onto the ELISA plate and incubated overnight at 4°C. Blocking was performed with 0.2% bovine serum albumin (Beijing Lanbolide Biotechnology Co., Ltd.) at room temperature for 1 hour. 100 μL of purified antibody AXL-4# (2 μg / mL) was added, along with a positive control of anti-mouse IgG2a isotype (BioLegend) and human GAS6 antibody (R&D, AF885); incubation was performed at room temperature for 1 hour. After washing the ELISA plate, 100 μL of HRP-goat anti-mouse IgG (Zhongshan Jinqiao) diluted 1:10000 with blocking buffer was added to each well, and incubation was performed at room temperature for 1 hour. 100 μL of TMB (ThermoFisher) was added to each well, and color development was performed for 2-10 minutes. The reaction was terminated by adding 50 μL of 2M H2SO4 to each well, and the OD value at 450 nm was measured using an ELISA reader (BioTek).

[0183] like Figure 2 As shown, antibody AXL-4# does not interact with GAS6 in the absence of AXL.

[0184] 2. Flow cytometry detection of the interaction between AXL-4# antibody and GAS6

[0185] A suspension of 293T-hGAS6 cells (i.e., the 293T-memGAS6 cells obtained in “4. Preparation of cells expressing membrane-bound GAS6 (MemGAS6)” of Example 1) digested with PBS containing 2 mM EDTA was added to a 96-well U-shaped culture plate. 4Cells / 100 μl / well. Centrifuge at 2200 rpm for 3 minutes, discard the supernatant, and resuspend in the presence of 5 μg / mL secretory AXL (purified sAXL-Fc or sAXL-his). Add 5 μg / mL AXL-4# antibody, 5 μg / mL human GAS6 antibody, or anti-IgG2a isotype control antibody, and incubate at 4°C for 30 minutes. Wash cells twice with FACS buffer (PBS containing 2% FBS and 2 mM EDTA). Then incubate with 100 μL / well of PE-labeled goat anti-mouse IgG antibody (Biolegend, Poly4053, 405307) (1:500 dilution) at 4°C in the dark for 30 minutes. Wash cells twice with FACS buffer. After final washing, analyze GFP and PE signals in cells using flow cytometry (Thermo Analyzer).

[0186] like Figure 3 As shown, antibody AXL-4# can bind to GAS6 in the presence of secretory AXL-his, but cannot bind to cells expressing human GAS6 in the absence of secretory AXL-his. This indicates that antibody AXL-4# can only bind to GAS6 in the presence of AXL, that is, antibody AXL-4# only binds to the AXL-GAS6 complex.

[0187] Example 6: AXL-4# antibody binds to AXL domain 1

[0188] To further understand the binding site of the antibody AXL-4# to the AXL-GAS6 complex, 293T-hAXL cells expressing full-length human AXL, 293T-hAXL-domain 1 cells expressing AXL domain 1, and 293T cells expressing AXL domain 234 (293T-hAXL-domain 234) prepared in Example 1 were used. The binding of the AXL-4# antibody to these cells was evaluated by flow cytometry (see Example 1). Cells and antibody were incubated at 4°C in the dark for 30 minutes in the presence of 1 μg / mL human GAS6 or in the absence of GAS6. PE-labeled goat anti-mouse IgG antibody (Biolegend, Poly4053, 405307) (1:500 dilution) was added and incubated at 4°C in the dark for 30 minutes. Cells were washed twice with FACS buffer. After the final wash, analysis was performed using flow cytometry (Thermo Analyzer).

[0189] Figure 5 The AXL-4# antibody is shown to bind to the full-length AXL and domain 1 in the presence of human GAS6, indicating that the antibody binds to domain 1 of the AXL-GAS6 complex.

[0190] Example 7: Binding of AXL-4# antibody to human hAXL-human GAS6 complex or human hAXL-mouse GAS6 complex

[0191] Using 293T cells expressing full-length human AXL and full-length mouse AXL, the binding of the AXL-4# antibody to cells in the presence and absence of GAS6 was evaluated by flow cytometry. Antibody AXL-4#, antibody AXL-4#+hGAS6, and antibody AXL-4#+mGAS6 were added to either 293T-hAXL cell suspension or 293T-mAXL cell suspension, respectively, and the binding assay was performed as described above. The cells were incubated at 4°C for 30 minutes. Then, PE-labeled goat anti-mouse IgG antibody (BioLegend, Poly4053, 405307) (1:500 dilution) was added, and the cells were incubated at 4°C in the dark for 30 minutes. Cells were washed twice with FACS buffer. After the final wash, GFP and PE signals were analyzed using flow cytometry (Thermo Analyzer).

