Antigen-binding protein targeting AXL and use thereof

Abstract

The present application relates to an antigen-binding protein targeting AXL, which comprises a heavy chain variable region VH and a light chain variable region VL, wherein the heavy chain variable region VH comprises HCDR1, HCDR2, and HCDR3, and the light chain variable region VL comprises LCDR1, LCDR2, and LCDR3. The antigen-binding protein of the present application exhibits good affinity for the tumor-specific AXL antigen and possesses significant internalization capability for cells expressing AXL. The present application further provides a method for preparing the antigen-binding protein and use thereof.

Description

An antigen-binding protein targeting AXL and its applications Technical Field

[0001] This application relates to the field of biomedicine, specifically to an antigen-binding protein that can specifically target AXL and its applications. Background Technology

[0002] The AXL protein (also known as Ark, UFO, Tyro-7) is a member of the Tyro-3 kinase family, which can be activated by binding to the ligand Gas6 (a 70-kDa protein homologous to the anticoagulation factor protein S). Gas6 binds to AXL to form an antigen-antibody complex, which is then phosphorylated. The phosphorylation site binds to the corresponding protein, activating downstream signal transduction pathways such as PI-3-kinase / Akt (Franke et al., Oncogene 22:8983-8998, 2003) and other major pathways such as Ras / Erk and β-linkin / TCF (Goruppi et al., Mol. CellBiol. 21:902-915, 2001).

[0003] Dysregulation of AXL or its ligand Gas6 is involved in the pathogenesis of a variety of human cancers. Overexpression of AXL is not only associated with poor prognosis, but also with increased invasiveness of 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 that AXL is highly expressed in various tumors, AXL inhibitors have been shown to enhance tumor cell apoptosis and inhibit migration and invasion. AXL-targeted drugs are becoming a hot topic for development by major pharmaceutical companies. Therapies targeting AXL are emerging as a new strategy for treating cancer.

[0005] Therefore, there is still a need in the field to provide therapeutic antibodies targeting AXL with improved performance. Summary of the Invention

[0006] This application provides an antigen-binding protein (e.g., an antibody or an antigen-binding fragment thereof) capable of specifically binding to the AXL protein. This application also provides nucleic acid molecules encoding the isolated antigen-binding protein, expression vectors, transformants, host cells, fusion proteins comprising the antigen-binding protein, pharmaceutical compositions, methods for preparing the antigen-binding protein, and the use of the antibody or antigen-binding fragment thereof described in this application.

[0007] On one hand, this application provides an isolated antigen-binding protein that can specifically bind to AXL protein, comprising a heavy chain variable region VH, wherein the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises an amino acid sequence as shown in SEQ ID NO:1, HCDR2 comprises an amino acid sequence as shown in SEQ ID NO:2 and HCDR3 comprises an amino acid sequence as shown in SEQ ID NO:3.

[0008] In some embodiments, the heavy chain variable region VH contains an amino acid sequence as shown in SEQ ID NO:7.

[0009] In some embodiments, the heavy chain variable region VH contains an amino acid sequence as shown in SEQ ID NO:8, 9, 10 or 11.

[0010] In some embodiments, the heavy chain variable region VH comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 7, 8, 9, 10, or 11; the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity maintains at least the same antigen-binding function as the original sequence; this identity is based on the aforementioned CDR combination being determined, i.e., the site of sequence change is the FR region.

[0011] In some embodiments, the antigen-binding protein includes a constant region of the antibody heavy chain.

[0012] In some embodiments, the antibody heavy chain constant region is derived from the human IgG constant region. In some embodiments, the antibody heavy chain constant region is derived from the human IgA constant region. In some embodiments, the antibody heavy chain constant region is derived from the human IgM constant region. In some embodiments, the antibody heavy chain constant region is derived from the human IgD constant region. In some embodiments, the antibody heavy chain constant region is derived from the human IgE constant region.

[0013] In some embodiments, the antibody heavy chain constant region is derived from the human IgG1 constant region. In some embodiments, the antibody heavy chain constant region is derived from the human IgG2 constant region. In some embodiments, the antibody heavy chain constant region is derived from the human IgG3 constant region. In some embodiments, the antibody heavy chain constant region is derived from the human IgG4 constant region.

[0014] In some implementations, the antibody heavy chain constant region contains LALA mutations or LALAPG mutations according to the EU numbering system.

[0015] In some embodiments, the antibody heavy chain constant region comprises an amino acid sequence as shown in SEQ ID NO:16.

[0016] In some embodiments, the constant region of the antibody heavy chain comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity as shown in SEQ ID NO:16; the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity maintains at least the same function as the original sequence.

[0017] In some embodiments, the antigen-binding protein comprises an antibody heavy chain, and the heavy chain comprises an amino acid sequence as shown in SEQ ID NO:18.

[0018] In some embodiments, the antigen-binding protein comprises an antibody heavy chain, and the heavy chain comprises an amino acid sequence as shown in SEQ ID NO:19, 20, 21 or 22.

[0019] In some embodiments, the antigen-binding protein includes a light chain variable region VL, the light chain variable region VL including LCDR1, LCDR2 and LCDR3, wherein LCDR1 includes the amino acid sequence shown in SEQ ID NO:4, LCDR2 includes the amino acid sequence shown in SEQ ID NO:5 and LCDR3 includes the amino acid sequence shown in SEQ ID NO:6.

[0020] In some embodiments, the light chain variable region VL comprises an amino acid sequence as shown in SEQ ID NO:12.

[0021] In some embodiments, the light chain variable region VL comprises an amino acid sequence as shown in SEQ ID NO:13, 14 or 15.

[0022] In some embodiments, the light chain variable region VL comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 12, 13, 14, or 15; the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity maintains at least equivalent antigen-binding function to the original sequence.

[0023] In some embodiments, the antigen-binding protein comprises a constant region of the antibody light chain.

[0024] In some embodiments, the antibody light chain constant region is derived from the human Igκ constant region. In some embodiments, the antibody light chain constant region is derived from the human Igλ constant region.

[0025] In some embodiments, the constant region of the antibody light chain contains an amino acid sequence as shown in SEQ ID NO:17.

