A separated protein binding to antigen AXL and its use

Abstract

Provided is an isolated antigen-binding protein comprising at least one CDR from a VH having an amino acid sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 46, and at least one CDR from a VL having an amino acid sequence as set forth in SEQ ID NO: 2. Also provided are immunoconjugates comprising the isolated antigen-binding protein, nucleic acids encoding the isolated antigen-binding protein, vectors comprising the isolated antigen-binding protein, cells comprising the nucleic acid or the vector, methods for preparing the isolated antigen-binding protein, and uses of the isolated antigen-binding protein.

Description

Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to an isolated protein binding to the antigen AXL and its use. Background Art

[0002] AXL (receptor tyrosine kinase) is a member of the Tyro-3 family of kinases that can be activated by binding of the ligand Gas6 (a 70-kDa protein homologous to anticoagulant protein S). AXL activation leads to signal transduction through PI-3-kinase / Akt (Franke et al., Oncogene 22:8983-8998, 2003) and other major pathways such as Ras / Erk and β-catenin / TCF (Goruppi et al., Mol. Cell Biol. 21:902-915, 2001).

[0003] In tumor cells, AXL plays an important role in regulating cell invasion and migration. Overexpression of AXL is not only associated with poor prognosis, but also with increased invasion in various human cancers reported in breast, colon, esophageal, hepatocellular, gastric, glioma, lung, melanoma, osteosarcoma, ovarian, prostate, rhabdomyosarcoma, kidney, thyroid and endometrial cancers (Linger R M Adv. Cancer Res. 2008, 100, 35-83 and Verma A. Mol. Cancer Ther. (2011). 10, 1763-1773).

[0004] Given the therapeutic potential of AXL, it is necessary to generate antibodies that specifically bind to the AXL protein. Summary of the Invention

[0005] In one aspect, the present application provides an isolated antigen-binding protein comprising at least one CDR in VH represented by the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 46; and at least one CDR in VL represented by the amino acid sequence of SEQ ID NO: 2.

[0006] In certain embodiments, the isolated antigen-binding proteins described herein have one or more of the following properties:

[0007] 1) Can be 1×10 -7 M or lower K D Binds to AXL protein;

[0008] 2) Ability to specifically recognize AXL protein expressed on the cell surface;

[0009] 3) It can mediate internalization after binding to AXL protein expressed on the cell surface.

[0010] In certain embodiments, the AXL protein comprises a human AXL protein.

[0011] In certain embodiments, the human AXL protein comprises the amino acid sequence shown in SEQ ID NO:39.

[0012] In certain embodiments, the AXL protein comprises an extracellular domain.

[0013] In certain embodiments, the extracellular domain comprises the amino acid sequence shown in SEQ ID NO:40.

[0014] In certain embodiments, the cells comprise tumor cells.

[0015] In certain embodiments, the tumor comprises an AXL-positive tumor.

[0016] In certain embodiments, the tumor is selected from the group consisting of lung cancer, skin cancer, kidney cancer, pancreatic cancer, hematological tumors, breast cancer, ovarian cancer, lymphoma, and myeloma.

[0017] In certain embodiments, the tumor is selected from the group consisting of non-small cell lung cancer, squamous cell carcinoma of the skin, clear cell renal adenocarcinoma, pancreatic cancer, erythroleukemia, acute T-cell leukemia, breast cancer, ovarian cancer, lymphoma, and myeloma.

[0018] In certain embodiments, the cells comprise human cells.

[0019] In certain embodiments, the cells are selected from the group consisting of human non-small cell lung cancer A549 cells, human skin squamous cell carcinoma A431 cells, renal clear cell adenocarcinoma 786-O cells, human pancreatic cancer MIA PaCa-2 cells, erythroleukemia K562 cells, acute T-cell leukemia Jurkat cells, human breast cancer MCF-7 cells, human breast cancer MDA-MB-231 cells, human breast cancer MDA-MB-468 cells, human breast cancer SKBR3 cells, human ovarian cancer SKOV3 cells, lymphoma U-937 cells, lymphoma Raji cells, human myeloma U266 cells, and human multiple myeloma RPMI8226 cells.

[0020] In certain embodiments, the VH comprises HCDR1, HCDR2, and HCDR3.

[0021] In certain embodiments, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:25.

[0022] In certain embodiments, the HCDR2 comprises the amino acid sequence shown in any one of SEQ ID NOs: 26, 44, and 45.

[0023] In certain embodiments, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:27.

[0024] In certain embodiments, the VL comprises LCDR1, LCDR2, and LCDR3.

[0025] In certain embodiments, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:28.

[0026] In certain embodiments, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:29.

[0027] In certain embodiments, the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:30.

[0028] In certain embodiments, the VH comprises framework regions H-FR1, H-FR2, H-FR3, and H-FR4.

[0029] In certain embodiments, the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1, and the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:7.

[0030] In certain embodiments, the H-FR1 comprises the amino acid sequence shown in any one of SEQ ID NOs: 11 and 15.

[0031] In certain embodiments, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:8.

[0032] In certain embodiments, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:12.

[0033] In certain embodiments, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:9.

[0034] In certain embodiments, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:13.

[0035] In certain embodiments, the N-terminus of the H-FR4 is connected to the C-terminus of the HCDR3, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:10.

[0036] In certain embodiments, the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:14.

[0037] In certain embodiments, the VH comprises the amino acid sequence shown in any one of SEQ ID NOs: 3, 5, 42, and 43.

[0038] In certain embodiments, the VL comprises framework regions L-FR1, L-FR2, L-FR3, and L-FR4.

[0039] In certain embodiments, the C-terminus of the L-FR1 is directly or indirectly connected to the N-terminus of the LCDR1, and the L-FR1 comprises the amino acid sequence shown in SEQ ID NO:16.

[0040] In certain embodiments, the L-FR1 comprises the amino acid sequence shown in any one of SEQ ID NOs: 20 and 24.

[0041] In certain embodiments, the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 comprises the amino acid sequence shown in SEQ ID NO:17.

[0042] In certain embodiments, the L-FR2 comprises the amino acid sequence shown in SEQ ID NO:21.

[0043] In certain embodiments, the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 comprises the amino acid sequence shown in SEQ ID NO:18.

[0044] In certain embodiments, the L-FR3 comprises the amino acid sequence shown in SEQ ID NO:22.

[0045] In certain embodiments, the N-terminus of the L-FR4 is connected to the C-terminus of the LCDR3, and the L-FR4 comprises the amino acid sequence shown in SEQ ID NO:19.

[0046] In certain embodiments, the L-FR4 comprises the amino acid sequence shown in SEQ ID NO:23.

[0047] In certain embodiments, the VL comprises the amino acid sequence shown in any one of SEQ ID NOs: 4 and 6.

[0048] In certain embodiments, the isolated antigen-binding protein described herein comprises an antibody heavy chain constant region, and the antibody heavy chain constant region is derived from a human IgG heavy chain constant region.

[0049] In certain embodiments, the antibody heavy chain constant region is derived from a human IgG1 heavy chain constant region or a human IgG4 heavy chain constant region.

[0050] In certain embodiments, the antibody heavy chain constant region comprises the amino acid sequence shown in any one of SEQ ID NOs: 33 and 41.

[0051] In certain embodiments, the isolated antigen-binding protein described herein comprises an antibody light chain constant region, and the antibody light chain constant region comprises a human Igκ constant region.

[0052] In certain embodiments, the antibody light chain constant region comprises the amino acid sequence shown in SEQ ID NO:34.

[0053] In certain embodiments, the isolated antigen-binding protein described herein comprises an antibody heavy chain, and the antibody heavy chain comprises the amino acid sequence shown in any one of SEQ ID NOs: 35 and 37.

[0054] In certain embodiments, the isolated antigen-binding protein described herein comprises an antibody light chain, and the antibody light chain comprises the amino acid sequence shown in any one of SEQ ID NOs: 36 and 38.

[0055] In certain embodiments, the isolated antigen-binding protein described herein comprises an antibody or an antigen-binding fragment thereof.

[0056] In certain embodiments, the antibody is selected from the group consisting of a monoclonal antibody, a single-chain antibody, a chimeric antibody, a multispecific antibody, a humanized antibody, and a fully human antibody.

[0057] In certain embodiments, the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab)2, Fv, F(ab')2, scFv, di-scFv, and dAb fragments.

[0058] In another aspect, the present application provides an immunoconjugate comprising the isolated antigen-binding protein described herein.

[0059] In certain embodiments, the immunoconjugate further comprises at least one other agent selected from the group consisting of a chemotherapeutic agent, a radioactive element, a cytostatic agent, and a cytotoxic agent.

[0060] In certain embodiments, the isolated antigen binding protein is linked to the other agent via a linker molecule.

[0061] In certain embodiments, the isolated antigen binding protein and the other agent are each covalently linked to the linker molecule.

[0062] In certain embodiments, the additional agent comprises maytansine or a derivative thereof.

[0063] In certain embodiments, the maytansine derivative includes the maytansine derivative DM1.

[0064] In another aspect, the present application provides isolated one or more nucleic acid molecules encoding the isolated antigen binding protein described herein.

[0065] In another aspect, the present application provides a vector comprising the nucleic acid molecule described herein.

[0066] In another aspect, the present application provides a cell comprising the nucleic acid molecule described herein or the vector described herein.

[0067] On the other hand, the present application provides a pharmaceutical composition comprising the isolated antigen-binding protein, the immunoconjugate, the nucleic acid molecule, the vector and / or the cell described herein, and optionally a pharmaceutically acceptable adjuvant.

[0068] In another aspect, the present application provides a method for preparing the isolated antigen-binding protein described herein, the method comprising culturing the cell under conditions allowing the isolated antigen-binding protein to be expressed.

[0069] On the other hand, the present application provides the use of the isolated antigen-binding protein, the immunoconjugate, the nucleic acid molecule, the vector, the cell and / or the pharmaceutical composition in the preparation of a drug for preventing, alleviating and / or treating tumors.

[0070] In certain embodiments, the tumor comprises an AXL-positive tumor.

[0071] In certain embodiments, the tumor is selected from the group consisting of lung cancer, skin cancer, kidney cancer, pancreatic cancer, hematological tumors, breast cancer, ovarian cancer, lymphoma, and myeloma.

[0072] In certain embodiments, the tumor is selected from the group consisting of non-small cell lung cancer, squamous cell carcinoma of the skin, clear cell renal adenocarcinoma, pancreatic cancer, erythroleukemia, acute T-cell leukemia, breast cancer, ovarian cancer, lymphoma, and myeloma.

[0073] In another aspect, the present application provides use of the isolated antigen-binding protein in preparing a diagnostic agent for diagnosing a disease or condition associated with the expression of an AXL protein.

[0074] In another aspect, the present application provides a method for diagnosing a disease or condition associated with AXL protein expression in a subject, the method comprising: contacting a sample derived from the subject with the isolated antigen-binding protein, and determining the presence and / or amount of a substance capable of specifically binding to the isolated antigen-binding protein in the sample.

[0075] In another aspect, the present application provides a method for detecting AXL in a sample, the method comprising administering the isolated antigen-binding protein.

[0076] Those skilled in the art can easily discern other aspects and advantages of the present application from the detailed description below. In the detailed description below, only exemplary embodiments of the present application are shown and described. As will be appreciated by those skilled in the art, the content of this application enables those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application relates. Accordingly, the descriptions in the drawings and specification of this application are merely exemplary and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] The specific features of the invention involved in this application are shown in the appended claims. The features and advantages of the invention involved in this application can be better understood by referring to the exemplary embodiments described in detail below and the accompanying drawings. A brief description of the drawings is as follows:

[0078] Figure 1 Demonstrate the ability of the isolated antigen-binding proteins described herein to bind to antigen.

[0079] Figure 2 The binding of the antigen binding protein 6G12M11 described in the present application to different target antigens is shown.

[0080] Figure 3 The binding of the antigen binding protein 6G12M21 described in the present application to different target antigens is shown.

[0081] Figure 4 The binding of the antigen binding protein 6G12M31 described in the present application to different target antigens is shown.

[0082] Figure 5 The binding of the antigen binding protein 6G12M41 described in the present application to different target antigens is shown.

[0083] Figure 6 The graph shows the binding of the isolated antigen-binding proteins 6G12M11 and 6G12M21 described in the present application to antigens on the surface of A549 cells.

[0084] Figure 7The graph shows the binding of the isolated antigen-binding proteins 6G12M11 and 6G12M21 described in the present application to antigens on the surface of MDA-MB-231 cells.

[0085] Figure 8 The graph shows the binding of the isolated antigen-binding proteins 6G12M11 and 6G12M21 described in the present application to antigens on the surface of 786-O cells.

[0086] Figure 9 The graph shows the binding of the isolated antigen-binding proteins 6G12M21, 6G12M31, and 6G12M41 described in the present application to antigens on the surface of MDA-MB-231 cells.

[0087] Figure 10 The graph shows the binding of the isolated antigen-binding proteins 6G12M21, 6G12M31, and 6G12M41 described in the present application to antigens on the surface of 786-O cells.

[0088] Figure 11 The internalization efficiency of the isolated antigen-binding proteins 6G12M11 and 6G12M21 described herein on A549 cells is shown.

[0089] Figure 12 The internalization efficiency of the isolated antigen-binding proteins 6G12M11 and 6G12M21 described herein on MDA-MB-231 cells is shown.

[0090] Figure 13 The internalization efficiency of the isolated antigen-binding proteins 6G12M11 and 6G12M21 described herein on 786-O cells is shown.

[0091] Figure 14 The internalization efficiency of the isolated antigen-binding proteins 6G12M21, 6G12M31, and 6G12M41 described herein on MDA-MB-231 cells is shown.

[0092] Figure 15 The internalization efficiency of the isolated antigen-binding proteins 6G12M21, 6G12M31, and 6G12M41 described herein on 786-O cells is shown.

[0093] Figure 16 Inhibition curves of MDA-MB-231 by the immunoconjugates described herein are shown.

[0094] Figure 17 A graph showing the trend of tumor (human breast cancer MDA-MB-231) volume changes after administration of the immunoconjugate described in the present application.

[0095] Figure 18A graph showing the trend of changes in mouse body weight after administration of the immunoconjugate described in the present application. DETAILED DESCRIPTION

[0096] The following describes the implementation of the present invention through specific embodiments. People familiar with this technology can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0097] The present application is further described below. Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are those commonly used in the respective fields. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0098] In this application, the term "isolated" generally refers to a substance obtained artificially from its natural state. If a substance or component is "isolated" in nature, it may be that its natural environment has been altered, or that the substance has been separated from its natural environment, or both. For example, a polynucleotide or polypeptide that is naturally present in a living animal and has not been separated is considered isolated. The term "isolated" does not exclude the presence of artificial or synthetic substances, nor does it exclude the presence of other impure substances that do not affect the activity of the substance.

[0099] In this application, the term "isolated antigen-binding protein" generally refers to a protein with antigen-binding ability obtained from a natural state by artificial means. The "isolated antigen-binding protein" may comprise a portion that binds to an antigen and, optionally, a scaffold or framework portion that allows the antigen-binding portion to adopt a conformation that promotes the binding of the antigen-binding portion to the antigen. The antigen-binding protein may comprise, for example, an antibody-derived protein scaffold or an alternative protein scaffold or an artificial scaffold having transplanted CDRs or CDR derivatives. Such scaffolds include, but are not limited to, antibody-derived scaffolds comprising mutations introduced, for example, to stabilize the three-dimensional structure of the antigen-binding protein and fully synthetic scaffolds comprising, for example, biocompatible polymers. See, for example, Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, 53(1): 121-129 (2003); Roque et al., Biotechnol. Prog. 20: 639-654 (2004). Additionally, peptide antibody mimetics ("PAMs") as well as antibody mimetic-based scaffolds utilizing fibronectin components can be used as scaffolds.

[0100] In this application, the term "K D ”(Similarly, “K D ” or “KD”) usually refers to the “affinity constant” or “equilibrium dissociation constant” and refers to the dissociation rate constant (k d ) divided by the binding rate constant (k a ) was obtained. The binding rate constant (k a ), dissociation rate constant (k d ) and the equilibrium dissociation constant (K D ) represents the binding affinity of a binding protein (e.g., an isolated antigen binding protein as described herein) for an antigen (e.g., an AXL protein). Methods for determining association and dissociation rate constants are well known in the art. The use of fluorescence-based techniques provides high sensitivity and the ability to examine samples at equilibrium in physiological buffers. For example, the K can be determined by Octet D The K value can also be determined using other experimental approaches and instruments such as BIAcore (biomolecular interaction analysis) (for example, an instrument available from BIAcore International AB, a GE Healthcare company, Uppsala, Sweden). In addition, the K value can also be determined using KinExA (Kinetic Exclusion Assay) available from Sap idyne Instruments (Boise, Idaho). D The K value can be measured by using a surface plasmon resonance (SPR) D value.

[0101] In this application, the term "EC50" or "EC 50 ", also known as half-maximal effect concentration, usually refers to the antibody concentration that causes 50% of the maximum effect.

[0102] In this application, the term "AXL protein" generally refers to the protein receptor tyrosine kinase encoded by the axl gene. AXL (Ark, UFO, Tyro-7) is a member of the Tyro-3 family of kinases and can be activated by binding to the ligand Gas6 (a 70-kDa protein homologous to anticoagulant protein S). In cells of some cancers (e.g., lung cancer, kidney cancer, or breast cancer), there may be overexpression of AXL protein. Human AXL protein is an 894-amino acid protein, the amino acid sequence of which can be shown in SEQ ID NO: 39; wherein amino acid residues 1-26 are a signal peptide; and positions 27-451 are the extracellular domain of the AXL protein (the amino acid sequence of which is shown in SEQ ID NO: 40).

[0103] In this application, the term "extracellular domain" generally refers to a polypeptide or protein domain located outside a cell. For example, the extracellular domain may be the extracellular domain of the AXL protein, whose amino acid sequence may be as shown in SEQ ID NO:40. The extracellular domain of the AXL protein may have a structure similar to that required for a cell adhesion molecule. The extracellular domain of the AXL protein may be a combination of two immunoglobulin-like domains, capable of binding to the Gas6 ligand (Sasaki T et al., EMBO J. (2006). 25, 80-87).