[0192] like Figure 6 A and Figure 6 As shown in B, antibody AXL-4# can bind to 293T-hAXL cells in the presence of human or mouse GAS6, but not to 293T-mAXL cells, indicating that antibody AXL-4# can bind to the human hAXL-human / mouse GAS6 complex, but not to the mouse AXL-human / mouse GAS6 complex.

[0193] Example 8: Binding of AXL-4# antibody to tumor cells

[0194] The binding of AXL-4# antibody to AXL in human breast cancer cells HS578T (purchased from ATCC), human renal cancer cells SN12C (purchased from Shanghai Yubo Biotechnology Co., Ltd.), human lung cancer cells Calu-1 (purchased from Shanghai Cell Bank of Chinese Academy of Sciences), and human breast cancer cells SK-BR-3 (purchased from Shanghai Cell Bank of Chinese Academy of Sciences) was determined by flow cytometry. All of these cells expressed GAS6 and AXL.

[0195] Single-cell suspensions of breast cancer cells HS578T, human renal cell carcinoma cells SN12C, human lung cancer cells Calu-1, and human breast cancer cells SK-BR-3 were obtained by digestion with PBS containing 2 mM EDTA. Cells were incubated with AXL-4# antibody at 4°C in the dark for 30 minutes. PE-labeled goat anti-mouse IgG antibody (BioLegend, Poly4053, 405307) (1:500 dilution) was added and incubated at 4°C in the dark for 30 minutes. Cells were washed twice with FACS buffer. After the final wash, the expression of AXL in the cells and the binding of AXL-4# antibody to the cells were analyzed using flow cytometry (Thermo Analyzer).

[0196] like Figure 7 As shown, the AXL-4# antibody can bind to cancer cells expressing AXL. Since these cancer cells also express GAS6, the antibody of the present invention can bind to the AXL-GAS6 complex and thus bind to the cancer cells; that is, the antibody of the present invention can bind to tumor cells that simultaneously express AXL and GAS6.

[0197] Example 9: Determination of AXL-4# antibody sequence

[0198] 1. RNA extraction

[0199] RNA was extracted from hybridoma cell line AXL-4# by lysing it with Trizol (Invitrogen). The specific steps for RNA extraction are as follows:

[0200] Add 200 μl of chloroform to each 1 ml of Trizol, vortex thoroughly, and let stand for 10 minutes; centrifuge at 13000 rpm / 4℃ / 15 minutes; add 400 μl of supernatant to 400 μl of pre-chilled isopropanol, mix well, and let stand overnight at -20℃; centrifuge at 13000 rpm / 4℃ / 15 minutes; remove the supernatant, add 70% ethanol; centrifuge at 13000 rpm / 4℃ / 10 ​​minutes; remove the supernatant, add 70% ethanol; remove the supernatant, add 40 μl of water to dissolve, and obtain the RNA solution.

[0201] 2. Obtaining cDNA

[0202] The RNA obtained in step 1 was reverse transcribed to obtain cDNA. The specific steps for reverse transcription are as follows: Take 16 μl of the RNA solution prepared in step 1, add 1 μl of 100 nM Oligo dT (Invitrogen); react at 70°C for 5 minutes; immediately place on ice; add 1 μl of RNase inhibitor (Takara), 1 μl of 10 mM dNTP (Takara), 1 μl of MLV reverse transcriptase and 5 μl of 5X buffer (Promega); react at 42°C for 60 minutes; treat at 80°C for 10 minutes.

[0203] 3. PCR amplification and sequencing

[0204] The subtype of antibody AXL-4# is mIgG2a (see Example 4). Based on the IgG2a sequence of Genbank accession number D78344.1, heavy chain primer R was designed; based on Genbank accession number LC522515.1, light chain primer R was designed.