[0026] In some embodiments, the constant region of the antibody light chain comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity as shown in SEQ ID NO:17; the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity maintains at least the same function as the original sequence.

[0027] In some embodiments, the antigen-binding protein comprises an antibody light chain, and the light chain comprises an amino acid sequence as shown in SEQ ID NO:23.

[0028] In some embodiments, the antigen-binding protein comprises an antibody light chain containing an amino acid sequence as shown in SEQ ID NO:24, 25, or 26.

[0029] In some embodiments, the antigen-binding protein comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:2, HCDR3 comprises the amino acid sequence shown in SEQ ID NO:3, LCDR1 comprises the amino acid sequence shown in SEQ ID NO:4, LCDR2 comprises the amino acid sequence shown in SEQ ID NO:5, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO:6.

[0030] In some embodiments, the antigen-binding protein comprises a heavy chain variable region VH and a light chain variable region VL, wherein the heavy chain variable region VH has the amino acid sequence shown in SEQ ID NO:7 and the light chain variable region VL has the amino acid sequence shown in SEQ ID NO:12.

[0031] In some embodiments, the antigen-binding protein comprises a heavy chain variable region VH and a light chain variable region VL, wherein the heavy chain variable region VH is the amino acid sequence shown in SEQ ID NO:8, 9, 10 or 11, and the light chain variable region VL is the amino acid sequence shown in SEQ ID NO:13, 14 or 15.

[0032] In this application, the antibody or its antigen-binding fragment may include VH and VL.

[0033] In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO:8, and the VL comprises the amino acid sequence shown in SEQ ID NO:13. In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO:9, and the VL comprises the amino acid sequence shown in SEQ ID NO:13. In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO:10, and the VL comprises the amino acid sequence shown in SEQ ID NO:13. In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO:11, and the VL comprises the amino acid sequence shown in SEQ ID NO:13.

[0034] In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO:8, and the VL comprises the amino acid sequence shown in SEQ ID NO:14. In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO:9, and the VL comprises the amino acid sequence shown in SEQ ID NO:14. In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO:10, and the VL comprises the amino acid sequence shown in SEQ ID NO:14. In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO:11, and the VL comprises the amino acid sequence shown in SEQ ID NO:14.

[0035] In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO:8, and the VL comprises the amino acid sequence shown in SEQ ID NO:15. In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO:9, and the VL comprises the amino acid sequence shown in SEQ ID NO:15. In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO:10, and the VL comprises the amino acid sequence shown in SEQ ID NO:15. In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO:11, and the VL comprises the amino acid sequence shown in SEQ ID NO:15.

[0036] In some embodiments, the antigen-binding protein comprises an antibody or an antigen-binding fragment thereof.

[0037] In some embodiments, the antibody is selected from one or more of the following groups: fully human antibodies, humanized antibodies, chimeric antibodies, proantibodies, monospecific antibodies, bispecific antibodies, multispecific antibodies, monoclonal antibodies, and polyclonal antibodies.

[0038] In some embodiments, the antigen-binding fragment includes Fab, Fab', F(ab')2, F(ab)2, scFv, di-scFv, and / or dAb.

[0039] In some embodiments, the antigen-binding protein is a full-length antibody. In this application, the antigen-binding protein comprises a heavy chain and a light chain.

[0040] In some embodiments, the antigen-binding protein comprises a heavy chain and a light chain, wherein the heavy chain has the amino acid sequence shown in SEQ ID NO:18 and the light chain has the amino acid sequence shown in SEQ ID NO:23.

[0041] In some embodiments, the antigen-binding protein comprises a heavy chain and a light chain, wherein the heavy chain is the amino acid sequence shown in SEQ ID NO:19, 20, 21 or 22, and the light chain is the amino acid sequence shown in SEQ ID NO:24, 25 or 26.

[0042] In some embodiments, the heavy chain comprises the amino acid sequence shown in SEQ ID NO:19, and the light chain comprises the amino acid sequence shown in SEQ ID NO:24. In some embodiments, the heavy chain comprises the amino acid sequence shown in SEQ ID NO:20, and the light chain comprises the amino acid sequence shown in SEQ ID NO:24. In some embodiments, the heavy chain comprises the amino acid sequence shown in SEQ ID NO:21, and the light chain comprises the amino acid sequence shown in SEQ ID NO:24. In some embodiments, the heavy chain comprises the amino acid sequence shown in SEQ ID NO:22, and the light chain comprises the amino acid sequence shown in SEQ ID NO:24.

[0043] In some embodiments, the heavy chain comprises the amino acid sequence shown in SEQ ID NO:19, and the light chain comprises the amino acid sequence shown in SEQ ID NO:25. In some embodiments, the heavy chain comprises the amino acid sequence shown in SEQ ID NO:20, and the light chain comprises the amino acid sequence shown in SEQ ID NO:25. In some embodiments, the heavy chain comprises the amino acid sequence shown in SEQ ID NO:21, and the light chain comprises the amino acid sequence shown in SEQ ID NO:25. In some embodiments, the heavy chain comprises the amino acid sequence shown in SEQ ID NO:22, and the light chain comprises the amino acid sequence shown in SEQ ID NO:25.

[0044] In some embodiments, the heavy chain comprises the amino acid sequence shown in SEQ ID NO:19, and the light chain comprises the amino acid sequence shown in SEQ ID NO:26. In some embodiments, the heavy chain comprises the amino acid sequence shown in SEQ ID NO:20, and the light chain comprises the amino acid sequence shown in SEQ ID NO:26. In some embodiments, the heavy chain comprises the amino acid sequence shown in SEQ ID NO:21, and the light chain comprises the amino acid sequence shown in SEQ ID NO:26. In some embodiments, the heavy chain comprises the amino acid sequence shown in SEQ ID NO:22, and the light chain comprises the amino acid sequence shown in SEQ ID NO:26.

[0045] In some embodiments, the antigen-binding protein is capable of specifically binding to human AXL, monkey AXL, or mouse AXL.

[0046] On the other hand, this application also provides a polypeptide molecule comprising the antigen-binding protein, such as an antibody or an antigen-binding fragment thereof. In some embodiments, the polypeptide molecule comprises a fusion protein.