[0104] In this application, the term "specific binding" or "specific" generally refers to a measurable and reproducible interaction, such as binding between a target and an antibody, which can be determined in the presence of a heterogeneous population of molecules (including biomolecules). For example, an antibody that specifically binds to a target (which can be an epitope) is one that binds to that target with greater affinity, avidity, more readily, and / or for a greater duration than it binds to other targets. In one embodiment, the extent to which the antibody binds to an unrelated target is less than about 10% of the binding of the antibody to the target, as measured, for example, by radioimmunoassay (RIA). For example, in this application, the isolated antigen binding protein is capable of binding to an unrelated target at a rate of <1x10 -7 The antibody binds to the AXL protein with a dissociation constant (KD) of M or less. In certain embodiments, the antibody specifically binds to an epitope on the protein that is conserved among proteins of different species. In another embodiment, specific binding may include but does not require exclusive binding.

[0105] In this application, the term "internalization" generally refers to the process by which an antibody or its antigen-binding fragment or polypeptide specifically binds to a receptor on the cell surface, forming a receptor-antibody complex, and then enters the cell via endocytosis mediated by the receptor. At this time, such an antibody or its antigen-binding fragment (such as a Fab fragment) can become an internalizing antibody. The internalizing antibody can be used as a carrier for the targeted delivery of drugs, enzymes, or DNA. In certain cases, the internalization can inhibit the proliferation of tumor cells. For example, the internalizing antibody can be used to couple anti-tumor chemotherapeutics, radioactive elements, cell growth inhibitors, and cytotoxic agents, and as a candidate molecule for tumor biotherapy.

[0106] In this application, the term "tumor" generally refers to a neoplasm or solid lesion formed by abnormal cell growth. In this application, a tumor can be a solid tumor or a hematological tumor. For example, in this application, the tumor can be an AXL-positive tumor, wherein the AXL-positive tumor can be selected from the group consisting of lung cancer, skin cancer, kidney cancer, pancreatic cancer, hematological tumors, breast cancer, ovarian cancer, lymphoma, and myeloma. In certain embodiments, the AXL-positive tumor can be selected from the group consisting of non-small cell lung cancer, squamous cell carcinoma of the skin, clear cell renal adenocarcinoma, pancreatic cancer, erythroleukemia, acute T-cell leukemia, breast cancer, ovarian cancer, lymphoma, and myeloma.

[0107] In this application, the term "variable domain" generally refers to the amino terminal domain of an antibody heavy or light chain. The variable domains of the heavy and light chains may be referred to as "VH" and "VL", respectively (or "V H ” and “V L These domains are generally the most variable parts of an antibody (relative to other antibodies of the same type) and contain the antigen-binding site.

[0108] In this application, the term "variable" generally refers to the fact that certain segments of the variable domains differ greatly in sequence between antibodies. The V domain mediates antigen binding and determines the specificity of a particular antibody for its specific antigen. However, variability is not evenly distributed throughout the variable domain. Instead, it is concentrated in three segments called hypervariable regions (CDRs or HVRs) in the light and heavy chain variable domains. The more highly conserved parts of the variable domains are called framework regions (FRs). The variable domains of natural heavy and light chains each contain four FR regions, most of which adopt a β-sheet configuration and are connected by three CDRs, which form a loop connection and, in some cases, form part of the β-sheet structure. The CDRs in each chain are held together in close proximity by the FR region, and the CDRs from the other chain together contribute to the formation of the antigen binding site of the antibody (see Kabat et al, Sequences of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). The constant domains are not directly involved in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity.

[0109] In this application, the term "antibody" generally refers to an immunoglobulin or its fragment or derivative thereof, encompassing any polypeptide comprising an antigen binding site, whether produced in vitro or in vivo. The term includes, but is not limited to, polyclonal, monoclonal, monospecific, multispecific, nonspecific, humanized, single-chain, chimeric, synthetic, recombinant, hybridized, mutated and transplanted antibodies. Unless otherwise modified by the term "complete", as in "complete antibody", for the purposes of the present invention, the term "antibody" also includes antibody fragments, such as Fab, F(ab')2, Fv, scFv, Fd, dAb and other antibody fragments that retain antigen binding function (e.g., specific binding to AXL). Typically, such fragments should include an antigen binding domain. The basic 4-chain antibody unit is a heterotetrameric glycoprotein consisting of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies are composed of five basic heterotetrameric units and an additional polypeptide called the J chain, containing 10 antigen-binding sites. IgA antibodies, on the other hand, consist of two to five basic four-chain units that can combine with the J chain to form multivalent combinations. For IgG, the four-chain unit is generally approximately 150,000 daltons. Each L chain is linked to an H chain by a covalent disulfide bond, while the two H chains are interconnected by one or more disulfide bonds that depend on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has a variable domain (VH) at its N-terminus, followed by three constant domains (CH) for each of the α and γ chains and four CH domains for the μ and ε isotypes. Each L chain has a variable domain (VL) at its N-terminus and a constant domain at its other end. The VL corresponds to the VH, and the CL corresponds to the first constant domain (CH1) of the heavy chain. Specific amino acid residues are believed to form the interface between the light and heavy chain variable domains. VH and VL pair together to form a single antigen binding site. For the structure and properties of different classes of antibodies, see, for example, Basic and Clinical Immunology, 8th Edition, Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw (eds), Appleton & Lange, Norwalk, Conn., 1994, page 71 and Chapter 6. The L chain from any vertebrate species can be divided into one of two distinct types, referred to as κ and λ, based on the amino acid sequence of its constant domain. Depending on the amino acid sequence of its heavy chain (CH) constant domain, immunoglobulins can be divided into different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with heavy chains designated α, δ, ε, γ, and μ, respectively.The gamma and alpha classes are further divided into subclasses based on relatively small differences in CH sequence and function, eg, humans express the following subclasses: IgGl, IgG2A, IgG2B, IgG3, IgG4, IgAl, and IgKl.

[0110] In this application, the term "CDR" generally refers to the region of the antibody variable domain, whose sequence is highly variable and / or forms a structural definition loop. Typically, an antibody includes six CDRs; three in VH (HCDR1, HCDR2, HCDR3), and three in VL (LCDR1, LCDR2, LCDR3). In natural antibodies, HCDR3 and LCDR3 show most of the diversity of the six CDRs, and HCDR3 in particular is considered to play a unique role in conferring fine specificity on antibodies. See, for example, Xu et al, Immunity 13:37-45 (2000); Johnson and Wu, in Methods in Molecular Biology 248:1-25 (Lo, ed., Human Press, Totowa, NJ, 2003). In fact, naturally occurring camel antibodies consisting only of heavy chains function normally and stably in the absence of light chains. See, eg, Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al, Nature Struct. Biol. 3:733-736 (1996).

[0111] In the present application, the term "FR" generally refers to the more highly conserved part of the antibody variable domain, which is referred to as the framework region. Typically, the variable domains of native heavy and light chains each include four FR regions, i.e., four (H-FR1, H-FR2, H-FR3, and H-FR4) in VH, and four (L-FR1, L-FR2, L-FR3, and L-FR4) in VL. For example, the VL of the antigen-binding proteins of the separation described herein can include framework regions L-FR1, L-FR2, L-FR3, and L-FR4. The VH of the antigen-binding proteins of the separation described herein can include framework regions H-FR1, H-FR2, H-FR3, and H-FR4.

[0112] As used herein, the term "antigen-binding fragment" generally refers to one or more fragments that specifically bind to an antigen (e.g., an AXL protein). As used herein, the antigen-binding fragment may include Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, and / or dAb.

[0113] In this application, the term "monoclonal antibody" or "monoclonal antibody" or "monoclonal antibody composition" generally refers to an antibody molecule preparation of a single molecular composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a specific epitope.

[0114] In this application, the term "single-chain antibody" generally refers to a molecule comprising an antibody heavy chain variable region and a light chain variable region. For example, the single-chain antibody can be formed by connecting the antibody heavy chain variable region and the light chain variable region through a linker (e.g., a connecting peptide).

[0115] In this application, the term "human antibody" generally refers to antibodies whose variable region framework and CDR regions are derived from human germline immunoglobulin sequences. In addition, if the antibody contains a constant region, it is also derived from human germline immunoglobulin sequences. The human antibodies of the present application may contain amino acid residues that are not encoded by human germline immunoglobulin sequences, such as mutations introduced by random mutations or point mutations in vitro or by somatic mutations in vivo. However, the term "human antibody" does not include antibodies in which CDR sequences derived from other mammalian species are inserted into human framework sequences.

[0116] In this application, the term "murine antibody" generally refers to antibodies whose variable region framework and CDR regions are derived from mouse germline immunoglobulin sequences. In addition, if the antibody contains a constant region, it is also derived from mouse germline immunoglobulin sequences. The murine antibody of the present application may contain amino acid residues not encoded by mouse germline immunoglobulin sequences, such as mutations introduced by in vitro random mutations or point mutations or by in vivo somatic mutations. However, the term "murine antibody" does not include antibodies in which CDR sequences derived from other mammalian species are inserted into the mouse framework sequence.

[0117] In this application, the term "chimeric antibody" generally refers to an antibody that is derived from a combination of non-human genetic material and human genetic material. Or more generally, a chimeric antibody refers to an antibody that combines genetic material from one species with genetic material from another species.

[0118] In this application, the term "multispecific antibody" generally refers to an antibody molecule that can simultaneously recognize two or more antigens or epitopes. The multispecific antibody can be obtained in a eukaryotic expression system or a prokaryotic expression system by chemical coupling, hybridization-hybridoma method, genetic engineering antibody preparation method, etc.

[0119] In this application, the term "humanized antibody" generally refers to an antibody derived from a non-human species but whose protein sequence has been modified to increase its similarity to naturally occurring antibodies in humans.

[0120] In this application, the term "fully human antibody" generally refers to a fully human antibody, i.e., an antibody whose constant and variable regions are both derived from humans. Such fully human antibodies can be achieved through phage antibody library technology, transgenic mouse production of humanized antibodies, ribosome display technology, EBV-transformed B cell cloning technology, single B cell cloning, and other technologies.

[0121] In this application, the terms "antibody that recognizes an antigen" and "antibody specific for an antigen" are used interchangeably herein with the term "antibody that specifically binds to an antigen."

[0122] In the present application, the term "directly connected" is contrasted with the term "indirectly connected", and the term "directly connected" generally refers to direct connection. For example, the direct connection can be a situation where the substances are directly connected without a spacer. The spacer can be a linker. For example, the linker can be a peptide linker. The term "indirectly connected" generally refers to a situation where the substances are not directly connected. For example, the indirect connection can be a situation where the substances are connected through a spacer. For example, in the isolated antigen-binding protein described in the present application, the C-terminus of the L-FR1 and the N-terminus of the LCDR1 can be directly or indirectly connected.

[0123] In the present application, the term "immunoconjugate" generally refers to a conjugate formed by conjugating the other agents (e.g., chemotherapeutic agents, radioactive elements, cell growth inhibitors, and cytotoxic agents) to the separated antigen-binding proteins (e.g., covalently linked by a linker molecule), which can be delivered to target cells (e.g., tumor cells) by the specific binding of the separated antigen-binding proteins to the antigens on the target cells. The immunoconjugate is then internalized and eventually enters the interior of the target cell (e.g., into vesicles such as lysosomes), whereupon the linker molecule in the immunoconjugate can be cracked, releasing the other agents to exert their cytotoxic effects. In addition, the antigen can also be secreted by the target cell and located in the gap outside the target cell.

[0124] In the present application, the term "chemotherapeutic agent" generally refers to a chemotherapeutic agent that can inhibit the proliferation of tumors and / or tumor cells. The chemotherapeutic agent can be selected from the following groups: mitotic inhibitors, kinase inhibitors, alkylating agents, antimetabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, histone deacetylase inhibitors, antisurvival agents, biological response modifiers, anti-hormones such as anti-androgens and anti-angiogenic agents. For example, the chemotherapeutic agent can be selected from the following groups: capecitabine, daunorubicin, daunorubicin, actinomycin D, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytarabine, bischloroethyl nitrosourea, busulfan, mitomycin C, actinomycin D, plicamycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil, methylcyclopentyl Hexylnitrosourea, nitrogen mustard, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine (CA), 5-azacytidine, hydroxyurea, deoxycoformycin, 4-hydroxyperoxycyclophosphamide, 5-fluorouracil (5-FU), 5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol, vincristine, vinblastine, etoposide, trimetrexate, teniposide, and / or diethylstilbestrol (DES).

[0125] In this application, the term "radioactive element" generally refers to an element used in radiotherapy that can inhibit the proliferation of tumors and / or tumor cells. The radioactive element can be selected from the following group: 3 H. 14 C. 15 N. 35 S. 90 Y. 99 Tc, 111 In, 125 I and / or 131 I.

[0126] In this application, the term "cytostatic" generally refers to an agent that inhibits tumors by inhibiting growth factors that promote the growth and replication of tumor cells. Growth factors activate intracellular signaling pathways after binding to receptors on the cell surface. Complex pathways may cause cells to grow out of control, resulting in excessive cell division and development into tumors. The cytostatic can inhibit the effects of these growth factors. The cytostatic can be selected from the following group: angiogenesis inhibitors, deacetylase (HDAC) inhibitors, Hedgehog signaling pathway blockers, mTOR inhibitors, p53 / mdm2 inhibitors, PARP inhibitors, proteasome inhibitors and / or tyrosine kinase inhibitors.

[0127] In the present application, the term "cytotoxic agent" generally refers to an agent that inhibits the proliferation of tumors and / or tumor cells by producing toxins to the cells on which it acts. The cytotoxic agent can be selected from the following groups: alkylating agents, such as busulfan, hexamethylmelamine, thiotepa, cyclophosphamide, mechlorethamine, uracil, melphalan, chlorambucil, carmustine, streptozotocin, dacarbazine, temozolomide, ifosfamide, etc.; antitumor agents, such as mitomycin C, etc.; antimetabolites, such as methotrexate, azathioprine, mercaptopurine, fludarabine, 5-fluorouracil, etc.; platinum-containing anticancer agents, such as cisplatin, carboplatin, etc.; anthracyclines, such as daunorubicin, doxorubicin, epirubicin, etc. Rubicin, idarubicin, mitoxantrone, etc.; plant alkaloids and terpenoids, such as vincristine, vinblastine, vinorelbine, vindesine, podophyllotoxin, docetaxel, etc.; topoisomerase inhibitors, such as irinotecan, amsacrine, topotecan, etoposide, teniposide, etc.; antibodies, such as rituximab, trastuzumab, bevacizumab, erlotinib, dactinomycin, etc.; finasteride; aromatase inhibitors; tamoxifen; goserelin; paclitaxel and / or imatinib mesylate. The cytotoxic agent can be administered orally, by injection, etc.

[0128] In this application, the term "linker molecule" generally refers to a functional molecule that connects or links two molecules. For example, the linker molecule can connect one molecule and another molecule (for example, one molecule is a protein molecule, the other molecule is also a protein molecule, or it can be a small molecule drug). The linker molecule can be used in the construction of the immunoconjugate. In the immunoconjugate, the linker molecule can have two functional characteristics: 1. It has circulatory system stability and cannot be cleaved in the circulatory system to release the other reagents before the immunoconjugate reaches the target cell, thereby avoiding toxic effects; 2. After entering the target cell, the linker molecule needs to be broken quickly and effectively so that the other reagents are effectively released to exert their due pharmacological activity. The linker molecule can be composed of polar or non-polar amino acids. The linker molecule can also be a carbon chain containing heteroatoms (such as nitrogen atoms, sulfur atoms, etc.). The length of the linker can be 2 to 100 atoms, for example, between 2 and 50 atoms, or 3, 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 atoms; for example, the length of the linker can be 20 to 26 (20, 21, 22, 23, 24, 25 or 26) atoms. The linker can include a substituent selected from the following groups: hydrogen, alkyl, alkenyl, alkynyl, amino, alkylamino, dialkylamino, trialkylamino, hydroxyl, alkoxy, halogen, aryl, heterocycle, aromatic heterocycle, cyano, amide, carbamoyl, carboxylic acid, ester, thioether, alkylthioether, sulfhydryl and urea. In addition, the linker can be selected from the following groups: pH sensitive linker, protease cleavable linker, nuclease sensitive linker, lipase sensitive linker, glycosidase linker, hypoxia linker, photocleavage linker, heat labile linker and ultrasound sensitive linker.

[0129] In this application, the term "covalent" generally refers to a covalent bond, that is, two or more atoms share a pair of electrons, reaching a state of electron saturation to form a relatively stable chemical structure. The formation of a covalent bond is that electrons with opposite spin directions between two adjacent atoms pair with each other. At this time, the atomic orbitals overlap with each other, and the electron cloud density between the two nuclei increases relatively, thereby increasing the attraction to the two nuclei. Covalent bonds can have saturation and directionality. Covalent bonds can be divided into non-polar covalent bonds, polar covalent bonds and coordinate bonds. Compounds containing only covalent bonds can be called covalent compounds.

[0130] In this application, the term "maytansine" generally refers to a compound isolated from the genus Maytansine (see U.S. Patent No. 3,896,111), which is an anti-mitotic cytotoxin with the structural formula:

[0131]

[0132] The CAS number of the maytansine is 35846-53-8. Maytansine has a significant therapeutic effect on various tumors, such as L-1210, P-388 leukemia, S-180, W-256, Lewis lung cancer and nasopharyngeal carcinoma in vitro. The maytansine derivatives may include compounds having a ring structure of maytansine and having one or more substituents on the ring, such as maytansine derivatives DM1 and DM4.

[0133] In this application, the term "maytansine derivative DM1" generally refers to a compound having the following structural formula:

[0134]

[0135] The CAS number is 139504-50-0. The maytansine derivative DM1 can be an anti-mitotic cytotoxin.

[0136] In this application, the term "disease or condition associated with expression of AXL protein" generally refers to a disease or condition associated with expression of AXL protein, or a disease or condition that may result from upregulated expression of AXL protein. For example, a disease or condition associated with expression of AXL protein may include lung cancer, skin cancer, kidney cancer, pancreatic cancer, hematological tumors, breast cancer, ovarian cancer, lymphoma, and / or myeloma.

[0137] As used herein, the term "isolated nucleic acid molecule" generally refers to isolated forms of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof, separated from their natural environment or artificially synthesized.