[0205] Using the cDNA obtained in step 2 as a template, PCR amplification was performed using heavy chain primers F and R, and light chain primers F and R, respectively, to obtain fragments encoding the heavy and light chains, which were then sequenced. The primer sequences are as follows:

[0206] Light chain primer F: GAYATTGTGMTSACMCARWCTMCA (SEQ ID NO:25);

[0207] Light chain primer R: GGATACAGTTGGTGCAGCATC (SEQ ID NO:26);

[0208] Heavy chain primer F: SARGTNMAGCTGSAGSAGTC (SEQ ID NO:27);

[0209] Heavy chain primer R: CTTGACCAGGCATCCTAGAGTCA (SEQ ID NO:28);

[0210] Among them, R=a,g; Y=c,t; M=a,c; K=g,t; S=c,g; W=a,t; V=a,c,g; N=a,c,g,t.

[0211] The PCR reaction conditions were as follows: pre-denaturation, 98℃, 2 min; denaturation, 98℃, 30 s; annealing, 54℃; extension, 72℃, 1 min; for a total of 35 cycles, with a final extension of 10 min.

[0212] The sequencing results are as follows:

[0213] The nucleotide and amino acid sequences of the heavy chain variable region of the AXL monoclonal antibody AXL-4# are shown in... Figure 8 In the study, the nucleotide sequence of the heavy chain variable region of the AXL monoclonal antibody AXL-4# is SEQ ID NO:9, and the amino acid sequence of the heavy chain variable region of the AXL monoclonal antibody AXL-4# is SEQ ID NO:7. The obtained sequences were analyzed using https: / / www.ncbi.nlm.nih.gov / igblast. According to the IMGT numbering scheme, the amino acid sequence shown at positions 26-33 of the heavy chain variable region of the AXL monoclonal antibody AXL-4# (SEQ ID NO:1) is named AXL-4#HCDR1, the amino acid sequence shown at positions 51-58 of the heavy chain variable region of the AXL monoclonal antibody AXL-4# (SEQ ID NO:2) is named AXL-4#HCDR2, and the amino acid sequence shown at positions 97-111 of the heavy chain variable region of the AXL monoclonal antibody AXL-4# (SEQ ID NO:3) is named AXL-4#HCDR3.

[0214] The nucleotide and amino acid sequences of the light chain variable region of the AXL monoclonal antibody AXL-4# are shown in... Figure 9 In the study, the nucleotide sequence of the light chain variable region of the AXL monoclonal antibody AXL-4# is SEQ ID NO:10, and the amino acid sequence of the light chain variable region of the AXL monoclonal antibody AXL-4# is SEQ ID NO:8. The obtained sequences were analyzed using https: / / www.ncbi.nlm.nih.gov / igblast. According to the IMGT numbering scheme, the amino acid sequence shown at positions 27-38 of the light chain variable region of the AXL monoclonal antibody AXL-4# (SEQ ID NO:4) was named AXL-4#LCDR1, the amino acid sequence shown at positions 56-58 of the light chain variable region of the AXL monoclonal antibody AXL-4# (SEQ ID NO:5) was named AXL-4#LCDR2, and the amino acid sequence shown at positions 95-103 of the light chain variable region of the AXL monoclonal antibody AXL-4# (SEQ ID NO:6) was named AXL-4#LCDR3.

[0215] 4. Antibody sequence analysis

[0216] Analysis of the nucleic acid fragment of the AXL-GAS6 monoclonal antibody AXL-4# using the antibody sequence analysis tool igBlast tool (http: / / www.ncbi.nlm.nih.gov / igblast / ) revealed that the V, D, and J genes encoding the heavy chain of the AXL-GAS6 monoclonal antibody AXL-4# correspond to the mouse IGHV5-17, IGHD1-2, and IGHJ4 genes, respectively. The comparison results between the amino acid sequence of the variable region of the heavy chain of the AXL-GAS6 monoclonal antibody AXL-4# (SEQ ID NO:7) and the amino acid sequence of the mouse VDJ region are as follows: Figure 10 As shown. The V and J genes encoding the light chain of the AXL monoclonal antibody AXL-4# correspond to the mouse IGKV8-30 and IGKJ2 genes, respectively. The comparison results between the amino acid sequence of the variable region of the light chain of the AXL monoclonal antibody AXL-4# (SEQ ID NO:8) and the amino acid sequence of the mouse VJ region are shown below. Figure 11 As shown.