[0047] On the other hand, this application also provides an immunoconjugate comprising the antigen-binding protein, such as an antibody or an antigen-binding fragment thereof.

[0048] On the other hand, this application also provides a nucleic acid molecule that encodes the antigen-binding protein, such as an antibody or its antigen-binding fragment or the polypeptide molecule.

[0049] On the other hand, this application also provides a carrier containing the nucleic acid molecule.

[0050] On the other hand, this application also provides a cell that contains the nucleic acid molecule or the carrier.

[0051] On the other hand, this application also provides a transformant comprising the nucleic acid molecule or the vector.

[0052] In some embodiments, the transformant is neither an animal nor a plant variety.

[0053] In some embodiments, the host cell of the transformant is a eukaryotic cell.

[0054] In some embodiments, the host cell of the transformant is a mammalian cell.

[0055] In some embodiments, the host cell of the transformant is a CHO cell or a derived cell line thereof.

[0056] In some embodiments, the host cell of the transformant is an Expi CHO cell.

[0057] On the other hand, this application also provides a pharmaceutical composition comprising the antigen-binding protein, the polypeptide molecule, the fusion protein, the immunoconjugate, the nucleic acid molecule, the carrier, the cell and / or the transformant, and optionally a pharmaceutically acceptable carrier.

[0058] On the other hand, this application also provides a method for preparing the antigen-binding protein, the method comprising culturing the transformant on a culture medium suitable for its growth under conditions that allow the antigen-binding protein to be expressed.

[0059] On the other hand, this application also provides the use of the antigen-binding protein, the polypeptide molecule, the fusion protein, the immunoconjugate, the nucleic acid molecule, the carrier, the cell, the transformant and / or the pharmaceutical composition in the preparation of a medicament for the prevention and / or treatment of diseases and / or conditions.

[0060] In some implementations, the disease and / or condition is cancer associated with AXL expression.

[0061] In some embodiments, the disease and / or condition includes cancers that overexpress and / or ectopically express AXL, such as solid tumors like non-small cell lung cancer, breast cancer, large cell lung cancer, and squamous cell lung cancer.

[0062] In some implementations, the disease and / or condition is breast cancer and / or non-small cell lung cancer.

[0063] On the other hand, this application also provides a method for detecting AXL in a sample, the method comprising using the antigen-binding protein, the polypeptide molecule, the fusion protein, the immunoconjugate, the nucleic acid molecule, the vector, the cell, the transformant and / or the pharmaceutical composition.

[0064] In some implementations, the detection is for non-diagnostic and / or therapeutic purposes.

[0065] On the other hand, this application also provides reagents or kits for detecting AXL in samples, which include the antigen-binding protein, the polypeptide molecule, the fusion protein, the immunoconjugate, the nucleic acid molecule, the carrier, the cell, the transformant and / or the pharmaceutical composition.

[0066] On the other hand, this application also provides the use of the antigen-binding protein, the polypeptide molecule, the fusion protein, the immunoconjugate, the nucleic acid molecule, the vector, the cell, the transformant and / or the pharmaceutical composition in the preparation of a kit for detecting the presence and / or content of AXL in a sample.

[0067] On the other hand, this application also provides a method for diagnosing, preventing and / or treating cancers associated with AXL expression, the method comprising administering to a subject in need the antigen-binding protein, the polypeptide molecule, the fusion protein, the immunoconjugate, the nucleic acid molecule, the carrier, the cell, the transformant and / or the pharmaceutical composition.

[0068] In some embodiments, the cancer is a cancer that overexpresses and / or ectopically expresses AXL, such as solid tumors like non-small cell lung cancer, breast cancer, large cell lung cancer, and squamous cell lung cancer.

[0069] In some implementations, the cancer is breast cancer and / or non-small cell lung cancer.

[0070] On the other hand, this application also provides a combination therapy, the method comprising administering to a subject in need the antigen-binding protein, the polypeptide molecule, the fusion protein, the immunoconjugate, the nucleic acid molecule, the carrier, the cell, the transformant and / or the pharmaceutical composition, and a second therapeutic agent, respectively.

[0071] In some embodiments, the second therapeutic agent comprises other antigen-binding proteins or pharmaceutical compositions comprising said other antigen-binding proteins, which are capable of specifically binding to the AXL protein, and / or other drugs for treating cancer.

[0072] In some implementations, the cancer is a cancer associated with AXL expression.

[0073] In some embodiments, the cancer includes cancers that overexpress and / or ectopically express AXL, such as solid tumors like non-small cell lung cancer, breast cancer, large cell lung cancer, and squamous cell lung cancer.

[0074] In some implementations, the cancer is breast cancer and / or non-small cell lung cancer.

[0075] The positive and progressive effects of this invention are found in any of the following:

[0076] The antigen-binding protein of this application has a high affinity for the AXL receptor and can bind tightly and specifically, ensuring that it can still function effectively at low concentrations.

[0077] The antigen-binding protein of this application exhibits excellent internalization ability, effectively binding to the AXL receptor and promoting the internalization of the receptor-antibody complex; and

[0078] The antigen-binding protein of this application can block the binding of the AXL receptor to its ligand GAS6, effectively inhibiting receptor self-activation and thus blocking downstream signaling pathways.

[0079] Other aspects and advantages of this application will readily be apparent to those skilled in the art from the detailed description below. Only exemplary embodiments of this application are shown and described in the following detailed description. As will be appreciated by those skilled in the art, the content of this application enables them to make modifications to the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application pertains. Accordingly, the descriptions in the accompanying drawings and specification of this application are merely exemplary and not restrictive. Attached Figure Description

[0080] The specific features of the invention involved in this application are shown in the appended claims. The features and advantages of the invention can be better understood by referring to the exemplary embodiments and drawings described in detail below. A brief description of the drawings is as follows:

[0081] Figures 1A-1C show the detection of the activity of human AXL monoclonal antibodies against immunogenic antigens using the ELISA method. Figure 1A shows the activity of human AXL monoclonal antibodies against human AXL antigen and monkey AXL immunogenic antigen; Figure 1B shows the binding activity of human AXL monoclonal antibodies against human AXL antigen and monkey AXL screening antigen protein; Figure 1C shows the binding activity of human AXL monoclonal antibodies against mouse AXL screening antigen protein.