[0138] In this application, the term "vector" generally refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a protein can be inserted and the protein expressed. A vector can be used to transform, transduce, or transfect host cells, allowing the genetic material elements it carries to be expressed in host cells. For example, vectors include: plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages such as lambda phage or M13 phage, and animal viruses. Types of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, a vector may also contain a replication initiation site. Vectors may also include components that assist in their entry into cells, such as viral particles, liposomes, or protein coats, but are not limited to these substances.

[0139] In this application, the term "cell" generally refers to a single cell, cell line or cell culture that may be or has been a recipient of a subject's plasmid or vector, including nucleic acid molecules of the present invention or vectors of the present invention. Cells may include the offspring of a single cell. Due to natural, accidental or intentional mutations, offspring may not necessarily be identical to the original parent cell (in the form of the total DNA complement or in the genome). Cells may include cells transfected in vitro with the vectors described herein. The cell can be a bacterial cell (e.g., Escherichia coli), a yeast cell, or other eukaryotic cell, such as a COS cell, a Chinese hamster ovary (CHO) cell, a CHO-K1 cell, a LNCAP cell, a HeLa cell, a HEK293 cell, a COS-1 cell, a NSO cell, a human non-small cell lung cancer A549 cell, a human skin squamous cell carcinoma A431 cell, a renal clear cell adenocarcinoma 786-0 cell, a human pancreatic cancer MIA PaCa-2 cell, an erythroleukemia K562 cell, an acute T-cell leukemia Jurkat cell, a human breast cancer MCF-7 cell, a human breast cancer MDA-MB-231 cell, a human breast cancer MDA-MB-468 cell, a human breast cancer SKBR3 cell, a human ovarian cancer SKOV3 cell, a lymphoma U-937 cell, a lymphoma Raji cell, a human myeloma U266 cell, or a human multiple myeloma RPMI8226 cell. In certain embodiments, the cell is a mammalian cell. In certain embodiments, the mammalian cell is a HEK293 cell.

[0140] In this application, the term "pharmaceutical composition" generally refers to a composition suitable for application to a patient, preferably a human patient. For example, the pharmaceutical composition described herein may include the antigen-binding proteins of the separation described herein, the immunoconjugates described herein, the nucleic acid molecules described herein, the carrier described herein and / or the cell described herein, and optionally a pharmaceutically acceptable adjuvant. In addition, the pharmaceutical composition may also include a suitable formulation of one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and / or preservatives. The acceptable ingredients of the composition are preferably nontoxic to the recipient at the dosage and concentration used. The pharmaceutical composition of the present invention includes but is not limited to liquid, frozen and lyophilized compositions.

[0141] As used herein, the term "pharmaceutically acceptable adjuvant" generally refers to any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like that are compatible with pharmaceutical administration and are generally safe, non-toxic, and neither biologically nor otherwise undesirable.

[0142] In this application, the term "subject" generally refers to a human or non-human animal, including but not limited to a cat, dog, horse, pig, cow, sheep, rabbit, mouse, rat, or monkey.

[0143] In this application, the term "comprising" generally means including the features specifically stated, but not excluding other elements.

[0144] In this application, the term "about" generally refers to a variation within a range of 0.5%-10% above or below the specified value, for example, a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the specified value.

[0145] Isolated antigen binding protein

[0146] On the one hand, the present application provides an isolated antigen-binding protein comprising at least one CDR in VH with an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 46; and at least one CDR in VL with an amino acid sequence as shown in SEQ ID NO: 2.

[0147] QVQL X1QSGPGLVKPSQSLSLTC X2V X3G X4SISSGYYWNWIRQ X5PG X6X7LEWMGYRSYDGSNNYNPSLKNRISITRDTSKNQFFLKLNSVT X8EDTATYYCARGWLLHYTMDYWGQGT X9VTVSS (SEQ ID NO: 1), wherein X1 can be K or V, X2 can be S or A, X3 can be T or S, X4 can be F, Y, D or S, X5 can be F or S, X6 can be N or Q, X7 can be K or G, X8 can be T or S, and X9 can be S or T.

[0148] QVQLVQSGPGLVKPSQSLSLTCSVTGFSISSGYYWNWIRQFPGQKLEWMGYRSYDGSNNYX1PSLKNRISITRDTSKNQFFLKLNSVTSEDTATYYCARGWLLHYTMDYWGQGTTVTVSS (SEQ ID NO: 46), where X1 can be D or G.

[0149] X1X2VMTQSP X3S X4X5VTLG X6SASISCRSSRSLLHSNGFTYLYWYX7QKPGQSPQLLIYQMSNLASGVPDRFS 10 GTKLEX 11K (SEQ ID NO: 2), wherein X1 can be E or D, X2 can be L or I, X3 can be F or S, X4 can be N or V, X5 can be A or S, X6 can be T or Q, X7 can be L or Q, X8 can be S or G, X9 can be R or K, X 10 Can be A or G, X 11 Can be L or I.

[0150] The VH of the isolated antigen-binding protein described herein can be obtained by amino acid mutation at one or more positions in the 6G12 antibody VH (SEQ ID NO: 31). Specifically, the isolated antigen-binding protein described herein can be obtained by amino acid mutation at one or more positions in the framework region (FR) of the 6G12 antibody VH, or by amino acid mutation at one or more positions in the variable region (CDR) of the 6G12 antibody VH. For example, in the 6G12 antibody VH, from the N-terminus to the C-terminus, K at position 5 can be mutated to V, S at position 23 can be mutated to A, T at position 25 can be mutated to S, F at position 27 can be mutated to Y, D or S, F at position 41 can be mutated to S, N at position 44 can be mutated to Q, K at position 45 can be mutated to G, T at position 88 can be mutated to S, and S at position 114 can be mutated to T.

[0151] For example, in the 6G12 antibody VH, from N-terminus to C-terminus, K at position 5 can be mutated to V, S at position 23 can be mutated to A, T at position 25 can be mutated to S, F at position 27 can be mutated to Y, D or S, F at position 41 can be mutated to S, N at position 44 can be mutated to Q, K at position 45 can be mutated to G, N at position 61 can be mutated to D or G, T at position 88 can be mutated to S, and S at position 114 can be mutated to T.

[0152] The VL of the isolated antigen-binding protein described in the present application can be obtained by amino acid mutation at one or more positions in the 6G12 antibody VL (SEQ ID NO: 32). Specifically, the isolated antigen-binding protein described in the present application can be obtained by amino acid mutation at one or more positions in the framework region (FR) of the 6G12 antibody VL.

[0153] For example, in the VL of the 6G12 antibody, from the N-terminus to the C-terminus, the E at position 1 can be mutated to D, the L at position 2 can be mutated to I, the F at position 9 can be mutated to S, the N at position 11 can be mutated to V, the A at position 12 can be mutated to S, the T at position 17 can be mutated to Q, the L at position 42 can be mutated to Q, the S at position 69 can be mutated to G, the R at position 79 can be mutated to K, the A at position 105 can be mutated to G, and the L at position 111 can be mutated to I.

[0154] Properties of the isolated antigen-binding protein

[0155] In the present application, the isolated antigen-binding protein may have one or more of the following properties:

[0156] 1) Can be 1×10 -7 M or lower KD binds to AXL protein;

[0157] 2) Ability to specifically recognize AXL protein expressed on the cell surface;

[0158] 3) It can mediate internalization after binding to AXL protein expressed on the cell surface.

[0159] In the present application, the isolated antigen binding protein can be expressed as 1×10 -7 M or lower K D Binds to AXL protein. For example, the isolated antigen binding protein described in the present application binds to the K of AXL protein derived from human. D The value can be ≤1×10 - 7 M, ≤9×10 -8 M, ≤8×10 -8 M, ≤7×10 -8 M, ≤6×10 -8 M, ≤5×10 -8 M, ≤4×10 -8 M, ≤3×10 -8 M, ≤2×10 -8 M, ≤1.5×10 -8 M, ≤1.2×10 -8 M, ≤1.15×10 -8 M, ≤1.1×10 -8 M, ≤1.05×10 -8 M, ≤1×10 -8 M, ≤5×10 -9 M or ≤1×10 -9 M. For another example, the isolated antigen binding protein described in the present application binds to the K of the AXL protein derived from mouse D The value can be ≤1×10 -7M, ≤9×10 -8 M, ≤8×10 -8 M, ≤7×10 -8 M, ≤6×10 -8 M, ≤5×10 -8 M, ≤4×10 -8 M, ≤3×10 -8 M, ≤2×10 -8 M, ≤1.5×10 -8 M, ≤1.2×10 -8 M, ≤1.15×10 -8 M, ≤1.1×10 -8 M, ≤1.05×10 -8 M, ≤1×10 -8 M, ≤5×10 -9 M or ≤1×10 -9 M. For another example, the isolated antigen binding protein described in the present application binds to the K of the AXL protein derived from monkeys. D The value can be ≤1×10 -7 M, ≤9×10 -8 M, ≤8×10 - 8 M, ≤7×10 -8 M, ≤6×10 -8 M, ≤5×10 -8 M, ≤4×10 -8 M, ≤3×10 -8 M, ≤2×10 -8 M, ≤1.5×10 - 8 M, ≤1.2×10 -8 M, ≤1.15×10 -8 M, ≤1.1×10 -8 M, ≤1.05×10 -8 M, ≤1×10 -8 M, ≤5×10 -9 M or ≤1×10 -9 M.

[0160] In this application, the K D The value can also be determined by ELISA, competitive ELISA or BIACORE or KINEXA.

[0161] In the present application, the competitive binding ability can be measured by measuring the dissociation equilibrium constant of the antibody-antigen interaction of the isolated antigen-binding protein. The method for detecting the dissociation equilibrium constant can be selected from the following group: enzyme-linked immunosorbent assay, surface plasmon resonance (SRP) method, potentiometric titration, spectrophotometry, capillary electrophoresis, fluorescence method and thin-layer chromatography pH method. For example, the isolated antigen-binding protein can be detected by the SRP method (e.g., using a biomacromolecule interaction instrument).

[0162] In the present application, the isolated antigen binding protein is capable of specifically binding to the AXL protein expressed on the cell surface. The specific binding can be determined by FACS. For example, the EC50 in the FACS assay can be used to reflect the specific binding of the isolated antigen binding protein described in the present application to the AXL protein on the cell surface. For example, the lower the EC50, the better the specific binding. For example, the EC50 value of the isolated antigen binding protein binding to the AXL protein on the surface of non-small cell lung cancer A549 cells in the FACS assay can be 0.01μg / ml~0.10μg / ml, 0.01μg / ml~0.15μg / ml, 0.01μg / ml~0.20μg / ml, 0.01μg / ml~0.25μg / ml, 0.01μg / ml~0.30μg / ml, 0.01μg / ml~0.35μg / ml, 0.0 1μg / ml~0.40μg / ml, 0.01μg / ml~0.45μg / ml, 0.01μg / ml~0.50μg / ml, 0.01μg / ml~0.55μg / ml, 0.01μg / ml~0 .60μg / ml, 0.01μg / ml~0.65μg / ml, 0.01μg / ml~0.70μg / ml, 0.01μg / ml~0.75μg / ml or 0.01μg / ml~0.80μg / ml. For another example, the EC50 value of the isolated antigen binding protein binding to AXL protein on the surface of human breast cancer MDA-MB-231 cells in FACS assay can be 0.01 μg / ml to 0.10 μg / ml, 0.01 μg / ml to 0.15 μg / ml, 0.01 μg / ml to 0.20 μg / ml, 0.01 μg / ml to 0.25 μg / ml, 0.01 μg / ml to 0.30 μg / ml, 0.01 μg / ml to 0.35 μg / ml, 0. .01μg / ml~0.40μg / ml, 0.01μg / ml~0.45μg / ml, 0.01μg / ml~0.50μg / ml, 0.01μg / ml~0.55μg / ml, 0.01μg / ml~ 0.60μg / ml, 0.01μg / ml~0.65μg / ml, 0.01μg / ml~0.70μg / ml, 0.01μg / ml~0.75μg / ml or 0.01μg / ml~0.80μg / ml.For another example, the EC50 value of the isolated antigen-binding protein binding to AXL protein on the surface of renal clear cell adenocarcinoma 786-O cells in a FACS assay can be 0.01 μg / ml to 0.10 μg / ml, 0.01 μg / ml to 0.15 μg / ml, 0.01 μg / ml to 0.20 μg / ml, 0.01 μg / ml to 0.25 μg / ml, 0.01 μg / ml to 0.30 μg / ml, 0.01 μg / ml to 0.35 μg / ml, 0. 01μg / ml~0.40μg / ml, 0.01μg / ml~0.45μg / ml, 0.01μg / ml~0.50μg / ml, 0.01μg / ml~0.55μg / ml, 0.01μg / ml~0 .60μg / ml, 0.01μg / ml~0.65μg / ml, 0.01μg / ml~0.70μg / ml, 0.01μg / ml~0.75μg / ml or 0.01μg / ml~0.80μg / ml.

[0163] In the present application, the isolated antigen-binding protein can mediate internalization after binding to AXL protein expressed on the cell surface. For example, the internalization can include the following steps: when the isolated antigen-binding protein can bind to the plasma membrane of a cell (e.g., a tumor cell), or can be released intracellularly in response to proteolytic activity in the cell microenvironment (e.g., the tumor cell microenvironment). Thus, the isolated antigen-binding protein can be engulfed by the cell membrane and absorbed into the cell. In certain embodiments, the isolated antigen-binding protein in the immunoconjugate, and / or the other agent conjugated thereto, can also be engulfed by the cell membrane and absorbed into the cell after the isolated antigen-binding protein binds to the cell plasma membrane.

[0164] In the present application, the AXL protein may be human AXL protein (NP_068713) or cynomolgus monkey AXL protein (Genbank accession number HB387229.1). For example, the AXL protein may be human AXL protein, the amino acid sequence of which is shown in SEQ ID NO:39.

[0165] The AXL protein may include variants of the AXL protein. For example, the variant may be: 1) a protein or polypeptide in which one or more amino acids are substituted, deleted, or added in the amino acid sequence of the AXL protein; and 2) a protein or polypeptide having at least about 85% (e.g., at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence identity with the AXL protein.

[0166] The AXL protein may also include a fragment of the AXL protein. For example, the AXL protein may be the extracellular domain of the human AXL protein, the amino acid sequence of which is shown in SEQ ID NO:40.

[0167] In the present application, the cell may include a tumor cell. For example, the tumor may be an AXL-positive tumor. For example, the tumor may be selected from the group consisting of lung cancer, skin cancer, kidney cancer, pancreatic cancer, hematological tumors, breast cancer, ovarian cancer, lymphoma, and myeloma. In another example, the tumor may be selected from the group consisting of non-small cell lung cancer, squamous cell carcinoma of the skin, clear cell renal adenocarcinoma, pancreatic cancer, erythroleukemia, acute T-cell leukemia, breast cancer, ovarian cancer, lymphoma, and myeloma.

[0168] In the present application, the cells may include human cells. For example, the cells may include cells selected from the group consisting of human non-small cell lung cancer A549 cells, human skin squamous cell carcinoma A431 cells, renal clear cell adenocarcinoma 786-O cells, human pancreatic cancer MIA PaCa-2 cells, erythroleukemia K562 cells, acute T-cell leukemia Jurkat cells, human breast cancer MCF-7 cells, human breast cancer MDA-MB-231 cells, human breast cancer MDA-MB-468 cells, human breast cancer SKBR3 cells, human ovarian cancer SKOV3 cells, lymphoma U-937 cells, lymphoma Raji cells, human myeloma U266 cells, and human multiple myeloma RPMI8226 cells.

[0169] Type of the isolated antigen-binding protein

[0170] In the present application, the isolated antigen-binding protein may include an antibody or an antigen-binding fragment thereof. For example, the isolated antigen-binding protein described herein may include, but is not limited to, a recombinant antibody, a monoclonal antibody, a human antibody, a murine antibody, a humanized antibody, a chimeric antibody, a bispecific antibody, a single-chain antibody, a diabody, a triabody, a tetrabody, an Fv fragment, a scFv fragment, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, and a camelized single-domain antibody.

[0171] In the present application, the antibody may be a humanized antibody. In other words, the isolated antigen-binding protein described herein may be an antibody or a variant, derivative, analog, or fragment thereof that immunospecifically binds to a relevant antigen (e.g., human AXL) and comprises a framework (FR) region having substantially the amino acid sequence of a human antibody and a complementarity determining region (CDR) having substantially the amino acid sequence of a non-human antibody. Here, "substantially" in the context of a CDR means that the amino acid sequence of the CDR is at least 80%, preferably at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of the CDR of a non-human antibody. The humanized antibody may comprise substantially all of at least one and typically two variable domains (Fab, Fab', F(ab')2, FabC, Fv), wherein all or substantially all of the CDR regions correspond to the CDR regions of a non-human immunoglobulin (i.e., an antibody) and all or substantially all of the framework regions are framework regions having a consensus sequence of a human immunoglobulin. Preferably, the humanized antibody further comprises at least a portion of an immunoglobulin constant region (e.g., Fc), typically a constant region of a human immunoglobulin. In some embodiments, the humanized antibody contains at least the variable domains of a light chain and a heavy chain. The antibody may also include the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. In some embodiments, the humanized antibody contains only a humanized light chain. In some embodiments, the humanized antibody contains only a humanized heavy chain. In specific embodiments, the humanized antibody contains only a humanized variable domain of a light chain and / or a humanized heavy chain.

[0172] In the present application, the antigen-binding fragment may include Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv and / or dAb.

[0173] CDR

[0174] The CDR of an antibody, also known as the complementarity determining region, is part of the variable region. The amino acid residues in this region contact the antigen or antigenic epitope. Antibody CDRs can be identified using various coding systems, such as CCG, Kabat, Chothia, IMGT, and Kabat / Chothia combined. These coding systems are known in the art; for details, see http: / / www.bioinf.org.uk / abs / index.html#kabatnum. Those skilled in the art can identify CDR regions using different coding systems based on the sequence and structure of the antibody. CDR regions may differ using different coding systems. The CDRs of the isolated antigen-binding proteins described herein can be identified using Kabat.

[0175] In the present application, the VH of the isolated antigen-binding protein may comprise HCDR1, HCDR2 and HCDR3.

[0176] In the present application, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 25.

[0177] In the present application, the HCDR2 may comprise the amino acid sequences shown in SEQ ID NOs: 26, 44 and 45.

[0178] In the present application, the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 27.