[0217] Example 10. Establishment of a method for detecting the AXL-GAS6 complex using AXL-4# antibody.

[0218] 1. Preparation of the AXL-GAS6 complex

[0219] The AXL-Gas6 complex was prepared according to publicly available information (PMID:20088931). 250 ng / ml Gas6 (HY-P70470, MCE) and 30 ng / ml AXL (DY154, R&D system) were mixed to a total volume of 20 μL and heated in a water bath at 37°C for 1 hour to obtain the AXL-Gas6 complex. The above standard mixture was then diluted to the working concentration with PBS (containing 0.1% BSA and 0.05% TWEEN20).

[0220] 2. Establishment of the sandwich ELISA method

[0221] In this embodiment, the capture sandwich ELISA method is used to detect the level of the AXL-GAS6 complex. The capture sandwich ELISA method is a conventional method in the prior art. In this embodiment, the AXL-4# antibody obtained by screening according to the present invention is used as the coating antibody for detection, as detailed below. 100 μL of AXL-4# antibody, initially at a concentration of 1 μg / mL and serially diluted 2-fold with PBS, is used to coat each well of the ELISA plate and incubated overnight at 4°C. The plate is washed twice with 200 μL of PBS. 200 μL of blocking buffer (PBS containing 0.05% Tween 20 and 0.1% BSA) is added, and the plate is incubated at room temperature for 1 hour, followed by 5 washes with PBST. The AXL-GAS6 complex (formed from AXL standards (DY154, R&D system) and GAS6 (HY-P70470, MCE)) is added as a control sample and incubated at room temperature for 2 hours. Wash the plate 5 times as above, add biotinylated goat anti-human AXL detection antibody (DY154, R&D system), and incubate at room temperature for 1 hour. Wash the plate 5 times as above, add 100 μL of HRP-streptavidin (DY154, R&D system), and incubate at room temperature for 1 hour. Wash the plate 5 times as above, add 100 μL of TMB to each well, and incubate for 15 min. Stop the reaction by adding 50 μL of 2M H2SO4 to each well, and measure the OD value at 450 nm using an ELISA reader (BioTek). Plot a standard curve based on the OD value and the concentration of the AXL-GAS6 complex (an exemplary standard curve can be found at [link to standard curve]). Figure 13 ).

[0222] When testing the concentration of the AXL-GAS6 complex in the sample, the OD value at 450 nm is measured according to the above ELISA method. Then, the OD value is input into the standard curve to obtain the corresponding concentration of the AXL-GAS6 complex.

[0223] Furthermore, to verify that the AXL-4# antibody of the present invention binds only to the AXL-Gas6 complex and not to free AXL or Gas6, i.e., that the above measurement method can accurately reflect the concentration of the AXL-Gas6 complex in the sample, the inventors conducted the following verification:

[0224] The results are as follows Figure 12A As shown, to verify at the ELISA level whether AXL-4# specifically binds to the AXL-Gas6 complex (i.e., not to free AXL or Gas6), the test sample contained only Gas6 (i.e., Figure 12AWhen the white transparent group ("AXL-0 μg / ml") was used, the AXL-4# antibody could not detect the concentration of GAS6, indicating that AXL-4# does not bind to Gas6. When a fixed concentration of AXL (1000 ng / ml) was added to the sample, the OD value increased linearly with the increase of Gas6 concentration (thus increasing the concentration of the AXL-Gas6 complex in the sample), indicating that AXL-4 can bind to the AXL-Gas6 complex at the ELISA level but not to the free form of GAS6.

[0225] In addition, such as Figure 12B As shown, when the sample to be tested contains only AXL (i.e., Figure 12B In the white transparent group (GAS6-0μg / ml, no Gas6 present), the AXL-4# antibody could not detect the concentration of AXL in the sample. This indicates that when AXL is 0 μg / ml, the OD value is extremely low, meaning that AXL-4# does not bind to AXL. When the sample contains a fixed concentration of GAS6 (0.25 μg / ml), Figure 12B (In the red group), as the concentration of AXL increases (i.e., the concentration of the AXL-Gas6 complex in the sample increases), the OD increases linearly, further demonstrating that at the ELISA level, AXL-4# can bind to the AXL-Gas6 complex but not to free AXL. Therefore, the above results indicate that the AXL-4# antibody of the present invention can be used to detect the level of the AXL-GAS6 complex.