[0082] Figures 2A-2B show the FACS method used to detect the binding activity of AXL monoclonal antibody to hAXL overexpression screening cell lines. Figure 2A shows the binding activity of AXL monoclonal antibody to HEK-293T-hu-AXL; Figure 2B shows the binding activity of AXL monoclonal antibody to CHOK1-hu-AXL.

[0083] Figures 3A-3B show the cell binding activity of the AXL monoclonal antibody detected using the FACS method. Figure 3A shows the cell binding activity of the AXL monoclonal antibody against MDA-MB-231. Figure 3B shows the cell binding activity of the AXL monoclonal antibody against CHOK1-hu-AXL.

[0084] Figure 4 shows the number of surface AXL antigens in cells Calu-1, LCLC-103H, MDA-MB-231, and PC-9.

[0085] Figures 5A-5D show the FACS method used to detect the binding activity of AXL monoclonal antibody against primary tumor cell lines. Figure 5A shows the binding activity of AXL monoclonal antibody against MDA-MB-231. Figure 5B shows the binding activity of AXL monoclonal antibody against PC-9. Figure 5C shows the binding activity of AXL monoclonal antibody against Calu-1. Figure 5D shows the binding activity of AXL monoclonal antibody against LCLC-103H.

[0086] Figures 6A-6B show the FACS method used to detect the binding activity of AXL monoclonal antibody to CHOK1-hu-AXL in AXL-overexpressing cells at different pH levels. Figure 6A shows the binding activity of AXL monoclonal antibody to CHOK1-hu-AXL at pH 5.6. Figure 6B shows the binding activity of AXL monoclonal antibody to CHOK1-hu-AXL at pH 7.4.

[0087] Figures 7A-7D show the endocytic activity assays of the AXL monoclonal antibody in AXL-overexpressing cell lines and primary tumor cell lines using the Incucyte method. Figure 7A shows the endocytic activity assay on MDA-MB-231 cells (48 hours). Figure 7B shows the endocytic activity assay on MDA-MB-231 cells (50 nM). Figure 7C shows the endocytic activity assay on CHOK1-hu-AXL cells (48 hours). Figure 7D shows the endocytic activity assay on CHOK1-hu-AXL cells (50 nM).

[0088] Figure 8 shows the detection of cross-reactivity between AXL monoclonal antibody and cynomolgus monkey AXL using the ELISA method.

[0089] Figures 9A-9B show the FACS method used to detect the binding ability of GAS6 to AXL-overexpressing cells and the ability of monoclonal antibodies to block the binding of GAS6 to AXL-overexpressing cells. Figure 9A shows the binding of GAS6 to AXL-overexpressing cells. Figure 9B shows the blocking of the binding of GAS6 to AXL-overexpressing cells by monoclonal antibodies.

[0090] Figures 10A-10C show the detection of the self-activation ability of AXL monoclonal antibodies using the Western blotting (WB) method. Figure 10A shows the detection results of the self-activation ability of AXL monoclonal antibodies using the WB method. Figure 10B shows the statistical analysis of the p-AKT / β-Actin ratio. Figure 10C shows the statistical analysis of the p-AKT / β-Actin ratio.

[0091] Figure 11 shows the detection of the binding ability of humanized AXL antibodies on AXL-overexpressing cells HEK-293T-hu-AXL using the FACS method.

[0092] Figures 12A-12B show the ability of the humanized AXL antibody to block the binding of AXL ligand GAS6 in AXL-overexpressing cells and primary tumor cells using the FACS method. Figure 12A shows the ability of the humanized AXL antibody to block the binding of AXL ligand GAS6 in AXL-overexpressing cells HEK-293T-hu-AXL. Figure 12B shows the ability of the humanized AXL antibody to block the binding of AXL ligand GAS6 in primary tumor cells Calu-1.

[0093] Figures 13A-13D show the detection of the self-activation ability of humanized AXL antibodies using Western blotting (WB). Figure 13A shows the results of the WB method for detecting the self-activation ability of humanized AXL antibodies. Figure 13B shows the statistical values ​​of the p-AXL / GAPDH ratio. Figure 13C shows the statistical values ​​of the p-AKT / GAPDH ratio. Figure 13D shows the statistical values ​​of the p-ERK1 / 2 / GAPDH ratio.

[0094] Figure 14 shows the detection of endocytic activity of anti-human AXL humanized antibody using the FACS method.

[0095] Figures 15A-15D show the identification of antigenic epitopes for human AXL antibodies. Figure 15A shows the minimum saturation binding concentration of VH2-VL3-AF647 to Calu-1 cells as detected by FACS. Figure 15B shows the minimum saturation binding concentration of Enapomab-AF647 to Calu-1 cells as detected by FACS. Figures 15C and 15D show the antigenic epitope competition assay for human AXL antibodies. Detailed Implementation

[0096] The following specific embodiments illustrate the implementation of the invention. Those skilled in the art can easily understand other advantages and effects of the invention from the content disclosed in this specification.

[0097] Terminology Definition

[0098] In this application, the letters in the amino acid sequence represent single-letter abbreviations of amino acids known in the art, such as those described in J. Biol. Chem, 243, p3558 (1968): Alanine: Ala-A, Arginine: Arg-R, Aspartic acid: Asp-D, Cysteine: Cys-C, Glutamine: Gln-Q, Glutamic acid: Glu-E, Histidine: His-H, Glycine: Gly-G, Asparagine: Asn-N, Tyrosine: Tyr-Y, Proline: Pro-P, Serine: Ser-S, Methionine: Met-M, Lysine: Lys-K, Valine: Val-V, Isoleucine: Ile-I, Phenylalanine: Phe-F, Leucine: Leu-L, Tryptophan: Trp-W, Threonine: Thr-T.

[0099] In this application, the term "and / or" should be understood to mean any one of the options or any combination of two or more of the options.

[0100] In this application, the term "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" should be interpreted inclusively, that is, including at least one in the quantity or element list, but also including more than one, and optionally, additional unlisted items. Only when explicitly indicated by the opposite terms, such as "only one" or "exactly one" or when used in the claims as "consisting of...", will it refer to only one number or one element of the list.