[0179] For example, the HCDR1 of the isolated antigen-binding protein described herein may comprise the amino acid sequence shown in SEQ ID NO: 25, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 26, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 27.

[0180] For example, the HCDR1 of the isolated antigen-binding protein described herein may comprise the amino acid sequence shown in SEQ ID NO: 25, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 44, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 27.

[0181] The HCDR1 of the isolated antigen-binding protein described herein may comprise the amino acid sequence shown in SEQ ID NO: 25, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 45, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 27.

[0182] In the present application, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 28.

[0183] In the present application, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 29.

[0184] In the present application, the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 30.

[0185] For example, the LCDR1 of the isolated antigen-binding protein described herein may comprise the amino acid sequence shown in SEQ ID NO: 28, LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 29, and LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 30.

[0186] For another example, the HCDR1 of the isolated antigen-binding protein described herein may comprise the amino acid sequence shown in SEQ ID NO: 25, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 26, the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 27, and the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 28, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 29, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 30.

[0187] For another example, the HCDR1 of the isolated antigen-binding protein described herein may comprise the amino acid sequence shown in SEQ ID NO: 25, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 44, the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 27, and the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 28, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 29, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 30.

[0188] For another example, the HCDR1 of the isolated antigen-binding protein described herein may comprise the amino acid sequence shown in SEQ ID NO: 25, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 45, the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 27, and the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 28, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 29, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 30.

[0189] FR

[0190] In the present application, the VH of the isolated antigen-binding protein may comprise framework regions H-FR1, H-FR2, H-FR3, and H-FR4.

[0191] In the present application, the C-terminus of the H-FR1 may be directly or indirectly connected to the N-terminus of the HCDR1, and the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:7.

[0192] QVQL X1QSGPGLVKPSQSLSLTC X2V X3G X4SIS (SEQ ID NO: 7), wherein X1 can be K or V, X2 can be S or A, X3 can be T or S, and X4 can be F, Y, D or S.

[0193] For example, in the present application, the H-FR1 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 11 and 15.

[0194] In the present application, the H-FR2 may be located between the HCDR1 and the HCDR2, and the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:8.

[0195] WIRQ X1PG X2X3LEWMG (SEQ ID NO: 8), wherein X1 can be F or S, X2 can be N or Q, and X3 can be K or G.

[0196] For example, in the present application, the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:12.

[0197] In the present application, the H-FR3 may be located between the HCDR2 and the HCDR3, and the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:9.

[0198] RISITRDTSKNQFFLKLNSVT X1EDTATYYCAR (SEQ ID NO: 9), wherein X1 can be T or S.

[0199] For example, in the present application, the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:13.

[0200] In the present application, the N-terminus of the H-FR4 may be connected to the C-terminus of the HCDR3, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:10.

[0201] WGQGT X1VTVSS (SEQ ID NO: 10), wherein X1 can be S or T.

[0202] For example, in the present application, the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:14.

[0203] For another example, H-FR1 of the isolated antigen-binding protein described herein may comprise the amino acid sequence shown in SEQ ID NO: 11, H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 12, H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 13, and H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 14.

[0204] For another example, H-FR1 of the isolated antigen-binding protein described in the present application may comprise the amino acid sequence shown in SEQ ID NO: 15, H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 12, H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 13, and H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 14.

[0205] In the present application, the VL of the isolated antigen-binding protein may comprise framework regions L-FR1, L-FR2, L-FR3, and L-FR4.

[0206] In the present application, the C-terminus of the L-FR1 may be directly or indirectly connected to the N-terminus of the LCDR1, and the L-FR1 may comprise the amino acid sequence shown in SEQ ID NO:16.

[0207] X1X2VMTQSP X3S X4X5VTLG X6SASISC (SEQ ID NO: 16), wherein X1 can be E or D, X2 can be L or I, X3 can be F or S, X4 can be N or V, X5 can be A or S, and X6 can be T or Q.

[0208] For example, in the present application, the L-FR1 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 20 and 24.

[0209] In the present application, the L-FR2 may be located between the LCDR1 and the LCDR2, and the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO:17.

[0210] WY X1QKPGQSPQLLIY (SEQ ID NO: 17), wherein X1 can be L or Q.

[0211] For example, in the present application, the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 21.

[0212] In the present application, the L-FR3 may be located between the LCDR2 and the LCDR3, and the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO:18.

[0213] GVPDRFS X1SGSGTDFTL X2ISRVEAEDVGVYYC (SEQ ID NO: 18), wherein X1 can be S or G, and X2 can be R or K.

[0214] For example, in the present application, the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 22.

[0215] In the present application, the N-terminus of the L-FR4 may be connected to the C-terminus of the LCDR3, and the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO:19.

[0216] FG X1GTKLE X2K (SEQ ID NO: 19), wherein X1 can be A or G, and X2 can be L or I.

[0217] For example, in the present application, the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 23.

[0218] For another example, L-FR1 of the isolated antigen-binding protein described herein may comprise the amino acid sequence shown in SEQ ID NO: 20, L-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 21, L-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 22, and L-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 23.

[0219] For another example, L-FR1 of the isolated antigen-binding protein described herein may comprise the amino acid sequence shown in SEQ ID NO: 24, L-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 21, L-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 22, and L-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 23.

[0220] For another example, H-FR1 of the isolated antigen-binding protein described herein may comprise the amino acid sequence shown in SEQ ID NO: 11, H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 12, H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 13, H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 14, and L-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 20, L-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 21, L-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 22, and L-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 23.

[0221] For another example, H-FR1 of the isolated antigen-binding protein described herein may comprise the amino acid sequence shown in SEQ ID NO: 15, H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 12, H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 13, H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 14, and L-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 24, L-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 21, L-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 22, and L-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 23.

[0222] VH and VL

[0223] The isolated antigen-binding protein described herein may comprise an antibody light chain variable region VH and an antibody heavy chain variable region VL.

[0224] In the present application, the VH may comprise the amino acid sequence shown in any one of SEQ ID NOs: 3, 5, 42 and 43.

[0225] In the present application, the VL may comprise the amino acid sequence shown in any one of SEQ ID NOs: 4 and 6.

[0226] For example, the VH may comprise the amino acid sequence shown in SEQ ID NO: 3, and the VL may comprise the amino acid sequence shown in SEQ ID NO: 4.

[0227] For another example, the VH may comprise the amino acid sequence shown in SEQ ID NO: 5, and the VL may comprise the amino acid sequence shown in SEQ ID NO: 6.

[0228] For another example, the VH may comprise the amino acid sequence shown in SEQ ID NO: 42, and the VL may comprise the amino acid sequence shown in SEQ ID NO: 6.

[0229] For another example, the VH may comprise the amino acid sequence shown in SEQ ID NO: 43, and the VL may comprise the amino acid sequence shown in SEQ ID NO: 6.

[0230] Constant regions, heavy and light chains

[0231] In the present application, the isolated antigen-binding protein may include an antibody heavy chain constant region, and the antibody heavy chain constant region may be derived from a human IgG heavy chain constant region.

[0232] In certain embodiments, the isolated antigen-binding protein may include an antibody heavy chain constant region, and the antibody heavy chain constant region may be derived from a human IgG1 heavy chain constant region. In other embodiments, the isolated antigen-binding protein may include an antibody heavy chain constant region, and the antibody heavy chain constant region may be derived from a human IgG4 heavy chain constant region.

[0233] For example, the antibody heavy chain constant region may comprise the amino acid sequence shown in any one of SEQ ID NOs: 33 and 41.

[0234] In the present application, the isolated antigen-binding protein may include an antibody light chain constant region, and the antibody light chain constant region may include a human Igκ constant region. For example, the antibody light chain constant region may include the amino acid sequence shown in SEQ ID NO: 34.

[0235] In the present application, the isolated antigen-binding protein may comprise an antibody heavy chain HC, and the HC may comprise the amino acid sequence shown in any one of SEQ ID NOs: 35 and 37.

[0236] In the present application, the isolated antigen-binding protein may comprise an antibody light chain LC, and the LC may comprise the amino acid sequence shown in any one of SEQ ID NOs: 36 and 38.

[0237] The isolated antigen-binding protein described herein may comprise an antibody heavy chain and an antibody light chain.

[0238] For example, the heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 35, and the light chain may comprise the amino acid sequence shown in SEQ ID NO: 36.

[0239] For example, the heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 37, and the light chain may comprise the amino acid sequence shown in SEQ ID NO: 38.

[0240] In the present application, the heavy chain of the isolated antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO: 35, and the light chain may comprise the amino acid sequence shown in SEQ ID NO: 36. The HCDR1 of the isolated antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO: 25, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 26, the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 27, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 28, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 29, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 30. In addition, the H-FR1 of the isolated antigen-binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 11, the H-FR2 may comprise the amino acid sequence set forth in SEQ ID NO: 12, the H-FR3 may comprise the amino acid sequence set forth in SEQ ID NO: 13, and the H-FR4 may comprise the amino acid sequence set forth in SEQ ID NO: 14. Furthermore, the L-FR1 may comprise the amino acid sequence set forth in SEQ ID NO: 20, the L-FR2 may comprise the amino acid sequence set forth in SEQ ID NO: 21, the L-FR3 may comprise the amino acid sequence set forth in SEQ ID NO: 22, and the L-FR4 may comprise the amino acid sequence set forth in SEQ ID NO: 23. Furthermore, the VH may comprise the amino acid sequence set forth in SEQ ID NO: 3, and the VL may comprise the amino acid sequence set forth in SEQ ID NO: 4. For example, the isolated antigen-binding protein may be the 6G12M11 antibody.

[0241] In the present application, the heavy chain of the isolated antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO: 37, and the light chain may comprise the amino acid sequence shown in SEQ ID NO: 38. The HCDR1 of the isolated antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO: 25, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 26, the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 27, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 28, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 29, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 30. In addition, the H-FR1 of the isolated antigen-binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 15, the H-FR2 may comprise the amino acid sequence set forth in SEQ ID NO: 12, the H-FR3 may comprise the amino acid sequence set forth in SEQ ID NO: 13, and the H-FR4 may comprise the amino acid sequence set forth in SEQ ID NO: 14. Furthermore, the L-FR1 may comprise the amino acid sequence set forth in SEQ ID NO: 24, the L-FR2 may comprise the amino acid sequence set forth in SEQ ID NO: 21, the L-FR3 may comprise the amino acid sequence set forth in SEQ ID NO: 22, and the L-FR4 may comprise the amino acid sequence set forth in SEQ ID NO: 23. Furthermore, the VH may comprise the amino acid sequence set forth in SEQ ID NO: 5, and the VL may comprise the amino acid sequence set forth in SEQ ID NO: 6. For example, the isolated antigen-binding protein may be the 6G12M21 antibody.

[0242] In the present application, the HCDR1 of the isolated antigen-binding protein may comprise the amino acid sequence of SEQ ID NO: 25, the HCDR2 may comprise the amino acid sequence of SEQ ID NO: 44, the HCDR3 may comprise the amino acid sequence of SEQ ID NO: 27, the LCDR1 may comprise the amino acid sequence of SEQ ID NO: 28, the LCDR2 may comprise the amino acid sequence of SEQ ID NO: 29, and the LCDR3 may comprise the amino acid sequence of SEQ ID NO: 30. In addition, the H-FR1 of the isolated antigen-binding protein may comprise the amino acid sequence of SEQ ID NO: 15, the H-FR2 may comprise the amino acid sequence of SEQ ID NO: 12, the H-FR3 may comprise the amino acid sequence of SEQ ID NO: 13, and the H-FR4 may comprise the amino acid sequence of SEQ ID NO: 14, and the L-FR1 may comprise the amino acid sequence of SEQ ID NO: 24, the L-FR2 may comprise the amino acid sequence of SEQ ID NO: 21, the L-FR3 may comprise the amino acid sequence of SEQ ID NO: 22, and the L-FR4 may comprise the amino acid sequence of SEQ ID NO: 23. Furthermore, the VH may comprise the amino acid sequence shown in SEQ ID NO: 42, and the VL may comprise the amino acid sequence shown in SEQ ID NO: 4. For example, the isolated antigen-binding protein may be the 6G12M31 antibody.

[0243] In the present application, the HCDR1 of the isolated antigen-binding protein may comprise the amino acid sequence of SEQ ID NO: 25, the HCDR2 may comprise the amino acid sequence of SEQ ID NO: 45, the HCDR3 may comprise the amino acid sequence of SEQ ID NO: 27, the LCDR1 may comprise the amino acid sequence of SEQ ID NO: 28, the LCDR2 may comprise the amino acid sequence of SEQ ID NO: 29, and the LCDR3 may comprise the amino acid sequence of SEQ ID NO: 30. In addition, the H-FR1 of the isolated antigen-binding protein may comprise the amino acid sequence of SEQ ID NO: 15, the H-FR2 may comprise the amino acid sequence of SEQ ID NO: 12, the H-FR3 may comprise the amino acid sequence of SEQ ID NO: 13, and the H-FR4 may comprise the amino acid sequence of SEQ ID NO: 14, and the L-FR1 may comprise the amino acid sequence of SEQ ID NO: 24, the L-FR2 may comprise the amino acid sequence of SEQ ID NO: 21, the L-FR3 may comprise the amino acid sequence of SEQ ID NO: 22, and the L-FR4 may comprise the amino acid sequence of SEQ ID NO: 23. Furthermore, the VH may comprise the amino acid sequence shown in SEQ ID NO: 43, and the VL may comprise the amino acid sequence shown in SEQ ID NO: 6. For example, the isolated antigen-binding protein may be the 6G12M41 antibody.

[0244] In addition, it should be noted that the isolated antigen-binding proteins described herein may include heavy and / or light chain sequences that have one or more conservative sequence modifications compared to the 6G12M11, 6G12M21, 6G12M31, and 6G12M41 antibodies. The so-called "conservative sequence modifications" refer to amino acid modifications that do not significantly affect or alter the binding properties of the antibody. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the isolated antigen-binding proteins described herein using standard techniques known in the art, such as point mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are the replacement of an amino acid residue with an amino acid residue having a similar side chain. Groups of amino acid residues having similar side chains are known in the art. These amino acid residue groups include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In certain embodiments, one or more amino acid residues in the CDR region of the isolated antigen-binding protein described herein can be replaced with other amino acid residues from the same side chain group. Those skilled in the art will appreciate that some conservative sequence modifications will not abolish antigen binding. For details, see, for example, Brummell et al., (1993) Biochem 32:1180-8; de Wildt et al., (1997) Prot. Eng. 10:835-41; Komissrov et al., (1997) J. Biol. Chem. 272:26864-26870; Hall et al., (1992) J. Immunol. 149:1605-12; Kelley and O'Connell (1993) Biochem. 32:6862-35; Adib-Conquy et al., (1998) Int. Immunol. 10:341-6 and Beers et al., (2000) Clin. Can. Res. 6:2835-43.

[0245] The proteins, polypeptides and / or amino acid sequences involved in this application should also be understood to include at least the following scope: variants or homologs that have the same or similar functions as the proteins or polypeptides.

[0246] In the present application, the variant may be a protein or polypeptide in which one or more amino acids have been substituted, deleted, or added in the amino acid sequence of the protein and / or polypeptide (e.g., the isolated antigen-binding protein described herein). For example, the variant may comprise a protein or polypeptide having an amino acid alteration through at least one, e.g., 1-30, 1-20, or 1-10, or further e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, and / or insertions. The functional variant may substantially retain the biological properties of the protein or polypeptide prior to the alteration (e.g., substitution, deletion, or addition). For example, the functional variant may retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (e.g., the ability to specifically bind to an AXL protein) of the protein or polypeptide prior to the alteration.

[0247] In the present application, the homolog can be a protein or polypeptide having at least about 80% (for example, at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence homology with the amino acid sequence of the protein and / or the polypeptide (for example, the antibody or antigen-binding fragment thereof described in the present application).

[0248] In the present application, described homology generally refers to the similarity between two or more sequences or the degree of association.Can calculate " sequence homology per-cent " in the following manner: two sequences to be compared are compared in comparison window, determine that there is identical nucleic acid base (for example, A, T, C, G, I) or identical amino acid residue (for example, Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) number in the position to obtain the number of matching positions, with the number of matching positions divided by the total number of positions (that is, window size) in the comparison window, and result is multiplied by 100, to produce sequence homology per-cent.Comparison carried out in order to determine the sequence homology per-cent, can realize by several ways known in the art, for example, use publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximum alignment over the full length of the sequences being compared or within a region of interest. Homology can also be determined by the following methods: FASTA and BLAST. A description of the FASTA algorithm can be found in W. R. Earson and D. J. Lipman, "Improved tools for biological sequence comparison," Proc. Natl. Acad. Sci., 85: 2444-2448, 1988; and D. J. Lipman and W. R. Earson, "Rapid and sensitive protein similarity search," Science, 227: 1435-1441, 1989. A description of the BLAST algorithm can be found in S. Altschul, W. Gish, W. Miller, E. W. Myers, and D. Lipman, "A basic local alignment search tool," J. Mol. Biol., 215: 403-410, 1990.

[0249] Immunoconjugates, nucleic acid molecules, vectors, cells and pharmaceutical compositions

[0250] Immunoconjugates

[0251] On the other hand, the present application provides an immunoconjugate comprising the isolated antigen-binding protein.

[0252] For example, the immunoconjugate may comprise at least one other agent selected from the group consisting of a chemotherapeutic agent, a radioactive element, a cytostatic agent, and a cytotoxic agent. In certain embodiments, the isolated antigen-binding protein and the at least one other agent in the immunoconjugate may be linked via a linker molecule. For example, the isolated antigen-binding protein and the at least one other agent in the immunoconjugate may be covalently linked to the linker molecule, respectively.

[0253] In the present application, the other agent may include maytansine or a derivative thereof. For example, the maytansine derivative may include maytansine derivative DM1.

[0254] Nucleic acid molecules

[0255] In another aspect, the present application also provides one or more isolated nucleic acid molecules that can encode the isolated antigen-binding proteins described herein. The one or more isolated nucleic acid molecules described herein can be nucleotides, deoxyribonucleotides or ribonucleotides, of any length in isolated form, or analogs isolated from their natural environment or synthesized artificially, but can encode the isolated antigen-binding proteins described herein.