[0226] Example 11. Detection of AXL-Gas6 complex levels in patients with myocardial infarction using AXL-4# antibody.

[0227] The AXL-Gas6 complex level in myocardial infarction patients was detected using the capture sandwich ELISA method established in Example 10 with the AXL-4# antibody, and the AXL-Gas6 complex level was detected by commercially available AXL antibody (R&D system) (which can bind not only AXL-Gas6 complex level but also free AXL).

[0228] Coat each well of the ELISA plate with 100 μL of 1 μg / mL AXL-4# antibody or a commercially available AXL capture antibody and incubate overnight at 4°C. Block with dilution buffer for 1 hour. Add 100 μL of plasma from healthy individuals (CAG), ST-segment elevation myocardial infarction patients (STEMI), or non-ST-segment elevation myocardial infarction patients (NSTEMI) and incubate at room temperature for 2 hours. After washing the ELISA plate, add biotin-labeled AXL detection antibody (i.e., commercially available AXL antibody) to each well and incubate at room temperature for 1 hour. After washing the ELISA plate, add 100 μL of HRP-streptavidin (DY154, R&D system) and incubate at room temperature for 1 hour. Add 100 μL of TMB to each well and allow for color development for 15 min. Stop the reaction by adding 50 μL of 2M H2SO4 to each well and measure the OD value at 450 nm using an ELISA reader (BioTek).

[0229] The results are as follows Figure 14 As shown, the AXL-Gas6 complex level was significantly higher in patients with ST-segment elevation myocardial infarction (STEMI) than in the healthy control group (CAG). However, using commercially available antibody testing (RD group), while AXL levels were higher in STEMI patients than in healthy controls, the difference was not statistically significant. No significant changes in sAXL-Gas6 were observed in patients with non-ST-segment elevation myocardial infarction, suggesting that the AXL-Gas6 complex is primarily involved in STEMI.

[0230] Example 12. Univariate and multivariate logistic regression analysis of risk factors for STEMI patients.

[0231] This invention further investigated 64 AMI patients (excluding those who underwent emergency or urgent PCI treatment at the Ninth Medical Center of the General Hospital of the Chinese People's Liberation Army (formerly the Strategic Support Force Characteristic Medical Center) from October 2014 to December 2016 (excluding those who underwent PCI after unsuccessful thrombolytic therapy, those with surgical contraindications, and those with incomplete clinical and laboratory data). This included 40 STEMI patients and 24 NSTEMI patients. A retrospective case-control study was constructed by selecting 24 patients with negative coronary angiography (stenosis <50%) who visited the hospital during the same period as the control group. Based on the collection of basic epidemiological information, medical history, echocardiography, laboratory indicators, and data on AXL-Gas6 complex and sAXL, the study explored the impact of myocardial infarction on basic information, medical history, echocardiography results, clinical indicators, and AXL-Gas6 complex and sAXL, using the occurrence of myocardial infarction as the independent variable. A logistic regression model was constructed using conventional methods. In the logistic regression model, univariate screening was first performed. The odds ratio (OR) was calculated for each variable and statistically tested. Variables with a p-value < 0.05 were included in the multivariate model. Using manual stepwise regression and the Akaike criterion, the likelihood ratio test (LRT) was employed to screen variables and determine the final model, thus obtaining the logistic regression model of factors influencing the incidence of myocardial infarction in patients.