[0101] In this application, the term "AXL" generally refers to the protein receptor tyrosine kinase encoded by the axl gene. This term includes full-length AXL or functional fragments of AXL, as well as naturally occurring, artificially extracted, artificially synthesized, or modified AXL, provided they retain the desired activity. Amino acid sequences of AXL molecules from human and non-human species (e.g., mice, monkeys, rabbits, dogs, pigs, etc.) are available from public resources. AXL can be isolated from cells or tissues expressing them or synthesized using techniques well known in the art.

[0102] In this application, the term "antibody" generally refers to an immunoglobulin that reacts to a specified protein or peptide or fragment thereof. Antibodies can be from any class, including but not limited to IgG, IgA, IgM, IgD, and IgE, and antibodies from any subclass (e.g., IgG1, IgG2, IgG3, and IgG4). Antibodies may have a heavy chain constant region selected from, for example, IgG1, IgG2, IgG3, or IgG4. Antibodies may also have a light chain selected from, for example, kappa (κ) or lambda (λ). The antibodies of this application may be derived from any species. The term "antibody" may include complete polyclonal antibodies, complete monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), chimeric antibodies, humanized antibodies, human antibodies, fusion proteins containing antibodies, and any other modified immunoglobulin molecules, provided that these antibodies exhibit the desired biological activity. The term "antibody" also includes antibody fragments such as Fab, F(ab')2, Fv, scFv, Fd, dAb, and other antibody fragments that maintain antigen-binding function (e.g., specific binding to an antigen target). Typically, such fragments should include an antigen-binding domain. A basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies consist of five basic heterotetrameric units plus a polypeptide chain called the J chain and contain 10 antigen-binding sites, while IgA antibodies consist of 2-5 basic 4-chain units that can bind to the J chain and polymerize to form a multivalent combination. For IgG, a 4-chain unit is typically about 150,000 Daltons. Each L chain is linked to an H chain by a covalent disulfide bond, and two H chains are linked to each other by one or more disulfide bonds depending on the type of H chain. 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 the α and γ chains, and four CH domains for the μ and ε isoforms. Each L chain has a variable domain (VL) at its N-terminus and a constant domain at its other end. VL corresponds to VH, and CL corresponds to the first constant domain (CH1) of the heavy chain. Specific amino acid residues are thought to form interfaces between the variable domains of the light and heavy chains. 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 classified into one of two distinct types, called κ and λ, based on the amino acid sequence of its constant domain. Immunoglobulins can also be classified into different classes or isotypes based on the amino acid sequence of their heavy chain (CH) constant domain. Currently, there are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with heavy chains named α, δ, ε, γ, and μ, respectively.

[0103] In this application, the term "antigen-binding fragment" generally refers to one or more fragments having the ability to specifically bind an antigen (e.g., AXL). In this application, the antigen-binding fragment may include Fab, Fab', F(ab)2, Fv fragments, F(ab')2, scFv, di-scFv, and / or dAb. The portion of an antigen that is specifically recognized and bound by an antibody is called an "epitope." As described above, the antigen-binding domain typically includes a variable region (VL) of the antibody light chain and a variable region (VH) of the antibody heavy chain. Examples of antigen-binding fragments of antibodies include (1) Fab fragments, monovalent fragments having VL, VH, constant light chain (CL), and CH1 domains; (2) F(ab')2 fragments, divalent fragments having two Fab fragments connected by disulfide bridges in the hinge region; (3) Fv fragments having VL and VH domains in a single antibody arm; and (4) single-chain Fv (scFv), for example derived from a scFV library. Although the two domains VL and VH of the Fv fragment are encoded by independent genes, they can be joined together using a recombination method via a synthetic linker. The synthetic linker allows it to be prepared as a single protein chain in which the VL and VH regions pair to form a monovalent molecule (called single-chain Fv (scFv)) (see, for example, Huston et al., “Protein Engineering of Antibody Binding Sites: Recovery of Specific Activity in an Anti-Digoxin Single-ChainFv Analogue Produced in Escherichia coli,” Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988)).

[0104] In this application, the terms "variable domain" and "variable region" are used interchangeably, generally referring to a portion of the antibody heavy chain and / or light chain. The variable domains of the heavy and light chains can be respectively referred to as "V..." H " and "V L (or referred to as "VH" and "VL" respectively). These domains are usually the most varied parts of the antibody (relative to other antibodies of the same type) and contain antigen-binding sites.

[0105] In this application, the term "variable" generally refers to the fact that certain segments of the variable domain may differ significantly in sequence between antibodies. The variable domain mediates antigen binding and determines the specificity of a particular antibody for its specific antigen. However, variability is not uniformly distributed across the entire variable domain. It is typically concentrated in three segments within the light and heavy chain variable domains, known as hypervariable regions (CDRs or HVRs). The more highly conserved portions of the variable domain are called framework regions (FRs). The variable domains of the natural heavy and light chains each contain four FR regions, most of which are β-sheet conformations linked by three CDRs forming a ring link, and in some cases, forming part of a β-sheet structure. The CDRs in each chain are held together closely by the FR regions, and CDRs from the other chain together promote 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)).

[0106] In this application, the term "CDR," also known as "complementarity-determining region," typically refers to a region within the variable structural domain of an antibody whose sequence is highly variable and / or forms a structurally defining loop. Generally, an antibody comprises six CDRs: three in the VH (HCDR1, HCDR2, HCDR3) and three in the VL (LCDR1, LCDR2, LCDR3). Antibody CDRs can be determined using various coding systems, such as CCG, Kabat, AbM, Chothia, IMGT, and a combination of Kabat / Chothia. These coding systems are known in the art and can be found, for example, at http: / / www.bioinf.org.uk / abs / index.html#kabatnum. For example, the amino acid sequence number of the antigen-binding protein can be in accordance with the IMGT numbering scheme (IMGT, the international ImMunoGeneTics information system @imgt.cines.fr; http: / / imgt.cines.fr; Lefranc et al., 1999, Nucleic Acids Res. 27:209-212; Ruiz et al., 2000, Nucleic Acids Res. 28:219-221; Lefranc et al., 2001, Nucleic Acids Res. 29:207-209; Lefranc et al., 2003, Nucleic Acids Res. 31:307-310; Lefranc et al., 2005, DevComp Immunol 29:185-203). For example, the CDR of the antigen-binding protein can be determined according to the Kabat numbering system (see, for example, Kabat EA & Wu TT (1971) Ann NY Acad Sci 190:382-391 and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242). Those skilled in the art will understand that, unless otherwise specified, the terms “CDR” and “complementarity-determining region” for a given antibody or its region (e.g., variable region) should be understood to encompass complementarity-determining regions defined by any of the known schemes described herein. While the scope of protection claimed in this application is based on the sequences shown in the Kabat definition rules, amino acid sequences corresponding to other CDR definition rules should also fall within the scope of protection of this application.Therefore, when referring to antibodies defined by a specific CDR sequence as defined in this application, the scope of said antibody also includes antibodies whose variable region sequence contains the specific CDR sequence, but whose claimed CDR boundaries differ from those defined in this application due to the application of different schemes (e.g., different assignment system rules or combinations).