[0256] carrier

[0257] On the other hand, the present application also provides vectors, which may contain the nucleic acid molecules described herein. The vectors can be transformed, transduced, or transfected into host cells, so that the genetic material elements they carry are expressed in the host cells. For example, vectors may include: plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages such as lambda phage or M13 phage; and animal viruses. Types of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). For another example, the vectors may contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vectors may also contain a replication initiation site. Furthermore, the vectors may also include components that facilitate entry into cells, such as viral particles, liposomes, or protein coats, but are not limited to these substances.

[0258] cell

[0259] On the other hand, the application also provides cells, which can comprise nucleic acid molecules described herein or vectors described herein. The cells can include the offspring of individual cells. Due to natural, accidental or intentional mutations, offspring may not necessarily be identical with the original parent cell (in the form of total DNA complement or on genome). In some embodiments, the cells can also include cells transfected in vitro with the vectors described herein. In certain embodiments, the cell can be a bacterial cell (e.g., Escherichia coli), a yeast cell, or other eukaryotic cell, such as a COS cell, a Chinese hamster ovary (CHO) cell, a CHO-K1 cell, a LNCAP cell, a HeLa cell, a HEK293 cell, a COS-1 cell, a NSO cell, a myeloma cell, a human non-small cell lung cancer A549 cell, a human skin squamous cell carcinoma A431 cell, a renal clear cell adenocarcinoma 786-0 cell, a human pancreatic cancer MIA PaCa-2 cell, an erythroleukemia K562 cell, an acute T-cell leukemia Jurkat cell, a human breast cancer MCF-7 cell, a human breast cancer MDA-MB-231 cell, a human breast cancer MDA-MB-468 cell, a human breast cancer SKBR3 cell, a human ovarian cancer SKOV3 cell, a lymphoma U-937 cell, a lymphoma Raji cell, a human myeloma U266 cell, or a human multiple myeloma RPMI8226 cell. In certain embodiments, the cell can be a mammalian cell. In certain embodiments, the mammalian cells may be HEK293 cells.

[0260] Pharmaceutical composition

[0261] On the other hand, the present application also provides a pharmaceutical composition, which may comprise the isolated antigen-binding protein described herein, the immunoconjugate described herein, the nucleic acid molecule described herein, the vector described herein and / or the cell described herein, and optionally a pharmaceutically acceptable adjuvant.

[0262] In certain embodiments, the pharmaceutical composition may also include one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and / or suitable formulations of preservatives. The acceptable ingredients of the composition are preferably non-toxic to the recipient at the dosage and concentration used. The pharmaceutical composition of the present invention includes but is not limited to liquid, frozen and lyophilized compositions.

[0263] In certain embodiments, the pharmaceutically acceptable adjuvant may include any and all solvents, dispersion media, coatings, isotonic agents, and absorption delaying agents that are compatible with pharmaceutical administration and are generally safe, non-toxic, and neither biologically nor otherwise undesirable.

[0264] In certain embodiments, the pharmaceutical composition can include parenteral, percutaneous, intracavitary, intra-arterial, intrathecal and / or intranasal administration or direct injection into tissue.For example, the pharmaceutical composition can be administered to a patient or subject by infusion or injection. In certain embodiments, the administration of the pharmaceutical composition can be carried out in different ways, such as intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration. In certain embodiments, the pharmaceutical composition can be administered uninterruptedly. The uninterrupted (or continuous) administration can be achieved by a small pump system worn by the patient to measure the therapeutic agent flowing into the patient's body, as described in WO2015 / 036583.

[0265] The dosage regimen of the pharmaceutical composition may be a rapid infusion, multiple divided doses may be administered over time, or the dose may be reduced or increased in proportion to the severity of the treatment situation. In certain embodiments, the treatment regimen may be administered once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every three months, or once every three to six months. In certain embodiments, the dosage regimen comprises intravenous administration of 1 mg / kg body weight or 3 mg / kg body weight, with the antibody administered at one of the following dosing schedules: (i) six doses every four weeks, then once every three months; (ii) once every three weeks; (iii) 3 mg / kg body weight once, then 1 mg / kg body weight once every three weeks. In certain embodiments, the dose is adjusted to achieve a blood concentration of about 1-1000 μg / ml, for example, about 25-300 μg / ml.

[0266] Preparation method and use

[0267] Preparation method

[0268] On the other hand, the present application also provides a method for preparing the isolated antigen-binding protein described herein, which may include culturing the cells described herein under conditions that allow the isolated antigen-binding protein described herein to be expressed.

[0269] use

[0270] On the other hand, the present application also provides the use of the isolated antigen-binding protein, the immunoconjugate, the nucleic acid molecule, the vector, the cell and / or the pharmaceutical composition in the preparation of a drug for preventing, alleviating and / or treating tumors.

[0271] On the other hand, the present application also provides a method for preventing, alleviating or treating tumors, which may include administering the isolated antigen-binding protein, the immunoconjugate, the nucleic acid molecule, the vector, the cell and / or the pharmaceutical composition described herein to a subject in need thereof. In the present application, the administration may be performed in various ways, such as intravenous, intratumoral, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.

[0272] On the other hand, the isolated antigen-binding protein, the immunoconjugate, the nucleic acid molecule, the vector, the cell and / or the pharmaceutical composition described in the present application can be used to prevent, alleviate or treat tumors.

[0273] In the present application, the tumor may be a solid tumor or a hematological tumor.

[0274] In the present application, the tumor may include an AXL-positive tumor. For example, the AXL-positive tumor may be selected from the group consisting of lung cancer, skin cancer, kidney cancer, pancreatic cancer, blood cancer, breast cancer, ovarian cancer, lymphoma, and myeloma. For another example, the AXL-positive tumor may be selected from the group consisting of non-small cell lung cancer, squamous cell carcinoma of the skin, clear cell renal adenocarcinoma, pancreatic cancer, erythroleukemia, acute T-cell leukemia, breast cancer, ovarian cancer, lymphoma, and myeloma.

[0275] In the present application, the subject may include humans and non-human animals. For example, the subject may include, but is not limited to, cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats, or monkeys.

[0276] In the present application, the isolated antigen-binding protein can be administered together with one or more other antibodies to effectively inhibit tumor growth in a subject. In certain embodiments, the isolated antigen-binding protein and one or more other antibodies, such as LAG-3 antibodies, PD-1 antibodies, and / or CTLA-4 antibodies, can be administered to a subject.

[0277] In the present application, the isolated antigen binding protein can be administered together with a chemotherapeutic agent, which can be a cytotoxic agent, for example, SN-38, epirubicin, oxaliplatin, and / or 5-FU.

[0278] In another aspect, the present application provides the isolated antigen-binding protein according to the invention, which is used for diagnosing a disease or condition associated with the expression of the AXL protein.

[0279] In another aspect, the present application provides use of the antibody or antigen-binding fragment thereof for preparing a diagnostic agent, wherein the diagnostic agent is used to diagnose a disease or condition associated with the expression of the AXL protein.

[0280] In this application, the diagnostic agent can be used alone or in combination with an instrument, apparatus, device, or system. In the prevention, diagnosis, treatment monitoring, prognosis observation, health status evaluation, and prediction of genetic diseases, the diagnostic agent can be used to perform in vitro detection on human samples (e.g., various body fluids, cells, tissue samples, etc.). The diagnostic agent can be selected from the following group: reagents, kits, calibrators, and quality control products.

[0281] The in vitro detection method can be selected from the group consisting of Western blotting, ELISA, and immunohistochemistry. For example, the reagent can include a reagent capable of measuring the expression level of the AXL protein. For example, the reagent can be selected from the group consisting of reagents for Western blotting, reagents for ELISA, and reagents for immunohistochemistry.

[0282] In another aspect, the present application provides a method for diagnosing a disease or condition associated with AXL protein expression in a subject, the method comprising: contacting a sample derived from the subject with the isolated antigen-binding protein, and determining the presence and / or amount of a substance capable of specifically binding to the isolated antigen-binding protein in the sample.

[0283] In another aspect, the present application provides a method for detecting AXL in a sample, comprising administering the isolated antigen-binding protein. In the present application, the administration can be performed by various routes, such as intravenous, intratumoral, intraperitoneal, subcutaneous, intramuscular, topical, or intradermal administration.

[0284] In the present application, the disease or condition associated with the expression of AXL protein can be selected from the following group: lung cancer, skin cancer, kidney cancer, pancreatic cancer, blood tumors, breast cancer, ovarian cancer, lymphoma and myeloma, or can be selected from the following group: non-small cell lung cancer, skin squamous cell carcinoma, renal clear cell adenocarcinoma, pancreatic cancer, erythroleukemia, acute T-cell leukemia, breast cancer, ovarian cancer, lymphoma and myeloma.

[0285] In addition, the present application may also include the following implementation methods:

[0286] 1. An isolated antigen-binding protein comprising at least one CDR of the VH represented by the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 46; and at least one CDR of the VL represented by the amino acid sequence of SEQ ID NO: 2.

[0287] 2. The isolated antigen-binding protein according to embodiment 1, which has one or more of the following properties:

[0288] 1) Can be 1×10 -7M or lower K D Binds to AXL protein;

[0289] 2) Ability to specifically recognize AXL protein expressed on the cell surface;

[0290] 3) It can mediate internalization after binding to AXL protein expressed on the cell surface.

[0291] 3. The isolated antigen-binding protein according to embodiment 2, wherein the AXL protein comprises a human AXL protein.

[0292] 4. The isolated antigen-binding protein according to embodiment 3, wherein the human AXL protein comprises the amino acid sequence shown in SEQ ID NO: 39.

[0293] 5. The isolated antigen binding protein according to any one of embodiments 2 to 4, wherein the AXL protein comprises an extracellular domain.

[0294] 6. According to the isolated antigen-binding protein of embodiment 5, the extracellular domain comprises the amino acid sequence shown in SEQ ID NO:40.

[0295] 7. According to the isolated antigen-binding protein of embodiment 2, the cells include tumor cells.

[0296] 8. According to the isolated antigen binding protein of embodiment 7, the tumor comprises an AXL-positive tumor.

[0297] 9. According to the isolated antigen-binding protein of embodiment 8, the tumor is selected from the group consisting of lung cancer, skin cancer, kidney cancer, pancreatic cancer, blood tumors, breast cancer, ovarian cancer, lymphoma and myeloma.

[0298] 10. According to the isolated antigen-binding protein of embodiment 9, the tumor is selected from the group consisting of non-small cell lung cancer, squamous cell carcinoma of the skin, clear cell renal adenocarcinoma, pancreatic cancer, erythroleukemia, acute T-cell leukemia, breast cancer, ovarian cancer, lymphoma and myeloma.

[0299] 11. According to the isolated antigen-binding protein of embodiment 2, the cells include human cells.

[0300] 12. The isolated antigen-binding protein according to embodiment 7 or 11, wherein the cells are selected from the group consisting of human non-small cell lung cancer A549 cells, human skin squamous cell carcinoma A431 cells, renal clear cell adenocarcinoma 786-O cells, human pancreatic cancer MIA PaCa-2 cells, erythroleukemia K562 cells, acute T-cell leukemia Jurkat cells, human breast cancer MCF-7 cells, human breast cancer MDA-MB-231 cells, human breast cancer MDA-MB-468 cells, human breast cancer SKBR3 cells, human ovarian cancer SKOV3 cells, lymphoma U-937 cells, lymphoma Raji cells, human myeloma U266 cells, and human multiple myeloma RPMI8226 cells.

[0301] 13. The isolated antigen-binding protein according to embodiment 1, wherein the VH comprises HCDR1, HCDR2 and HCDR3.

[0302] 14. According to the isolated antigen-binding protein of embodiment 13, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 25.

[0303] 15. According to the isolated antigen-binding protein of embodiment 13, the HCDR2 comprises the amino acid sequence shown in any one of SEQ ID NO: 26, 44 and 45.

[0304] 16. According to the isolated antigen-binding protein of embodiment 13, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 27.

[0305] 17. According to the isolated antigen-binding protein of embodiment 1, the VL comprises LCDR1, LCDR2 and LCDR3.

[0306] 18. According to the isolated antigen-binding protein of embodiment 17, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 28.

[0307] 19. According to the isolated antigen-binding protein of embodiment 17, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 29.

[0308] 20 According to the isolated antigen-binding protein of embodiment 17, the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:30.

[0309] 21. The isolated antigen-binding protein according to embodiment 1 or 13, wherein the VH comprises framework regions H-FR1, H-FR2, H-FR3, and H-FR4.

[0310] 22 According to the isolated antigen-binding protein of embodiment 21, the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1, and the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:7.

[0311] 23. According to the isolated antigen-binding protein of embodiment 22, the H-FR1 comprises the amino acid sequence shown in any one of SEQ ID NOs: 11 and 15.

[0312] 24. According to the isolated antigen-binding protein of embodiment 21, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:8.

[0313] 25. According to the isolated antigen-binding protein of embodiment 24, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:12.

[0314] 26. According to the isolated antigen-binding protein of embodiment 21, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:9.

[0315] 27. According to the isolated antigen-binding protein of embodiment 26, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:13.

[0316] 28. According to the isolated antigen-binding protein of embodiment 21, the N-terminus of the H-FR4 is connected to the C-terminus of the HCDR3, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:10.

[0317] 29. According to the isolated antigen-binding protein of embodiment 28, the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:14.

[0318] 30 According to any one of embodiments 1, 13-16 and 22-29, the isolated antigen-binding protein, the VH comprises the amino acid sequence shown in any one of SEQ ID NO: 3, 5, 42 and 43.

[0319] 31. The isolated antigen-binding protein according to embodiment 1 or 17, wherein the VL comprises framework regions L-FR1, L-FR2, L-FR3, and L-FR4.

[0320] 32. According to the isolated antigen-binding protein of embodiment 31, the C-terminus of the L-FR1 is directly or indirectly connected to the N-terminus of the LCDR1, and the L-FR1 comprises the amino acid sequence shown in SEQ ID NO:16.

[0321] 33. According to the isolated antigen-binding protein of embodiment 32, the L-FR1 comprises the amino acid sequence shown in any one of SEQ ID NOs: 20 and 24.

[0322] 34. According to the isolated antigen-binding protein of embodiment 31, the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 comprises the amino acid sequence shown in SEQ ID NO:17.

[0323] 35. According to the isolated antigen-binding protein of embodiment 34, the L-FR2 comprises the amino acid sequence shown in SEQ ID NO:21.

[0324] 36. According to the isolated antigen-binding protein of embodiment 31, the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 comprises the amino acid sequence shown in SEQ ID NO:18.

[0325] 37. According to the isolated antigen-binding protein of embodiment 36, the L-FR3 comprises the amino acid sequence shown in SEQ ID NO:22.

[0326] 38. According to the isolated antigen-binding protein of embodiment 31, the N-terminus of the L-FR4 is connected to the C-terminus of the LCDR3, and the L-FR4 comprises the amino acid sequence shown in SEQ ID NO:19.

[0327] 39. According to the isolated antigen-binding protein of embodiment 38, the L-FR4 comprises the amino acid sequence shown in SEQ ID NO:23.

[0328] 40 According to any one of embodiments 1, 17-20 and 32-39, the isolated antigen-binding protein, the VL comprises the amino acid sequence shown in any one of SEQ ID NO: 4 and 6.

[0329] 41. The isolated antigen-binding protein of any one of embodiments 1-4, 6-11, 13-20, 22-29 and 32-39, which comprises an antibody heavy chain constant region, and wherein the antibody heavy chain constant region is derived from a human IgG heavy chain constant region.

[0330] 42. According to the isolated antigen-binding protein of embodiment 41, the antibody heavy chain constant region is derived from the human IgG1 heavy chain constant region or the human IgG4 heavy chain constant region.

[0331] 43. According to the isolated antigen-binding protein of embodiment 42, the antibody heavy chain constant region comprises the amino acid sequence shown in any one of SEQ ID NO: 33 and 41.

[0332] 44. The isolated antigen-binding protein of any one of embodiments 1-4, 6-11, 13-20, 22-29, 32-39, and 42-43, which comprises an antibody light chain constant region, and the antibody light chain constant region comprises a human Igκ constant region.

[0333] 45. According to the isolated antigen-binding protein of embodiment 44, the antibody light chain constant region comprises the amino acid sequence shown in SEQ ID NO: 34.

[0334] 46. ​​An isolated antigen-binding protein according to any one of embodiments 1-4, 6-11, 13-20, 22-29, 32-39, 42-43 and 45, comprising an antibody heavy chain, and the antibody heavy chain comprises the amino acid sequence shown in any one of SEQ ID NOs: 35 and 37.

[0335] 47. An isolated antigen-binding protein according to any one of embodiments 1-4, 6-11, 13-20, 22-29, 32-39, 42-43 and 45, comprising an antibody light chain, and the antibody light chain comprises the amino acid sequence shown in any one of SEQ ID NOs: 36 and 38.

[0336] 48. An isolated antigen-binding protein according to any one of embodiments 1-4, 6-11, 13-20, 22-29, 32-39, 42-43 and 45, which comprises an antibody or an antigen-binding fragment thereof.

[0337] 49. According to the isolated antigen-binding protein of embodiment 48, the antibody is selected from the group consisting of a monoclonal antibody, a single-chain antibody, a chimeric antibody, a multispecific antibody, a humanized antibody, and a fully human antibody.

[0338] 50 According to the isolated antigen-binding protein of embodiment 48, the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab)2, Fv, F(ab')2, scFv, di-scFv and dAb fragments.

[0339] 51. An immunoconjugate comprising the isolated antigen-binding protein of any one of embodiments 1-50.

[0340] 52. The immunoconjugate of embodiment 51, further comprising at least one other agent selected from the group consisting of a chemotherapeutic agent, a radioactive element, a cytostatic agent, and a cytotoxic agent.

[0341] 53. According to the immunoconjugate of embodiment 52, the isolated antigen binding protein is connected to the other agent via a linker molecule.

[0342] 54 According to the immunoconjugate of embodiment 53, the separated antigen binding protein and the other reagents are covalently linked to the linking molecule respectively.

[0343] 55. According to any one of embodiments 52-54, the other agent comprises maytansine or a derivative thereof.

[0344] 56. According to the immunoconjugate of embodiment 55, the maytansine derivative includes the maytansine derivative DM1.

[0345] 57. One or more isolated nucleic acid molecules encoding the isolated antigen-binding protein of any one of embodiments 1-50.

[0346] 58. A vector comprising the nucleic acid molecule of embodiment 57.

[0347] 59 cells, comprising the nucleic acid molecule of embodiment 57 or the vector of embodiment 58.