[0232] The results of the logistic regression analysis are shown in Table 4. Univariate analysis showed that AXL-Gas6 complex, AXL, Gas6, hyperlipidemia, left ventricular ejection fraction (LVEF), white blood cell count, neutrophil percentage, blood glucose, ALT, AST, LDL, HDL, and BNP were associated with myocardial infarction (P<0.05), while other factors were not statistically significant (P>0.05). Further multivariate logistic regression analysis incorporating the aforementioned variables revealed a correlation between myocardial infarction and three factors: AXL-Gas6 complex levels, hyperlipidemia, and left ventricular ejection fraction (LVEF). Hyperlipidemia and AXL-Gas6 complex levels showed positive correlations, indicating risk factors, with odds ratios (ORs) of 157.045 (7.171–3439.520, P < 0.001) and 3.307 (1.209–9.051, P = 0.020), respectively. LVEF showed a negative correlation, indicating a protective factor, with an OR of 0.660 (0.508–0.856, P = 0.002). Therefore, this model demonstrates that AXL-Gas6 complex levels are positively correlated with ST-segment elevation myocardial infarction (STEMI) and are a risk factor for STEMI.

[0233] Table 4 Univariate and Multivariate Logistic Regression in Patients with Myocardial Infarction

[0234]

[0235]

[0236]

[0237] To validate the effectiveness of this logistic regression model, the receiver operating characteristic (ROC) curve method was used to calculate the area under the receiver operating characteristic (AUC) (see [link to relevant documentation]). Figure 15 For the model constructed above, the AUC value was 97.50% according to ROC curve analysis, which, according to the AUC discrimination criteria, indicates that the model has excellent efficacy. In addition, the model's sensitivity was 92.50% and its specificity was 91.30%, showing that the model has good reliability. That is, the model reliably verifies that the level of AXL-Gas6 complex can reflect the risk of ST-segment elevation myocardial infarction, and that detecting the level of AXL-Gas6 complex can assess the risk of ST-segment elevation myocardial infarction.

[0238] To further investigate the threshold of AXL-Gas6 complex levels in the diagnosis of ST-segment elevation myocardial infarction (STEMI), we employed ROC analysis and logistic regression modeling. Based on the mean AXL-Gas6 complex level of 1.7 in the STEMI group and at intervals of 0.1 ng / ml, we manually performed stepwise regression, binarized the AXL-Gas6 complex levels, and re-included them in the model analysis. We then used the Akaike criterion for model selection and evaluation. The final AXL-Gas6 complex level was defined as having an interval of 1.8 (>1.8 and ≤1.8 as grouping intervals), indicating that an AXL-Gas6 level greater than 1.8 ng / ml suggests an increased risk of myocardial infarction.

[0239] The inventors further verified the accuracy of this threshold in clinical diagnosis by detecting the AXL-Gas6 complex level in the aforementioned 64 patients using the ELISA method described above and grouping them according to the AXL-Gas6 complex level (i.e., >1.8 for STMEI myocardial infarction, ≤1.8 for NSTMEI myocardial infarction). The results were then compared with the electrocardiogram results, the gold standard for diagnosis in the prior art. The results showed that the diagnostic method of the present invention had a sensitivity of 90.00%, a specificity of 86.96%, and an AUC of 97.66%, indicating that the diagnostic method of the present invention can accurately and sensitively diagnose STMEI.

[0240] Example 13. Correlation analysis of the sAXL-Gas6 complex with other factors in STEMI patients.

[0241] Further analysis of the correlation between sAXL-Gas6 complex or free sAXL in STMEI patients and patient general information, medical history, echocardiography, and laboratory indicators revealed that the level of free sAXL was positively correlated with creatinine. Figure 16 A). Unlike free sAXL, the sAXL-Gas6 complex is positively correlated with currently used clinical diagnostic biochemical indicators for STMEI myocardial infarction, such as CMKB, white blood cell count (WBC), and platelet count (PLT), and negatively correlated with the time from first onset to medical attention (So-FMC) and high-density lipoprotein (HDL) (see [link]). Figure 16 B-16F) suggests that the sAXL-Gas6 complex has certain advantages over free sAXL in assessing disease severity.

[0242] In summary, this invention enables the specific detection of the AXL-Gas6 complex in plasma using antibody AXL-4#, and the AXL-Gas6 complex has certain significance in disease assessment. In the plasma of patients with ST-segment elevation myocardial infarction (STMEI), the elevation of the AXL-Gas6 complex is more sensitive and significant compared to free AXL detected by conventional methods. Elevated AXL-Gas6 complex is one of the important risk factors for ST-segment elevation myocardial infarction, and its elevation is significantly positively correlated with infarct size and decreased cardiac function, and negatively correlated with the protective HDL. It can be used to diagnose ST-segment elevation myocardial infarction or assess the risk of having ST-segment elevation myocardial infarction.