[0107] The sequence identity between sequences is calculated as follows. To determine the percentage of identity between two amino acid sequences, the sequences are aligned for optimal comparison purposes (e.g., vacancies may be introduced in the first and second amino acid sequences for optimal alignment, or non-homologous sequences may be discarded for comparison purposes). In a preferred embodiment, for comparison purposes, the length of the reference sequence being compared is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, or 100% of the reference sequence length. The amino acid residues at corresponding amino acid positions are then compared. When a position in the first sequence is occupied by the same amino acid residue at the corresponding position in the second sequence, the molecules are identical at that position. Sequence comparison and the calculation of the percentage of identity between two sequences can be performed using mathematical algorithms. In a preferred embodiment, the Needlema and Wunsch ((1970) J. Mol. Biol. 48: 444-453) algorithm (available at http: / / www.gcg.com) is used in the GAP program, which is integrated into the GCG software package. The Blossum 62 matrix or PAM250 matrix and vacancy weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6 are used to determine the percentage of identity between two amino acid sequences. A particularly preferred set of parameters (and one set of parameters that should be used unless otherwise specified) is a Blossum 62 scoring matrix with a vacancy penalty of 12, a vacancy extension penalty of 4, and a frameshift vacancy penalty of 5. Alternatively, the PAM120 weighted remainder table, gap length penalty of 12, and gap penalty of 4 can be used, along with the E. Meyers and W. Miller algorithm ((1989) CABIOS, 4:11-17) which has been incorporated into the ALIGN program (version 2.0), to determine the percentage of identity between two amino acid sequences. Additionally or alternatively, the protein sequence described in this application can be further used as a "query sequence" to perform a search against public databases to, for example, identify other family member sequences or related sequences.

[0108] In this application, the term "full-length antibody" is used interchangeably to refer to a glycoprotein comprising at least two heavy chains (HC) and two light chains (LC) linked together by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated as VH in this application) and a heavy chain constant region. Mammalian heavy chains are classified as α, δ, ε, γ, and μ. Immunoglobulins containing α, δ, ε, γ, and μ heavy chains are classified as immunoglobulin (Ig) A, IgD, IgE, IgG, and IgM. A complete antibody forms a "Y" shape. The stem of the Y is formed by the second and third constant regions of the two heavy chains (and, for IgE and IgM, a fourth constant region) linked together, and disulfide bonds (inter-chain) are formed in the hinge (IgM and IgE lack hinge regions). Heavy chains γ, α, and δ have a constant region consisting of three tandem (in a row) Ig domains and a hinge region for increased flexibility; heavy chains μ and ε have a constant region consisting of four immunoglobulin domains. The first, second, third, and fourth constant regions are referred to as the "CH1 domain," "CH2 domain," "CH3 domain," and "CH4 domain," respectively. Each light chain consists of a light chain variable region (abbreviated as VL in this application) and a light chain constant region (abbreviated as CL in this application). The light chain constant region consists of one domain CL. Mammalian light chains are classified as λ or κ. Each arm of Y includes a variable region of a single heavy chain and a first constant region that bind to the variable and constant regions of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding.

[0109] In this application, the term "FR" generally refers to the more conserved portion of the antibody variable domain, which is referred to as the frame region. Typically, the variable domains of the natural heavy and light chains each contain four FR regions: four in VH (H-FR1, H-FR2, H-FR3, and H-FR4) and four in VL (L-FR1, L-FR2, L-FR3, and L-FR4).

[0110] In this application, the term "Fab" generally refers to the antigen-binding fragment of an antibody. As described above, an intact antibody can be digested using papain. After papain digestion, the antibody produces two identical antigen-binding fragments, namely the "Fab" fragment, and a residual "Fc" fragment (i.e., the Fc region, as above). The Fab fragment can consist of a complete L chain with a variable region of a heavy chain and the H chain (V... H The first constant region (C) H 1) Composition.

[0111] In this application, the term "Fab' fragment" generally refers to a monovalent antigen-binding fragment of a human monoclonal antibody, which is slightly larger than the Fab fragment. For example, the Fab' fragment may include all light chains, all heavy chain variable regions, and all or part of the first and second constant regions of the heavy chain. For example, the Fab' fragment may also include part or all of the 220-330 amino acid residues of the heavy chain.

[0112] In this application, the term "F(ab')2" generally refers to an antibody fragment produced by digesting an intact antibody with pepsin. The F(ab')2 fragment contains two Fab fragments held together by disulfide bonds and a partial hinge region. The F(ab')2 fragment has bivalent antigen-binding activity and is capable of cross-linking antigens.

[0113] In this application, the term "Fv fragment" generally refers to a monovalent antigen-binding fragment of a human monoclonal antibody, comprising all or part of the heavy chain variable region and light chain variable region, and lacking the heavy chain constant region and light chain constant region. The heavy chain variable region and light chain variable region include, for example, CDRs. For example, an Fv fragment comprises all or part of the amino-terminal variable region of about 110 amino acids of the heavy and light chains.