[0348] 60. A pharmaceutical composition comprising the isolated antigen-binding protein of any one of embodiments 1-50, the immunoconjugate of any one of embodiments 51-56, the nucleic acid molecule of embodiment 57, the vector of embodiment 58 and / or the cell of embodiment 59, and optionally a pharmaceutically acceptable adjuvant.

[0349] 61. A method for preparing the isolated antigen-binding protein of any one of embodiments 1-50, the method comprising culturing the cell of embodiment 59 under conditions such that the isolated antigen-binding protein of any one of embodiments 1-50 is expressed.

[0350] 62 Use of the isolated antigen-binding protein described in any one of embodiments 1-50, the immunoconjugate described in any one of embodiments 51-56, the nucleic acid molecule described in embodiment 57, the vector described in embodiment 58, the cell described in embodiment 59 and / or the pharmaceutical composition described in embodiment 60 in the preparation of a drug for preventing, alleviating and / or treating tumors.

[0351] 63. The use according to embodiment 62, wherein the tumor comprises an AXL-positive tumor.

[0352] 64. The use according to embodiment 63, wherein the tumor is selected from the group consisting of lung cancer, skin cancer, kidney cancer, pancreatic cancer, blood tumors, breast cancer, ovarian cancer, lymphoma and myeloma.

[0353] 65. According to the use described in embodiment 64, the tumor is selected from the following group: non-small cell lung cancer, squamous cell carcinoma of the skin, renal clear cell adenocarcinoma, pancreatic cancer, erythroleukemia, acute T-cell leukemia, breast cancer, ovarian cancer, lymphoma and myeloma.

[0354] 66. Use of the isolated antigen-binding protein of any one of embodiments 1-50 in the preparation of a diagnostic agent for diagnosing a disease or condition associated with the expression of an AXL protein.

[0355] 67. A method for diagnosing a disease or condition associated with AXL protein expression in a subject, the method comprising: contacting a sample derived from the subject with the isolated antigen-binding protein of any one of embodiments 1-50, and determining the presence and / or amount of a substance capable of specifically binding to the isolated antigen-binding protein in the sample.

[0356] 68. A method for detecting AXL in a sample, the method comprising administering the isolated antigen binding protein of any one of embodiments 1-50.

[0357] Without intending to be bound by any theory, the following examples are intended only to illustrate the protein molecules, preparation methods, and uses of the present invention and are not intended to limit the scope of the present invention. The examples do not include detailed descriptions of conventional methods, such as those used to construct vectors and plasmids, insert protein-encoding genes into such vectors and plasmids, or introduce plasmids into host cells. Such methods are well known to those skilled in the art and are described in many publications, including Sambrook, J., Fritsch, E.F. and Maniais, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press.

[0358] Example

[0359] Example 1. Antibody Recognition of Target Antigen

[0360] ELISA plates were coated with the target antigen AXL-His (Beijing Sino-Biotech Co., Ltd.) at 1 μg / ml overnight at 4°C. After washing with PBST, the plates were blocked with 10% fetal bovine serum at 37°C for 1 hour. Different concentrations of 6G12M11, 6G12M21, 6G12M31, and 6G12M41 antibodies were added and reacted at 37°C for 1 hour. After washing with PBST, horseradish peroxidase-conjugated goat anti-mouse IgG HRP (Abcam) secondary antibody was added and reacted at 37°C for 30 minutes. The plates were washed five times with PBST, and any remaining droplets were patted dry on absorbent paper. 100 μl of TMB (eBioscience) was added to each well and the plates were incubated at room temperature (20 ± 5°C) in the dark for 1.5 minutes. The substrate reaction was terminated by adding 100 μl of 2N H2SO4 stop solution to each well, and the OD values ​​were read at 450 nm on a microplate reader to analyze the binding ability of the antibodies to the target antigen AXL. Among them, the heavy chain amino acid sequence of the 6G12M11 antibody is shown in SEQ ID NO: 35, and the light chain amino acid sequence is shown in SEQ ID NO: 36; the heavy chain amino acid sequence of the 6G12M21 antibody is shown in SEQ ID NO: 37, and the light chain amino acid sequence is shown in SEQ ID NO: 38; the VH of the 6G12M31 antibody is shown in SEQ ID NO: 42, and the VL is shown in SEQ ID NO: 6; the VH of the 6G12M41 antibody is shown in SEQ ID NO: 43, and the VL is shown in SEQ ID NO: 6.

[0361] The test results are as follows Figure 1 A and Figure 1 As shown in B, it can be seen that both 6G12M11 and 6G12M21 antibodies can specifically recognize the target antigen AXL; the recognition activity is significantly dose-dependent, and the EC of 6G12M11 is 50 The value was 27.03 ng / mL, and the EC of 6G12M21 50 The value was 25.29 ng / mL, and the EC of 6G12M31 50 The value was 21.11 ng / mL, and the EC of 6G12M41 50 The value is 17.56ng / mL.

[0362] Example 2. Antibody affinity determination

[0363] In this experiment, surface plasmon resonance (SPR) technology was used to determine the affinity of AXL-His for the 6G12M11, 6G12M21, 6G12M31, and 6G12M41 antibodies. The assay was performed using a BIACOR biomacromolecular interaction instrument (GE Healthcare, T-200). 6G12M11, 6G12M21, 6G12M31, and 6G12M41 antibodies were immobilized on the chip surface using an antibody capture method at a concentration of 0.5 μg / mL. Samples were injected at a flow rate of 10 μL / min for 60 seconds. The interaction was then measured by flowing prepared AXL-His (6×His tag, molecular weight 47.5 kDa, Sino-Biotech Co., Ltd., Beijing) over the chip surface using a five-step mobile phase (8, 4, 2, 1, and 0.5 μg / mL). AXL-His bound for 120 seconds and dissociated for 1800 seconds at a flow rate of 30 μL / min. The results of antigen-antibody affinity are shown in Table 1. It can be seen that the isolated antigen-binding protein described in the present application can be used to bind to the antigen at a rate of 1×10 -7 The AXL protein was bound with a KD of M or lower.

[0364] Table 1. Antibody affinity determination results

[0365] Antibody Binding constant Ka(1 / Ms) Dissociation constant Kd (1 / s) Relative affinity KD(M) 6G12M11 9.742E+04 1.136E-03 1.166E-08 6G12M21 1.119E+05 1.137E-03 1.016E-08 6G12M31 3.97E+04 1.16E-03 2.92E-08 6G12M41 4.15E+04 1.19E-03 2.87E-08

[0366] Example 3. Antibody specific recognition of target antigen

[0367] AXL, TRYO3, and MER belong to the TYRO3 receptor tyrosine kinase subfamily. To verify the recognition specificity of 6G12M11, 6G12M21, 6G12M31, and 6G12M41 antibodies, the ELISA method was used to detect the binding ability of the antibodies to other members of the AXL family.

[0368] ELISA plates were coated with TYRO3-Fc (ACRO Biosystems), MER-His (ACRO Biosystems), and AXL-His (Beijing Sino Biological Technology Co., Ltd.) at 1 μg / ml overnight at 4°C. After washing with PBST, the plates were blocked with 10% fetal bovine serum at 37°C for 1 hour. 6G12M11, 6G12M21, 6G12M31, and 6G12M41 antibodies were added and reacted at 37°C for 1 hour. After washing with PBST, horseradish peroxidase-conjugated goat anti-human IgG Fab secondary antibody (Goat Anti-Human IgG(Fab')2(HRP), Abcam) was added and reacted at room temperature for 30 minutes. The plates were washed five times with PBST, and any remaining droplets were patted dry on absorbent paper. 100 μl of TMB (eBioscience, #85-00-420) was added to each well and incubated at room temperature (20±5°C) in the dark for 1.5 minutes. 100 μl of 2N The substrate reaction was terminated with H2SO4 stop solution, and the OD value was read at 450nm on a microplate reader to analyze the binding ability of the antibody and protein.

[0369] The results are as follows Figure 2-5 As shown, it can be seen that 6G12M11, 6G12M21, 6G12M31 and 6G12M41 antibodies can specifically recognize the target antigen AXL, but do not bind to other proteins in the same family of AXL, such as TYRO3 and MER.

[0370] Example 4. Antibody specific recognition of cell surface antigens

[0371] Flow cytometry was used to detect the binding of AXL to 6G12M11, 6G12M21, 6G12M31, and 6G12M41 antibodies on the surface of human non-small cell lung cancer A549 cells, human breast cancer MDA-MB-231 cells, and renal clear cell adenocarcinoma 786-O cells. Cells were collected during logarithmic growth phase and the cell density was adjusted to 5×10 6Pre-chill on ice. Dilute 6G12M11, 6G12M21, 6G12M31, and 6G12M41 antibodies to 20 μg / ml in pre-chilled saline containing 2% FBS. Add an equal volume of the aforementioned diluted antibodies to 100 μl of cells and incubate at 4°C in the dark for 30 min. Wash cells twice with pre-chilled saline containing 2% FBS (6000 rpm, 45 s). Resuspend cells in 100 μL of secondary antibody, PE Mouse Anti-Human IgG (BD Pharmingen), diluted 1:5 in pre-chilled saline containing 2% FBS and incubate at 4°C in the dark for 30 min. Wash cells twice with pre-chilled saline containing 2% FBS (6000 rpm, 45 s). Resuspend cells in 400 μl of 1% paraformaldehyde. Analyze antibody binding to cell surface antigens using flow cytometry (BD Calibur).

[0372] The results are as follows Figure 6 、 Figure 7 and Figure 8 As shown in the figure, it can be seen that the 6G12M11 and 6G12M21 antibodies can specifically and dose-dependently recognize AXL on the surface of human non-small cell lung cancer A549 cells, human breast cancer MDA-MB-231 cells, and renal clear cell adenocarcinoma 786-O cells. The recognition abilities of the 6G12M11 and 6G12M21 antibodies are similar. Among them, the EC50 values ​​of the 6G12M11 and 6G12M21 antibodies for binding to A549 cells are 0.46μg / mL and 0.78μg / mL, respectively; the EC50 values ​​for binding to MDA-MB-231 cells are 0.59μg / mL and 0.39μg / mL, respectively; and the EC50 values ​​for binding to 786-O cells are 0.43μg / mL and 0.31μg / mL, respectively.

[0373] The results are as follows Figure 9 and Figure 10 The results showed that the 6G12M21, 6G12M31, and 6G12M41 antibodies specifically and dose-dependently recognized AXL on the surfaces of human breast cancer MDA-MB-231 cells and renal clear cell adenocarcinoma 786-O cells. The recognition abilities of the 6G12M21, 6G12M31, and 6G12M41 antibodies were similar. The EC50 values ​​for binding to MDA-MB-231 cells were 0.64 μg / mL for 6G12M21, 0.60 μg / mL for 6G12M31, and 0.54 μg / mL for 6G12M41. The EC50 values ​​for binding to 786-O cells were 0.40 μg / mL for 6G12M21, 0.53 μg / mL for 6G12M31, and 0.47 μg / mL for 6G12M41.

[0374] Example 5. Internalization activity of antibodies

[0375] Flow cytometry was used to detect the internalization efficiency of 6G12M11, 6G12M21, 6G12M31, and 6G12M41 antibodies in human non-small cell lung cancer A549 cells, human breast cancer MDA-MB-231 cells, and renal clear cell adenocarcinoma 786-O cells. Cells in the logarithmic growth phase were collected and the cell density was adjusted to 5×10 6 100 μL of secondary antibody, PE Mouse Anti-Human IgG (BD Pharmingen), was diluted 1:5 in saline containing 2% FBS and resuspended in 100 μL. The cells were incubated at 4°C in the dark for 30 minutes. After the reaction, the cells were washed three times with 400 μL of 1% paraformaldehyde. The fluorescence intensity of the antibodies on the surface of cells incubated at different temperatures was analyzed by flow cytometry (BD Calibur). The internalization efficiency of the antibodies was calculated according to the following formula.

[0376] Internalization efficiency = (total MFI at 4°C - total MFI at 37°C) / total MFI at 4°C × 100%.

[0377] The results are as follows Figure 11 、 Figure 12 and Figure 13 As shown, it can be seen that 6G12M11 and 6G12M21 antibodies can be effectively internalized in human non-small cell lung cancer A549 cells, human breast cancer MDA-MB-231 cells, and renal clear cell adenocarcinoma 786-O cells, with similar internalization efficiencies, indicating that 6G12M11 and 6G12M21 antibodies have good internalization activity.

[0378] like Figure 14 and Figure 15 As shown in the figure, it can be seen that 6G12M21, 6G12M31, and 6G12M41 antibodies can be effectively internalized in human breast cancer MDA-MB-231 cells and renal clear cell adenocarcinoma 786-O cells with similar internalization efficiencies. It can be seen that 6G12M21, 6G12M31, and 6G12M41 antibodies have good internalization activity.

[0379] Example 6. Immunoconjugates inhibit tumor cell proliferation

[0380] The immunoconjugate 6G12M41-ADC was constructed using a small molecule linker and MMAF as an example ADC molecule. The biological activity of the 6G12M41-based immunoconjugate was evaluated, and the potential of the 6G12M41 antibody to construct immunoconjugates such as antibody-small molecule drug conjugates (ADCs) was analyzed.

[0381] A certain number of cells (human breast cancer MDA-MB-231 cells) in logarithmic growth phase were seeded into 96-well culture plates and allowed to adhere for 24 hours. Different concentrations of drugs were then added for 72 hours. After the drug treatment period, 10 μl of CCK-8 (Dojindo, Dojindo Chemical Research Institute, Japan) was added to the culture plates and incubated in a 37°C, 5% CO2 incubator for 3-5 hours. OD values ​​were measured at a wavelength of 450 nm using a microplate reader, and cell growth inhibition rates were calculated according to the following formula:

[0382] Inhibition rate = (OD value of control well - OD value of drug treatment well) / OD value of control well × 100%

[0383] According to the inhibition rate of each concentration, the half inhibition concentration IC50 was calculated.

[0384] The results are as follows Figure 16 The results showed that 6G12M41 antibody coupled with MMAF (6G12M41-ADC) exhibited a significant proliferation inhibitory effect on breast cancer MDA-MB-231, with an IC50 of 8.298 nM.

[0385] Example 7. In vivo tumor inhibition activity of immunoconjugates

[0386] Taking the immunoconjugate 6G12M41-ADC as an example, in vivo tumor inhibition activity analysis was performed.

[0387] A subcutaneous breast cancer model was established by subcutaneously inoculating B-NDG mice with MDA-MB-231 breast cancer cells to evaluate the in vivo tumor inhibition activity of 6G12M41-ADC. Female, 8-week-old B-NDG mice (Jiangsu Biotechnology Co., Ltd.) were selected and MDA-MB-231 cells were cultured in GIBCO Leibovitz's L-15 medium containing 10% inactivated fetal bovine serum. MDA-MB-231 cells were collected in the exponential growth phase and plated at 1×10 7 The concentration of 0.1 mL was inoculated into the right side of B-NDG mice. 3At the same time, the mice were randomly divided into 4 groups according to the tumor size, with 6 mice in each group, and G1 PBS, G2 6G12M41 (10 mg / kg), G3 6G12M41-ADC (10 mg / kg) and G4 6G12M41-ADC (5 mg / kg) were administered by intraperitoneal injection once a week for 2 consecutive times; the experiment was ended on the 25th day of group administration. The body weight and tumor volume of the mice were measured twice a week during the administration and observation period, and the measured values ​​were recorded. At the end of the experiment, the animals were euthanized, and the mouse tumors were dissected and weighed. The efficacy was evaluated according to the relative tumor inhibition rate (TGI), and the safety was evaluated according to the changes in animal weight and death. TGI TV (%) = [1-(Ti-T0) / (Vi-V0)] × 100% (Ti: mean tumor volume of the treatment group on the i-th day of administration, T0: mean tumor volume of the treatment group on the 0-th day of administration; Vi: mean tumor volume of the solvent control group on the i-th day of administration, V0: mean tumor volume of the solvent control group on the 0-th day of administration).

[0388] The results are as follows Figure 17 and Figure 18 As shown in the figure, during the experiment, the animals in the drug-treated group were active and ate well, and their weight increased to a certain extent, indicating that the experimental animals had good tolerance to the test product. On the 25th day of group administration, the average tumor volume of the PBS control group was 668±45mm 3 The average tumor volume of G2-G4 treatment groups was 458±14mm 3 、311±26mm 3 and 395±14mm 3 , TGI TV They are 36.5%, 62.0% and 47.5% respectively.

[0389] Each test product significantly inhibited the growth of subcutaneous MDA-MB-231 cell xenografts without causing significant toxic side effects, demonstrating a good safety profile. Furthermore, the tumor inhibition effect of the test product 6G12M41-ADC was concentration-dependent.