[0243] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0244] sequence list

[0245] HCDR1

[0246] SEQ ID NO:1:

[0247] GFTFSSFG

[0248] HCDR2

[0249] SEQ ID NO:2:

[0250] INSGSTLI

[0251] HCDR3

[0252] SEQ ID NO:3:

[0253] ARSPLLRLRGDAMDY

[0254] LCDR1

[0255] SEQ ID NO:4:

[0256] QSLLYSTNQKNY

[0257] LCDR2

[0258] SEQ ID NO:5:

[0259] WAS

[0260] LCDR3

[0261] SEQ ID NO:6:

[0262] QQYYRFPYT

[0263] SEQ ID NO:7:

[0264] The amino acid sequence of the heavy chain variable region

[0265] DVQLVESGGGLVQPGGSRKLSCAASGFTFSSFGMHWVRQPPEKGLEWVAYINSGSTLIYYADTVKGRFTISRDNPKNTLFLQLTSLRSEDTAMYYCARSPLLRLRGDAMDYWGQGTSVTVSS

[0266] SEQ ID NO:8

[0267] Amino acid sequence of the light chain variable region

[0268] DIVMSQSPSSLAVSVGEKVTMNCKSSQSLLYSTNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISTVEAEDLAVYYCQQYYRFPYTFGGGTKL

[0269] SEQ ID NO:9

[0270] Nucleotide sequence of the heavy chain variable region

[0271] gatgtgcagctggtggagtctgg

[0272] gggaggcttagtgcagcctggagggtcccggaaactctcctgtgcagcctctggattcactttcagtagctttggaatgcactgggttcgtcagcctccagagaaggggctggagtgggtcgcatacattaatagtggcagtactcttatctactatgcagacacagtgaagggccgattcaccatctccagagacaatcccaagaacaccctgttcctgcaattgaccagtctaaggtctgaggacacggccatgtattactgtgcaagatccccattactacggctacgaggcgatgctatggactactggggtcaaggaacctcagtcaccgtctcctca

[0273] SEQ ID NO:10

[0274] Nucleotide sequence of the light chain variable region

[0275] gacattgtgatgtcacagtc

[0276] tccatcctccctagctgtgtcagttggagagaaggttactatgaactgcaagtccagtcagagccttttatatagtaccaatcaaaagaactacttggcctggtaccagcagaaaccagggcagtctcctaaactgttgatttactgggcatccactagggaatctggggtccctgatcgcttcacaggcagtggatctgggacagatttcactctcaccatcagcactgtggaggctgaagacctggcagtttattactgtcagcaatattataggtttccgtacacattcggaggggggaccaagctg

[0277] SEQ ID NO:11

[0278] hAXL-F:5’-ccgaattcgccaccatggcgtggcggtgccccaggatgggc-3’

[0279] SEQ ID NO:12

[0280] hAXL-R:5’-ccagcagccccagggcaggaggatggtgcctgaggatccgcc-3’

[0281] SEQ ID NO:13

[0282] P1u-EcoRI:5’-gcgaattcGCCACCatggcgtggcggtgcccca-3’

[0283] SEQ ID NO:14

[0284] P2d6s,5’-CCCTGGGCGCCAcaagccctccagcccaacatagcc-3’

[0285] SEQ ID NO:15

[0286] P6u-5’-TGGCGCCCAGGGCAAGCAC-3’

[0287] SEQ ID NO:16

[0288] p6d-BamHI:5’-cgGGATCCTCAGGCACCATCCTCCTGCCCT-3’

[0289] SEQ ID NO:17

[0290] P1u:5’-gcGAATTCGCCACCatggcgtggcggtgcccca-3’

[0291] SEQ ID NO:18

[0292] P1d-3s:5’-CAGCCCAACATAGCCcgtgcccctgggggccatgc-3’(SEQ ID NO:18)

[0293] SEQ ID NO:19

[0294] P3u:5’-GGCTATGTTGGGCTGGAGGGCTTG-3’(SEQ ID NO:19)