[0114] In this application, the term "scFv" generally refers to a fusion protein comprising at least one antibody fragment including a variable region comprising a light chain and at least one antibody fragment including a variable region comprising a heavy chain, wherein the light and heavy chain variable regions are adjacent (e.g., via a synthetic linker, such as a short, flexible peptide linker) and are capable of being expressed as a single-chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise specified, as used in this application, the scFv may have the VL and VH variable regions in any order (e.g., relative to the N-terminus and C-terminus of the polypeptide), and the scFv may comprise VL-linker-VH or may comprise VH-linker-VL.

[0115] In this application, the term "dAb" generally refers to an antigen-binding fragment having a VH domain, a VL domain, or having either a VH domain or a VL domain, as seen in, for example, Ward et al. (Nature, 1989 Oct 12; 341(6242): 544-6), Holt et al., Trends Biotechnol., 2003, 21(11): 484-490; and other published patent applications, such as WO 06 / 030220, WO 06 / 003388, and Domantis Ltd. The term "dAb" generally includes sdAb. The term "sdAb" generally refers to a single-domain antibody. A single-domain antibody generally refers to an antibody fragment consisting only of the variable region (VH domain) of the antibody heavy chain or the variable region (VL) of the antibody light chain.

[0116] In this application, the term "monoclonal antibody" generally refers to an antibody molecule preparation consisting of a single molecule. Monoclonal antibodies typically exhibit high specificity against a single antigenic site. Moreover, unlike conventional polyclonal antibody preparations (which usually contain different antibodies targeting different determinants), each monoclonal antibody targets a single determinant on the antigen. In addition to their specificity, monoclonal antibodies have the advantage that they can be synthesized through hybridoma culture without contamination by other immunoglobulins. The modifier "monoclonal" indicates the characteristic of antibodies obtained from a substantially homogeneous group of antibodies and is not to be interpreted as requiring the production of antibodies through any particular method. For example, the monoclonal antibodies used in this application can be prepared in hybridoma cells or through recombinant DNA methods.

[0117] In this application, the term "multispecific antibody" is used in its broadest sense to encompass antibodies that have multi-epitope specificity. These multispecific antibodies include, but are not limited to: antibodies comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH-VL unit has multi-epitope specificity; antibodies having two or more VL and VH regions, each VH-VL unit binding to a different target or a different epitope of the same target; antibodies having two or more single variable regions, each single variable region binding to a different target or a different epitope of the same target; full-length antibodies, antibody fragments, bispecific antibodies, and trispecific antibodies, antibody fragments covalently or non-covalently linked together, etc.

[0118] In this application, the term “LALA” is mutated to L234A and L235A, also referred to as “Ala-Ala”; the term “LALAPG” is mutated to L234A, L235A, and P329G. The numbering is based on the EU index of Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991. These mutations reduce C1q and FcγR binding, resulting in decreased effector function.

[0119] In this application, the term "chimeric antibody" generally refers to an antibody in which the variable region is derived from one species and the constant region is derived from another species. Typically, the variable region is derived from an antibody from a laboratory animal such as a rodent ("parental antibody") and the constant region is derived from a human antibody, such that the resulting chimeric antibody is less likely to induce an adverse immune response in human individuals compared to parental (e.g., alpaca-derived) antibodies.

[0120] In this application, the term "humanized antibody" generally refers to an antibody in which some or all of the amino acids outside the CDR region of a non-human antibody are replaced by corresponding amino acids derived from human immunoglobulins. Small additions, deletions, insertions, substitutions, or modifications of amino acids in the CDR region are also permissible, as long as they retain the antibody's ability to bind to a specific antigen. Humanized antibodies may optionally contain at least a portion of the constant region of human immunoglobulins. "Humanized antibodies" retain antigen specificity similar to the original antibody. The "humanized" form of a non-human antibody may minimally contain a chimeric antibody with a sequence derived from a non-human immunoglobulin. In some cases, CDR region residues in a human immunoglobulin (recipient antibody) may be replaced with CDR region residues from a non-human species (donor antibody) (such as alpaca, mouse, rat, rabbit, or non-human primate) having the desired properties, affinity, and / or capabilities. In some cases, FR region residues in a human immunoglobulin may be replaced with corresponding non-human residues. Furthermore, humanized antibodies may contain amino acid modifications not present in the recipient antibody or the donor antibody. These modifications can be made to further improve antibody performance, such as binding affinity.

[0121] In this application, the term "fully human antibody" generally refers to an antibody that contains only the protein sequence of human immunoglobulins. If it is produced in mice, in mouse cells, or in hybridomas derived from mouse cells, then a fully human antibody may contain mouse glycans. Similarly, "mouse antibody" or "rat antibody" refers to an antibody that contains only mouse or rat immunoglobulin sequences, respectively. Fully human antibodies can be generated in humans or in transgenic animals with human immunoglobulin germline sequences by phage display or other molecular biology methods. Exemplary techniques that can be used to manufacture antibodies are described in U.S. Patents: 6,150,584, 6,458,592, and 6,420,140. Other techniques, such as the use of libraries, are known in the art.

[0122] In this application, the term "proantibody" refers to a "probody drug," a special antibody drug design derived from the combination of a "prodrug" and an "antibody." Probody drugs are initially inactive, only becoming active drugs after undergoing specific chemical reactions or enzymatic transformation within the organism. This design allows probody drugs to remain inactive in normal tissues, but are activated in specific microenvironments, such as the tumor microenvironment, through the action of proteases, thereby reducing toxicity to normal tissues and improving drug targeting.

[0123] In this application, "affinity" or "binding affinity" refers to the inherent binding affinity that reflects the interaction between members of a binding pair. The affinity of molecule X for its partner Y can generally be represented by the equilibrium dissociation constant (KD), which is the ratio of the dissociation rate constant to the binding rate constant (kdis and kon, respectively). Affinity can be measured by common methods known in the art. In some embodiments of this application, surface plasmon resonance (SPR) techniques are used to measure affinity, such as the affinity between the antibody and antigen in this application. In some preferred embodiments of this application, a specific method for measuring affinity is the BIAcore method described herein.

[0124] In this application, the terms "peptide molecule," "polypeptide," and "peptide" are used interchangeably and generally refer to polymers of amino acid residues. The term "fusion protein" generally refers to a polypeptide having at least two parts covalently linked together. Each part can be a polypeptide with different properties. These properties can be biological properties, such as in vitro or in vivo activity. They can also be simple chemical or physical properties, such as binding to target molecules, catalysis of reactions, etc. The two parts can be directly linked by a single peptide bond or through a peptide linker.