[0390] The foregoing detailed description is provided by way of explanation and example and is not intended to limit the scope of the appended claims. Various changes to the embodiments listed in the present application are obvious to those skilled in the art and are intended to be within the scope of the appended claims and their equivalents. Sequence Listing <110> Shangjian Monoclonal Antibody (Beijing) Biotechnology Co., Ltd.; Hangzhou Shangjian Biotechnology Co., Ltd. <120> A separated protein binding to antigen AXL and its use <130> 0070-PA-022CN <160> 46 <170> PatentIn version 3.5 <210> 1 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> VH general formula <220> <221> X <222> (5)..(5) <223> X can be K or V <220> <221> X <222> (23)..(23) <223> X can be S or A <220> <221> X <222> (25)..(25) <223> X can be T or S <220> <221> X <222> (27) <223> X can be F, Y, D or S <220> <221> X <222> (41)..(41) <223> X can be F or S <220> <221> X <222> (44)..(44) <223> X can be N or Q <220> <221> X <222> (45)..(45) <223> X can be K or G <220> <221> X <222> (88)..(88) <223> X can be T or S <220> <221> X <222> (114)..(114) <223> X can be S or T <400> 1 Gln Val Gln Leu Xaa Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Xaa Val Xaa Gly Xaa Ser Ile Ser Ser Gly 20 25 30 Tyr Tyr Trp Asn Trp Ile Arg Gln Xaa Pro Gly Xaa Xaa Leu Glu Trp 35 40 45 Met Gly Tyr Arg Ser Tyr Asp Gly Ser Asn Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Asn Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80 Leu Lys Leu Asn Ser Val Thr Xaa Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Arg Gly Trp Leu Leu His Tyr Thr Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Xaa Val Thr Val Ser Ser 115 <210> 2 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> VL pass <220> <221> X <222> (1)..(1) <223> X can be E or D <220> <221> X <222> (2)..(2) <223> X can be L or I <220> <221> X <222> (9)..(9) <223> X can be F or S <220> <221> X <222> (11)..(11) <223> X can be N or V <220> <221> X <222> (12)..(12) <223> X can be A or S <220> <221> X <222> (17) <223> X can be T or Q <220> <221> X <222> (42)..(42) <223> X can be L or Q <220> <221> X <222> (69)..(69) <223> X can be S or G <220> <221> X <222> (79)..(79) <223> X can be R or K <220> <221> X <222> (105)..(105) <223> X can be A or G <220> <221> X <222> (111)..(111) <223> X can be L or I <400> 2 Xaa Xaa Val Met Thr Gln Ser Pro Xaa Ser Xaa Xaa Val Thr Leu Gly 1 5 10 15 Xaa Ser Ala Ser Ile Ser Cys Arg Ser Ser Arg Ser Leu Leu His Ser 20 25 30 Asn Gly Phe Thr Tyr Leu Tyr Trp Tyr Xaa Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Xaa Ser Gly Ser Gly Thr Asp Phe Thr Leu Xaa Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gly Gln Asn 85 90 95 Leu Glu Leu Pro Leu Thr Phe Gly Xaa Gly Thr Lys Leu Glu Xaa Lys 100 105 110 <210> 3 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> 6G12M11's VH <400> 3 Gln Val Gln Leu Val Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Ala Val Thr Gly Phe Ser Ile Ser Ser Gly 20 25 30 Tyr Tyr Trp Asn Trp Ile Arg Gln Phe Pro Gly Gln Lys Leu Glu Trp 35 40 45 Met Gly Tyr Arg Ser Tyr Asp Gly Ser Asn Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Asn Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80 Leu Lys Leu Asn Ser Val Thr Ser Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Arg Gly Trp Leu Leu His Tyr Thr Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115 <210> 4 <并211> 112 <212> PRT <213> Artificial Sequence <220> <223> VL of 6G12M11 <400> 4 Glu Ile Val Met Thr Gln Ser Pro Ser Ser Val Ser Val Thr Leu Gly 1 5 10 15 Gln Ser Ala Ser Ile Ser Cys Arg Ser Ser Arg Ser Leu Leu His Ser Note: There seems to be a mistake in tag where it says "<并211>" which is likely incorrect. I translated it as best as possible based on the context but this might need to be corrected in the original. 20 25 30 Asn Gly Phe Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gly Gln Asn 85 90 95 Leu Glu Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 5 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> VH of 6G12M21 <400> 5 Gln Val Gln Leu Val Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Ser Val Thr Gly Phe Ser Ile Ser Ser Gly 20 25 30 Tyr Tyr Trp Asn Trp Ile Arg Gln Phe Pro Gly Gln Lys Leu Glu Trp​​​Met Gly Tyr Arg Ser Tyr Asp Gly Ser Asn Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Asn Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80 Leu Lys Leu Asn Ser Val Thr Ser Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Arg Gly Trp Leu Leu His Tyr Thr Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115 <210> 6 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> VL of 6G12M21 / 6G12M31 / 6G12M41 <400> 6 Asp Ile Val Met Thr Gln Ser Pro Ser Ser Val Ser Val Thr Leu Gly 1 5 10 15 Gln Ser Ala Ser Ile Ser Cys Arg Ser Ser Arg Ser Leu Leu His Ser 20 25 30 Asn Gly Phe Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gly Gln Asn 85 90 95 Leu Glu Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 7 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> H-FR1 general formula <220> <221> X <222> (5)..(5) <223> X can be K or V <220> <221> X <222> (23)..(23) <223> X can be S or A <220> <221> X <222> (25)..(25) <223> X can be T or S <220> <221> X <222> (27) <223> X can be F, Y, D or S <400> 7 Gln Val Gln Leu Xaa Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Xaa Val Xaa Gly Xaa Ser Ile Ser 20 25 30 <210> 8 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> H-FR2 general formula <220> <221> X <222> (5)..(5) <223> X can be F or S <220> <221> X <222> (8) <223> X can be N or Q <220> <221> X <222> (9)..(9) <223> X can be K or G <400> 8 Trp Ile Arg Gln Xaa Pro Gly Xaa Xaa Leu Glu Trp Met Gly 1 5 10 <210> 9 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> H-FR3 general formula <220> <221> X <222> (22)..(22) <223> X can be T or S <400> 9 Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe Leu Lys 1 5 10 15 Leu Asn Ser Val Thr Xaa Glu Asp Thr Ala Thr Tyr Tyr Cys Ala Arg 20 25 30 <210> 10 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> H-FR4 general formula <220> <221> X <222> (6) <223> X can be S or T <400> 10 Trp Gly Gln Gly Thr Xaa Val Thr Val Ser Ser 1 5 10 <210> 11 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> H-FR1 of 6G12M11 <400> 11 Gln Val Gln Leu Val Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Ala Val Thr Gly Phe Ser Ile Ser 20 25 30 <210> 12 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> H-FR2 for 6G12M11 / 6G12M121 / 6G12M31 / 6G12M41 <400> 12 Trp Ile Arg Gln Phe Pro Gly Gln Lys Leu Glu Trp Met Gly 1 5 10 <210> 13 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> H-FR3 for 6G12M11 / 6G12M21 / 6G12M31 / 6G12M41 <400> 13 Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe Leu Lys 1 5 10 15 Leu Asn Ser Val Thr Ser Glu Asp Thr Ala Thr Ala Thr Tyr Tyr Cys Ala Arg 20 25 30 <210> 14 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> H-FR4 for 6G12M11 / 6G12M21 / 6G12M31 / 6G12M41 <400> 14 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 1 5 10 <210> 15 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> H-FR1 for 6G12M21 / 6G12M31 / 6G12M41 <400> 15 Gln Val Gln Leu Val Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Ser Val Thr Gly Phe Ser Ile Ser 20 25 30 <210> 16 <211> twenty three <212> PRT <213> Artificial Sequence <220> <223> L-FR1 general formula <220> <221> X <222> (1)..(1) <223> X can be E or D <220> <221> X <222> (2)..(2) <223> X can be L or I <220> <221> X <222> (9)..(9) <223> X can be F or S <220> <221> X <222> (11)..(11) <223> X can be N or V <220> <221> X <222> (12)..(12) <223> X can be A or S <220> <221> X <222> (17) <223> X can be T or Q <400> 16 Xaa Xaa Val Met Thr Gln Ser Pro Xaa Ser Xaa Xaa Val Thr Leu Gly 1 5 10 15 Xaa Ser Ala Ser Ile Ser Cys 20 <210> 17 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> L-FR2 general formula <220> <221> X <222> (3) <223> X can be L or Q <400> 17 Trp Tyr Xaa Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr 1 5 10 15 <210> 18 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> L-FR3 general formula <220> <221> X <222> (8) <223> X can be S or G <220> <221> X <222> (18) <223> X can be R or K <400> 18 Gly Val Pro Asp Arg Phe Ser Xaa Ser Gly Ser Gly Thr Asp Phe Thr 1 5 10 15 Leu Xaa Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys 20 25 30 <210> 19 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> L-FR4 general formula <220> <221> X <222> (3) <223> X can be A or G <220> <221> X <222> (9)..(9) <223> X can be L or I <400> 19 Phe Gly Xaa Gly Thr Lys Leu Glu Xaa Lys 1 5 10 <210> 20 <211> twenty three <212> PRT <213> Artificial Sequence <220> <223> L-FR1 for 6G12M11 <400> 20 Glu Ile Val Met Thr Gln Ser Pro Ser Ser Val Ser Val Thr Leu Gly 1 5 10 15 Gln Ser Ala Ser Ile Ser Cys 20 <210> twenty one <211> 15 <212> PRT <213> Artificial Sequence <220> <223> L-FR2 for 6G12M11 / 6G12M21 / 6G12M31 / 6G12M41 <400> twenty one Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr 1 5 10 15 <210> twenty two <211> 32 <212> PRT <213> Artificial Sequence <220> <223> L-FR3 for 6G12M11 / 6G12M21 / 6G12M31 / 6G12M41 <400> twenty two Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 1 5 10 15 Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys 20 25 30 <210> twenty three <211> 10 <212> PRT <213> Artificial Sequence <220> <223> L-FR4 for 6G12M11 / 6G12M21 / 6G12M31 / 6G12M41 <400> twenty three Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 1 5 10 <210> twenty four <211> twenty three <212> PRT <213> Artificial Sequence <220> <223> L-FR1 for 6G12M21 / 6G12M31 / 6G12M41 <400> twenty four Asp Ile Val Met Thr Gln Ser Pro Ser Ser Val Ser Val Thr Leu Gly 1 5 10 15 Gln Ser Ala Ser Ile Ser Cys 20 <210> 25 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> HCDR1 of 6G12M11 / 6G12M21 / 6G12 / 6G12M31 / 6G12M41 <400> 25 Ser Gly Tyr Tyr Trp Asn 1 5 <210> 26 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> HCDR2 of 6G12M11 / 6G12M21 / 6G12 <400> 26 Tyr Arg Ser Tyr Asp Gly Ser Asn Asn Tyr Asn Pro Ser Leu Lys Asn 1 5 10 15 <210> 27 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> HCDR3 of 6G12M11 / 6G12M21 / 6G12 / 6G12M31 / 6G12M41 <400> 27 Gly Trp Leu Leu His Tyr Thr Met Asp Tyr 1 5 10 <210> 28 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> LCDR1 of 6G12M11 / 6G12M21 / 6G12 / 6G12M31 / 6G12M41 <400> 28 Arg Ser Ser Arg Ser Leu Leu His Ser Asn Gly Phe Thr Tyr Leu Tyr 1 5 10 15 <210> 29 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> LCDR2 of 6G12M11 / 6G12M21 / 6G12 / 6G12M31 / 6G12M41 <400> 29 Gln Met Ser Asn Leu Ala Ser 1 5 <210> 30 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> LCDR3 of 6G12M11 / 6G12M21 / 6G12 / 6G12M31 / 6G12M41 <400> 30 Gly Gln Asn Leu Glu Leu Pro Leu Thr 1 5 <210> 31 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> 6G12 VH <400> 31 Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Ser Val Thr Gly Phe Ser Ile Ser Ser Gly 20 25 30 Tyr Tyr Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp 35 40 45 Met Gly Tyr Arg Ser Tyr Asp Gly Ser Asn Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Asn Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80 Leu Lys Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Arg Gly Trp Leu Leu His Tyr Thr Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Ser Val Thr Val Ser Ser 115 <210> 32 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> VL of 6G12 <400> 32 Glu Leu Val Met Thr Gln Ser Pro Phe Ser Asn Ala Val Thr Leu Gly 1 5 10 15 Thr Ser Ala Ser Ile Ser Cys Arg Ser Ser Arg Ser Leu Leu His Ser 20 25 30 Asn Gly Phe Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Ser Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gly Gln Asn 85 90 95 Leu Glu Leu Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 110 <210> 33 <211> 330 <212> PRT <213> Homo sapiens <400> 33 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 34 <211> 107 <212> PRT <213> Homo sapiens <400> 34 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 35 <211> 449 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain of 6G12M11 <400> 35 Gln Val Gln Leu Val Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Ala Val Thr Gly Phe Ser Ile Ser Ser Gly 20 25 30 Tyr Tyr Trp Asn Trp Ile Arg Gln Phe Pro Gly Gln Lys Leu Glu Trp 35 40 45 Met Gly Tyr Arg Ser Tyr Asp Gly Ser Asn Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Asn Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80 Leu Lys Leu Asn Ser Val Thr Ser Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Arg Gly Trp Leu Leu His Tyr Thr Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Light <210> 36 <211> 219 <212> PRT <213> Artificial Sequence <220> <223> 6G12M11's chain <400> 36 Glu Ile Val Met Thr Gln Ser Pro Ser Ser Val Ser Val Thr Leu Gly 1 5 10 15 Gln Ser Ala Ser Ile Ser Cys Arg Ser Ser Arg Ser Leu Leu His Ser 20 25 30 Asn Gly Phe Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gly Gln Asn 85 90 95 Leu Glu Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 37 <211> 449 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain of 6G12M21 <400> 37 Gln Val Gln Leu Val Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Ser Val Thr Gly Phe Ser Ile Ser Ser Gly 20 25 30 Tyr Tyr Trp Asn Trp Ile Arg Gln Phe Pro Gly Gln Lys Leu Glu Trp 35 40 45 Met Gly Tyr Arg Ser Tyr Asp Gly Ser Asn Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Asn Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80 Leu Lys Leu Asn Ser Val Thr Ser Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90​​​​​Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lys <210> 38 <211> 219 <212> PRT <213> Artificial Sequence <220> <223> Light chain of 6G12M21 <400> 38 Asp Ile Val Met Thr Gln Ser Pro Ser Ser Val Ser Val Thr Leu Gly 1 5 10 15 Gln Ser Ala Ser Ile Ser Cys Arg Ser Ser Arg Ser Leu Leu His Ser 20 25 30 Asn Gly Phe Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gly Gln Asn 85 90 95 Leu Glu Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 39 <211> 894 <212> PRT <213> Homo sapiens <400> 39 Met Ala Trp Arg Cys Pro Arg Met Gly Arg Val Pro Leu Ala Trp Cys 1 5 10 15 Leu Ala Leu Cys Gly Trp Ala Cys Met Ala Pro Arg Gly Thr Gln Ala 20 25 30 Glu Glu Ser Pro Phe Val Gly Asn Pro Gly Asn Ile Thr Gly Ala Arg 35 40 45 Gly Leu Thr Gly Thr Leu Arg Cys Gln Leu Gln Val Gln Gly Glu Pro 50 55 60 Pro Glu Val His Trp Leu Arg Asp Gly Gln Ile Leu Glu Leu Ala Asp 65 70 75 80 Ser Thr Gln Thr Gln Val Pro Leu Gly Glu Asp Glu Gln Asp Asp Trp 85 90 95 Ile Val Val Ser Gln Leu Arg Ile Thr Ser Leu Gln Leu Ser Asp Thr 100 105 110 Gly Gln Tyr Gln Cys Leu Val Phe Leu Gly His Gln Thr Phe Val Ser 115 120 125 Gln Pro Gly Tyr Val Gly Leu Glu Gly Leu Pro Tyr Phe Leu Glu Glu 130 135 140 Pro Glu Asp Arg Thr Val Ala Ala Asn Thr Pro Phe Asn Leu Ser Cys 145 150 155 160 Gln Ala Gln Gly Pro Pro Glu Pro Val Asp Leu Leu Trp Leu Gln Asp 165 170 175 Ala Val Pro Leu Ala Thr Ala Pro Gly His Gly Pro Gln Arg Ser Leu 180 185 190 His Val Pro Gly Leu Asn Lys Thr Ser Ser Phe Ser Cys Glu Ala His 195 200 205 Asn Ala Lys Gly Val Thr Thr Ser Arg Thr Ala Thr Ile Thr Val Leu 210 215 220 Pro Gln Gln Pro Arg Asn Leu His Leu Val Ser Arg Gln Pro Thr Glu 225 230 235 240 Leu Glu Val Ala Trp Thr Pro Gly Leu Ser Gly Ile Tyr Pro Leu Thr 245 250 255 His Cys Thr Leu Gln Ala Val Leu Ser Asp Asp Gly Met Gly Ile Gln 260 265 270 Ala Gly Glu Pro Asp Pro Pro Glu Glu Pro Leu Thr Ser Gln Ala Ser 275 280 285 Val Pro Pro His Gln Leu Arg Leu Gly Ser Leu His Pro His Thr Pro 290 295 300 Tyr His Ile Arg Val Ala Cys Thr Ser Ser Gln Gly Pro Ser Ser Trp 305 310 315 320 Thr His Trp Leu Pro Val Glu Thr Pro Glu Gly Val Pro Leu Gly Pro 325 330 335 Pro Glu Asn Ile Ser Ala Thr Arg Asn Gly Ser Gln Ala Phe Val His 340 345 350 Trp Gln Glu Pro Arg Ala Pro Leu Gln Gly Thr Leu Leu Gly Tyr Arg 355 360 365 Leu Ala Tyr Gln Gly Gln Asp Thr Pro Glu Val Leu Met Asp Ile Gly 370 375 380 Leu Arg Gln Glu Val Thr Leu Glu Leu Gln Gly Asp Gly Ser Val Ser 385 390 395 400 Asn Leu Thr Val Cys Val Ala Ala Tyr Thr Ala Ala Gly Asp Gly Pro 405 410 415 Trp Ser Leu Pro Val Pro Leu Glu Ala Trp Arg Pro Gly Gln Ala Gln 420 425 430 Pro Val His Gln Leu Val Lys Glu Pro Ser Thr Pro Ala Phe Ser Trp 435 440 445 Pro Trp Trp Tyr Val Leu Leu Gly Ala Val Val Ala Ala Ala Cys Val 450 455 460 Leu Ile Leu Ala Leu Phe Leu Val His Arg Arg Lys Lys Glu Thr Arg 465 470 475 480 Tyr Gly Glu Val Phe Glu Pro Thr Val Glu Arg Gly Glu Leu Val Val 485 490 495 Arg Tyr Arg Val Arg Lys Ser Tyr Ser Arg Arg Thr Thr Glu Ala Thr 500 505 510 Leu Asn Ser Leu Gly Ile Ser Glu Glu Leu Lys Glu Lys Leu Arg Asp 515 520 525 Val Met Val Asp Arg His Lys Val Ala Leu Gly Lys Thr Leu Gly Glu 530 535 540 Gly Glu Phe Gly Ala Val Met Glu Gly Gln Leu Asn Gln Asp Asp Ser 545 550 555 560 Ile Leu Lys Val Ala Val Lys Thr Met Lys Ile Ala Ile Cys Thr Arg 565 570 575 Ser Glu Leu Glu Asp Phe Leu Ser Glu Ala Val Cys Met Lys Glu Phe 580 585 590 Asp His Pro Asn Val Met Arg Leu Ile Gly Val Cys Phe Gln Gly Ser 595 600 605 Glu Arg Glu Ser Phe Pro Ala Pro Val Val Ile Leu Pro Phe Met Lys 610 615 620 His Gly Asp Leu His Ser Phe Leu Leu Tyr Ser Arg Leu Gly Asp Gln 625 630 635 640 Pro Val Tyr Leu Pro Thr Gln Met Leu Val Lys Phe Met Ala Asp Ile 645 650 655 Ala Ser Gly Met Glu Tyr Leu Ser Thr Lys Arg Phe Ile His Arg Asp 660 665 670 Leu Ala Ala Arg Asn Cys Met Leu Asn Glu Asn Met Ser Val Cys Val 675 680 685 Ala Asp Phe Gly Leu Ser Lys Lys Ile Tyr Asn Gly Asp Tyr Tyr Arg 690 695 700 Gln Gly Arg Ile Ala Lys Met Pro Val Lys Trp Ile Ala Ile Glu Ser 705 710 715 720 Leu Ala Asp Arg Val Tyr Thr Ser Lys Ser Asp Val Trp Ser Phe Gly 725 730 735 Val Thr Met Trp Glu Ile Ala Thr Arg Gly Gln Thr Pro Tyr Pro Gly 740 745 750 Val Glu Asn Ser Glu Ile Tyr Asp Tyr Leu Arg Gln Gly Asn Arg Leu 755 760 765 Lys Gln Pro Ala Asp Cys Leu Asp Gly Leu Tyr Ala Leu Met Ser Arg 770 775 780 Cys Trp Glu Leu Asn Pro Gln Asp Arg Pro Ser Phe Thr Glu Leu Arg 785 790 795 800 Glu Asp Leu Glu Asn Thr Leu Lys Ala Leu Pro Pro Ala Gln Glu Pro 805 810 815 Asp Glu Ile Leu Tyr Val Asn Met Asp Glu Gly Gly Gly Tyr Pro Glu 820 825 830 Pro Pro Gly Ala Ala Gly Gly Ala Asp Pro Pro Thr Gln Pro Asp Pro 835 840 845 Lys Asp Ser Cys Ser Cys Leu Thr Ala Ala Glu Val His Pro Ala Gly 850 855 860 Arg Tyr Val Leu Cys Pro Ser Thr Thr Pro Ser Pro Ala Gln Pro Ala 865 870 875 880 Asp Arg Gly Ser Pro Ala Ala Pro Gly Gln Glu Asp Gly Ala 885 890 <210> 40 <211> 425 <212> PRT <213> Artificial Sequence <220> <223> Extracellular domain of human AXL protein <400> 40 Pro Arg Gly Thr Gln Ala Glu Glu Ser Pro Phe Val Gly Asn Pro Gly 1 5 10 15 Asn Ile Thr Gly Ala Arg Gly Leu Thr Gly Thr Leu Arg Cys Gln Leu 20 25 30 Gln Val Gln Gly Glu Pro Pro Glu Val His Trp Leu Arg Asp Gly Gln 35 40 45 Ile Leu Glu Leu Ala Asp Ser Thr Gln Thr Gln Val Pro Leu Gly Glu 50 55 60 Asp Glu Gln Asp Asp Trp Ile Val Val Ser Gln Leu Arg Ile Thr Ser 65 70 75 80 Leu Gln Leu Ser Asp Thr Gly Gln Tyr Gln Cys Leu Val Phe Leu Gly 85 90 95 His Gln Thr Phe Val Ser Gln Pro Gly Tyr Val Gly Leu Glu Gly Leu 100 105 110 Pro Tyr Phe Leu Glu Glu Pro Glu Asp Arg Thr Val Ala Ala Asn Thr 115 120 125 Pro Phe Asn Leu Ser Cys Gln Ala Gln Gly Pro Pro Glu Pro Val Asp 130 135 140 Leu Leu Trp Leu Gln Asp Ala Val Pro Leu Ala Thr Ala Pro Gly His 145 150 155 160 Gly Pro Gln Arg Ser Leu His Val Pro Gly Leu Asn Lys Thr Ser Ser 165 170 175 Phe Ser Cys Glu Ala His Asn Ala Lys Gly Val Thr Thr Ser Arg Thr 180 185 190 Ala Thr Ile Thr Val Leu Pro Gln Gln Pro Arg Asn Leu His Leu Val 195 200 205 Ser Arg Gln Pro Thr Glu Leu Glu Val Ala Trp Thr Pro Gly Leu Ser 210 215 220 Gly Ile Tyr Pro Leu Thr His Cys Thr Leu Gln Ala Val Leu Ser Asp 225 230 235 240 Asp Gly Met Gly Ile Gln Ala Gly Glu Pro Asp Pro Pro Glu Glu Pro 245 250 255 Leu Thr Ser Gln Ala Ser Val Pro Pro His Gln Leu Arg Leu Gly Ser 260 265 270 Leu His Pro His Thr Pro Tyr His Ile Arg Val Ala Cys Thr Ser Ser 275 280 285 Gln Gly Pro Ser Ser Trp Thr His Trp Leu Pro Val Glu Thr Pro Glu 290 295 300 Gly Val Pro Leu Gly Pro Pro Glu Asn Ile Ser Ala Thr Arg Asn Gly 305 310 315 320 Ser Gln Ala Phe Val His Trp Gln Glu Pro Arg Ala Pro Leu Gln Gly 325 330 335 Thr Leu Leu Gly Tyr Arg Leu Ala Tyr Gln Gly Gln Asp Thr Pro Glu 340 345 350 Val Leu Met Asp Ile Gly Leu Arg Gln Glu Val Thr Leu Glu Leu Gln 355 360 365 Gly Asp Gly Ser Val Ser Asn Leu Thr Val Cys Val Ala Ala Tyr Thr 370 375 380 Ala Ala Gly Asp Gly Pro Trp Ser Leu Pro Val Pro Leu Glu Ala Trp 385 390 395 400 Arg Pro Gly Gln Ala Gln Pro Val His Gln Leu Val Lys Glu Pro Ser 405 410 415 Thr Pro Ala Phe Ser Trp Pro Trp Trp 420 425 <210> 41 <211> 229 <212> PRT <213> Homo sapiens <400> 41 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe 1 5 10 15 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 20 25 30 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 35 40 45 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 50 55 60 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 65 70 75 80 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 85 90 95 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 100 105 110 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 115 120 125 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 130 135 140 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 145 150 155 160 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 165 170 175 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 180 185 190 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 195 200 205 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 210 215 220 Leu Ser Leu Gly Lys 225 <210> 42 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> VH of 6G12M31 <400> 42 Gln Val Gln Leu Val Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Ser Val Thr Gly Phe Ser Ile Ser Ser Gly 20 25 30 Tyr Tyr Trp Asn Trp Ile Arg Gln Phe Pro Gly Gln Lys Leu Glu Trp 35 40 45 Met Gly Tyr Arg Ser Tyr Asp Gly Ser Asn Asn Tyr Asp Pro Ser Leu 50 55 60 Lys Asn Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80 Leu Lys Leu Asn Ser Val Thr Ser Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Arg Gly Trp Leu Leu His Tyr Thr Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115 <210> 43 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> VH of 6G12M41 <400> 43 Gln Val Gln Leu Val Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Ser Val Thr Gly Phe Ser Ile Ser Ser Gly 20 25 30 Tyr Tyr Trp Asn Trp Ile Arg Gln Phe Pro Gly Gln Lys Leu Glu Trp 35 40 45 Met Gly Tyr Arg Ser Tyr Asp Gly Ser Asn Asn Tyr Gly Pro Ser Leu 50 55 60 Lys Asn Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80 Leu Lys Leu Asn Ser Val Thr Ser Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Arg Gly Trp Leu Leu His Tyr Thr Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115 <210> 44 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> HCDR2 of 6G12M31 <400> 44 Tyr Arg Ser Tyr Asp Gly Ser Asn Asn Tyr Asp Pro Ser Leu Lys Asn 1 5 10 15 <210> 45 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> HCDR2 of 6G12M41 <400> 45 Tyr Arg Ser Tyr Asp Gly Ser Asn Asn Tyr Gly Pro Ser Leu Lys Asn 1 5 10 15 <210> 46 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> VH of 6G12M31; VH of 6G12M41 <220> <221> misc_feature <222> (61)..(61) <223> X can be D or G <400> 46 Gln Val Gln Leu Val Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Ser Val Thr Gly Phe Ser Ile Ser Ser Gly 20 25 30 Tyr Tyr Trp Asn Trp Ile Arg Gln Phe Pro Gly Gln Lys Leu Glu Trp 35 40 45 Met Gly Tyr Arg Ser Tyr Asp Gly Ser Asn Asn Tyr Xaa Pro Ser Leu 50 55 60 Lys Asn Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80 Leu Lys Leu Asn Ser Val Thr Ser Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Arg Gly Trp Leu Leu His Tyr Thr Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115