[0295] SEQ ID NO:20

[0296] p6d-BamHI:5’-cgGGATCCTCAGGCACCATCCTCCTGCCCT-3’

[0297] SEQ ID NO:21

[0298] ATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGTGAC

[0299] SEQ ID NO:22

[0300] AATGCTGTGGGCCAGGACACGCAGGAGGTCATCGTGGTGCCACACTCCTTGCCCTTTAAGGTGGTGGTGATCTCAGCCATCCTGGCCCTGGTGGTGCTCACCATCATCTCCCTTATCATCCTCATCATGCTTTGGCAGAAGAAGCCACGTTAG

[0301] SEQ ID NO:23

[0302] GCGAATTCGCCACCATGGCGTGGCGGTGCCCCAGGATGGGCAGGGTC-3’

[0303] SEQ ID NO:24

[0304] CGGGATCCTTAGTGATGGTGGTGGTGATGTGGGCGCCAGGCCTCCAGG

[0305] SEQ ID NO:25

[0306] GAYATTGTGMTSACMCARWCTMCA

[0307] SEQ ID NO:26

[0308] GGATACAGTTGGTGCAGCATC

[0309] SEQ ID NO:27

[0310] SARGTNMAGCTGSAGSAGTC

[0311] SEQ ID NO:28

[0312] CTTGACCAGGCATCCTAGAGTCA。

Claims

1. A method for detecting the level of the AXL-GAS6 complex in a sample, including (a) Contacting the sample with anti-AXL-GAS6 antibody or its antigen-binding fragment; and (b) Detect the binding of the AXL-GAS6 antibody or its antigen-binding fragment to the AXL-GAS complex and determine the level of the AXL-GAS6 complex in the sample; The anti-AXL-GAS6 antibody comprises three complementarity-determining regions (HCDRs) in the heavy chain variable region and three complementarity-determining regions (LCDRs) in the light chain variable region, wherein: The amino acid sequence of HCDR1 is shown in SEQ ID NO:1; The amino acid sequence of HCDR2 is shown in SEQ ID NO:2; The amino acid sequence of HCDR3 is shown in SEQ ID NO:3; The amino acid sequence of LCDR1 is shown in SEQ ID NO:4; The amino acid sequence of LCDR2 is shown in SEQ ID NO:5; and The amino acid sequence of LCDR3 is shown in SEQ ID NO:

6.

2. The method of claim 1, wherein the detection of the binding level of the antibody or its antigen-binding fragment to the AXL-GAS complex in step (b) is performed by enzyme-linked immunosorbent assay (ELISA).

3. The method of claim 2, wherein the ELISA is a capture sandwich ELISA.

4. The method of claim 1, wherein the sample is plasma.

5. Use of anti-AXL-GAS6 antibody or its antigen-binding fragment in the preparation of medicines or kits for the diagnosis of myocardial infarction or the assessment of myocardial infarction risk.

6. The use according to claim 5, wherein the anti-AXL-GAS6 antibody comprises three complementarity-determining regions (HCDRs) of the heavy chain variable region and three complementarity-determining regions (LCDRs) of the light chain variable region, wherein: The amino acid sequence of HCDR1 is shown in SEQ ID NO:1; The amino acid sequence of HCDR2 is shown in SEQ ID NO:2; The amino acid sequence of HCDR3 is shown in SEQ ID NO:3; The amino acid sequence of LCDR1 is shown in SEQ ID NO:4; The amino acid sequence of LCDR2 is shown in SEQ ID NO:5; and The amino acid sequence of LCDR3 is shown in SEQ ID NO:

6.

7. The use according to claim 5, wherein the myocardial infarction is ST-segment elevation myocardial infarction.

8. The use according to claim 5, wherein, The kit also includes ELISA detection reagents.

9. The use according to claim 5, wherein, The kit also includes an instruction manual, wherein the instruction manual indicates: When the AXL-GAS complex level is >1.8 ng / ml, it indicates a high risk of myocardial infarction or myocardial infarction. When the AXL-GAS complex level is ≤1.8 ng / ml, it indicates no myocardial infarction or low risk of myocardial infarction. The myocardial infarction mentioned above is ST-segment elevation myocardial infarction.

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