Claims

1. An isolated antigen-binding protein capable of binding to an AXL protein, the isolated antigen-binding protein comprising three complementarity-determining regions (HCDR1, HCDR2, and HCDR3) of a heavy chain variable region (VH), and three complementarity-determining regions (LCDR1, LCDR2, and LCDR3) of a light chain variable region (VL), wherein the amino acid sequence of HCDR1 is as shown in SEQ ID NO: 25, the amino acid sequence of HCDR2 is as shown in SEQ ID NO: 44 or SEQ ID NO: 45, the amino acid sequence of HCDR3 is as shown in SEQ ID NO: 27, the amino acid sequence of LCDR1 is as shown in SEQ ID NO: 28, the amino acid sequence of LCDR2 is as shown in SEQ ID NO: 29, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO:

30.

2. The isolated antigen-binding protein of claim 1, wherein the AXL protein is expressed on the cell surface.

3. The isolated antigen binding protein of claim 1, wherein the AXL protein comprises a human AXL protein.

4. The isolated antigen-binding protein according to claim 3, wherein the human AXL protein comprises the amino acid sequence shown in SEQ ID NO:

39.

5. The isolated antigen binding protein according to any one of claims 1 to 4, wherein the AXL protein comprises an extracellular domain.

6. The isolated antigen-binding protein of claim 5, wherein the extracellular domain comprises the amino acid sequence shown in SEQ ID NO:

40.

7. The isolated antigen binding protein of claim 2, wherein the cell comprises a tumor cell.

8. The isolated antigen binding protein of claim 7, wherein the tumor comprises an AXL-positive tumor.

9. The isolated antigen binding protein of claim 8, wherein the tumor is selected from the group consisting of lung cancer, skin cancer, kidney cancer, pancreatic cancer, hematological tumors, breast cancer, ovarian cancer, lymphoma, and myeloma.

10. The isolated antigen binding protein of claim 9, wherein the tumor is selected from the group consisting of non-small cell lung cancer, squamous cell carcinoma of the skin, clear cell renal adenocarcinoma, pancreatic cancer, erythroleukemia, acute T-cell leukemia, breast cancer, ovarian cancer, lymphoma, and myeloma.

11. The isolated antigen binding protein of claim 2, wherein the cell comprises a human cell.

12. The isolated antigen-binding protein according to claim 7 or 11, wherein the cells are selected from the group consisting of human non-small cell lung cancer A549 cells, human skin squamous cell carcinoma A431 cells, renal clear cell adenocarcinoma 786-0 cells, human pancreatic cancer MIA PaCa-2 cells, erythroleukemia K562 cells, acute T-cell leukemia Jurkat cells, human breast cancer MCF-7 cells, human breast cancer MDA-MB-231 cells, human breast cancer MDA-MB-468 cells, human breast cancer SKBR3 cells, human ovarian cancer SKOV3 cells, lymphoma U-937 cells, lymphoma Raji cells, human myeloma U266 cells, and human multiple myeloma RPMI8226 cells.

13. The isolated antigen-binding protein of claim 1, wherein the VH comprises framework regions H-FR1, H-FR2, H-FR3, and H-FR4. 14 . The isolated antigen-binding protein according to claim 13 , wherein the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1, and the amino acid sequence of the H-FR1 is as shown in SEQ ID NO:

7. 15 . The isolated antigen-binding protein according to claim 14 , wherein the amino acid sequence of H-FR1 is as shown in any one of SEQ ID NOs: 11 and 15. 16 . The isolated antigen-binding protein according to claim 13 , wherein the H-FR2 is located between the HCDR1 and the HCDR2, and the amino acid sequence of the H-FR2 is shown in SEQ ID NO:

8.

17. The isolated antigen-binding protein according to claim 16, wherein the amino acid sequence of H-FR2 is shown in SEQ ID NO:

12.

18. The isolated antigen-binding protein according to claim 13, wherein the H-FR3 is located between the HCDR2 and the HCDR3, and the amino acid sequence of the H-FR3 is shown in SEQ ID NO:

9.

19. The isolated antigen-binding protein according to claim 18, wherein the amino acid sequence of H-FR3 is shown in SEQ ID NO:

13.

20. The isolated antigen-binding protein according to claim 13, wherein the N-terminus of the H-FR4 is connected to the C-terminus of the HCDR3, and the amino acid sequence of the H-FR4 is shown in SEQ ID NO:

10.

21. The isolated antigen-binding protein according to claim 20, wherein the amino acid sequence of H-FR4 is shown in SEQ ID NO:

14.

22. The isolated antigen-binding protein according to any one of claims 1 and 13-21, wherein the VH comprises the amino acid sequence shown in any one of SEQ ID NOs: 42 and 43.

23. The isolated antigen-binding protein of claim 1, wherein the VL comprises framework regions L-FR1, L-FR2, L-FR3, and L-FR4. 24 . The isolated antigen-binding protein according to claim 23 , wherein the C-terminus of the L-FR1 is directly or indirectly connected to the N-terminus of the LCDR1, and the amino acid sequence of the L-FR1 is shown in SEQ ID NO:

16.

25. The isolated antigen-binding protein according to claim 24, wherein the amino acid sequence of L-FR1 is shown in any one of SEQ ID NOs: 20 and 24.

26. The isolated antigen-binding protein according to claim 23, wherein the L-FR2 is located between the LCDR1 and the LCDR2, and the amino acid sequence of the L-FR2 is shown in SEQ ID NO:

17.

27. The isolated antigen-binding protein according to claim 26, wherein the amino acid sequence of L-FR2 is shown in SEQ ID NO:

21.

28. The isolated antigen-binding protein of claim 23, wherein the L-FR3 is located between the LCDR2 and the LCDR3, and the amino acid sequence of the L-FR3 is shown in SEQ ID NO:

18.

29. The isolated antigen-binding protein according to claim 28, wherein the amino acid sequence of L-FR3 is shown in SEQ ID NO:

22.

30. The isolated antigen-binding protein of claim 23, wherein the N-terminus of the L-FR4 is connected to the C-terminus of the LCDR3, and the amino acid sequence of the L-FR4 is shown in SEQ ID NO:

19.

31. The isolated antigen-binding protein according to claim 30, wherein the amino acid sequence of L-FR4 is shown in SEQ ID NO:

23.

32. The isolated antigen-binding protein according to any one of claims 1 and 23-31, wherein the amino acid sequence of the VL is shown in SEQ ID NO:

6.

33. The isolated antigen binding protein of any one of claims 1-4, 6-11, 13-21 and 23-31, comprising an antibody heavy chain constant region, and wherein the antibody heavy chain constant region is derived from a human IgG heavy chain constant region.

34. The isolated antigen-binding protein of claim 33, wherein the antibody heavy chain constant region is derived from a human IgG1 heavy chain constant region or a human IgG4 heavy chain constant region.

35. The isolated antigen-binding protein according to claim 34, wherein the amino acid sequence of the antibody heavy chain constant region is shown in any one of SEQ ID NOs: 33 and 41.

36. The isolated antigen binding protein of any one of claims 1-4, 6-11, 13-21, 23-31, and 34-35, comprising an antibody light chain constant region, wherein the antibody light chain constant region comprises a human Ig kappa constant region.

37. The isolated antigen-binding protein according to claim 36, wherein the amino acid sequence of the antibody light chain constant region is shown in SEQ ID NO:

34.

38. The isolated antigen-binding protein according to any one of claims 1-4, 6-11, 13-21, 23-31, 34-35 and 37, comprising an antibody light chain, wherein the antibody light chain comprises the amino acid sequence shown in SEQ ID NO:

38.

39. The isolated antigen binding protein of any one of claims 1-4, 6-11, 13-21, 23-31, 34-35, and 37, which comprises an antibody or antigen-binding fragment thereof.

40. The isolated antigen binding protein of claim 39, wherein the antibody is selected from the group consisting of a monoclonal antibody, a chimeric antibody, a multispecific antibody, a humanized antibody, and a fully human antibody.

41. The isolated antigen binding protein of claim 39, wherein the antigen binding fragment is selected from the group consisting of Fab, Fab', F(ab)2, Fv, F(ab')2, scFv, and di-scFv.

42. An immunoconjugate comprising the isolated antigen binding protein of any one of claims 1-41.

43. The immunoconjugate of claim 42, further comprising at least one additional agent selected from the group consisting of a chemotherapeutic agent, a radioactive element, a cytostatic agent, and a cytotoxic agent. The immunoconjugate of claim 43 , wherein the isolated antigen binding protein and the other agent are linked via a linker molecule. The immunoconjugate of claim 44 , wherein the isolated antigen binding protein and the other agent are each covalently linked to the linker molecule.

46. ​​The immunoconjugate according to any one of claims 43 to 45, wherein the other agent comprises maytansine or a derivative thereof, wherein the maytansine derivative is the maytansine derivative DM1.

47. An isolated nucleic acid molecule or molecules encoding the isolated antigen binding protein of any one of claims 1-41.

48. A vector comprising the nucleic acid molecule of claim 47.

49. A cell comprising the nucleic acid molecule of claim 47 or the vector of claim 48.

50. A pharmaceutical composition comprising the isolated antigen binding protein of any one of claims 1-41, the immunoconjugate of any one of claims 42-46, the nucleic acid molecule of claim 47, the vector of claim 48 and / or the cell of claim 49, and optionally a pharmaceutically acceptable adjuvant.

51. A method of making the isolated antigen binding protein of any one of claims 1-41, the method comprising culturing the cell of claim 49 under conditions such that the isolated antigen binding protein of any one of claims 1-41 is expressed.

52. Use of the isolated antigen-binding protein of any one of claims 1-41, the immunoconjugate of any one of claims 42-46, the nucleic acid molecule of claim 47, the vector of claim 48, the cell of claim 49 and / or the pharmaceutical composition of claim 50 in the preparation of a medicament for preventing, alleviating and / or treating tumors, wherein the tumors are breast cancer and renal cancer.

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