[0125] In this application, the term "isolated" antigen-binding protein generally refers to an antigen-binding protein that has been identified, isolated, and / or recovered from components of its production environment (e.g., natural or recombinant). The contaminating components of its production environment are typically substances that interfere with its research, diagnostic, or therapeutic use and may include enzymes, hormones, and other protein or non-protein solutes. Isolated antigen-binding proteins or antibodies are typically prepared through at least one purification step.

[0126] In this application, the term "nucleic acid" generally refers to a nucleotide, deoxyribonucleotide or ribonucleotide, modified nucleotide or base of any length in isolated form, any substrate capable of being incorporated into the chain by DNA or RNA polymerase, or an analogue isolated from its natural environment or synthesized artificially.

[0127] In this application, the term "vector" generally refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a protein is inserted, thereby enabling the protein to be expressed. A vector can be used to transform, transduce, or transfect host cells, allowing the genetic material elements it carries to be expressed within the host cells. Suitable vectors include those designed for amplification and expansion, or for expression, or both of the above. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, granules, or phage vectors.

[0128] In this application, the term "cell" or "host cell" generally refers to a single cell, cell line, or cell culture that may be or is already a recipient of a subject plasmid or vector, including the nucleic acid molecules or vectors described in this invention. Cells may include the progeny of a single cell. Due to natural, accidental, or intentional mutations, the progeny may not necessarily be identical to the original parent cell (in terms of the morphology of the total DNA complement or in the genome). In one embodiment, the cell is a eukaryotic cell, such as a plant, animal, fungus, or algae; or it may be a prokaryotic cell, such as a bacterium or protozoan. In one embodiment, the cell is a cell originating from or obtained from an individual. In one embodiment, the cell is derived from or obtained from a mammal.

[0129] In this application, the methods and conditions for culturing the resulting transformants and for recovering the resulting antibody molecules are known to those skilled in the art and can be modified or optimized based on methods known in this specification and the prior art, depending on the specific expression vector and mammalian host cell used.

[0130] In this application, the term "immunoconjugate" generally refers to a conjugate formed by conjugating the other reagent (e.g., chemotherapeutic agents, radioactive elements, cell growth inhibitors, and cytotoxic agents) with the antibody or its antigen-binding fragment (e.g., covalently linked by a linker molecule), which can deliver the other reagent to the target cell (e.g., tumor cell) by specifically binding the antibody or its antigen-binding fragment to the antigen on the target cell.

[0131] In this application, applications for detection not intended for diagnosis and / or treatment include: detecting the presence or absence of AXL protein in a laboratory; screening other antibodies targeting AXL as a positive antibody; or competing with other antibodies targeting AXL to detect whether there is competition between antibodies, i.e., whether the antigenic epitopes are the same or similar.

[0132] In this application, the term "pharmaceutical composition" generally refers to a composition for the prevention / treatment of a disease or condition. The pharmaceutical composition may comprise the isolated antigen-binding protein described in this application, the nucleic acid molecule described in this application, the carrier described in this application, and / or the cell described in this application, and optionally a pharmaceutically acceptable adjuvant. Furthermore, the pharmaceutical composition may also comprise suitable formulations of one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and / or preservatives. The pharmaceutical composition may be in the form of a sterile injectable aqueous or oil suspension for intramuscular and subcutaneous administration. The pharmaceutical compositions of the present invention include, but are not limited to, liquid, freeze-dried, and lyophilized compositions.

[0133] In this application, the terms "pharmaceutically acceptable salt" or "pharmaceutically usable salt" generally refer to the salt of the compound or antibody-drug conjugate of this application, or the salt of the compound or antibody-drug conjugate described in this application, which may be safe and / or effective when used in mammals and may have the desired biological activity. The compound or antibody-drug conjugate of this application may form salts with acids.

[0134] In this application, the term "pharmaceutically acceptable carrier" generally includes pharmaceutically acceptable carriers, excipients, or stabilizers that are non-toxic to cells or mammals exposed thereto at the doses and concentrations employed. Physiologically acceptable carriers may include, for example, buffers, antioxidants, low molecular weight (less than about 10 residues) peptides, proteins, hydrophilic polymers, amino acids, monosaccharides, disaccharides and other carbohydrates, chelating agents, sugar alcohols, salt-forming anti-charge ions; and / or nonionic surfactants. In this application, a pharmaceutically acceptable carrier is any of those conventionally used carriers and is limited only by physicochemical considerations (such as solubility and lack of reactivity with antibodies targeting AXL) and by route of administration. The pharmaceutically acceptable carriers described herein, such as mediators, adjuvants, excipients, and diluents, are well known to those skilled in the art and are readily available to the public.

[0135] In this application, the terms "specific binding" or "specific" generally refer to measurable and reproducible interactions, such as binding between a target and an antibody, where the presence of the target is determined in the presence of a heterogeneous population of molecules (including biomolecules). For example, an antibody that specifically binds to a target (which may be an epitope) may be an antibody that binds to the target with greater affinity, strength, ease, and / or duration than it binds to other targets. In some embodiments, the antibody specifically binds to an epitope on a protein that is conserved across proteins of different species. In some embodiments, specific binding may include, but is not required to be, exclusive binding.

[0136] In this application, the term "effective amount" means the amount of a drug or agent that elicits a biological or pharmaceutical response in a tissue, system, animal, or human, as sought by, for example, an investigator or clinician. Furthermore, the term "therapeuticly effective amount" means the amount that causes improved treatment, cure, prevention, or reduction of disease, symptom, or side effects, or reduces the rate of progression of a disease or condition, compared to a corresponding subject who did not receive that amount. The term also includes, within its scope, amounts that effectively enhance normal physiological function.

[0137] In this application, "cancer disease" or "cancer" includes diseases characterized by abnormally regulated cell growth, proliferation, differentiation, adhesion, and / or migration. Cancer cells are abnormal cells that grow through rapid, uncontrolled cell proliferation and continue to grow after the stimulus that initiated new growth has ceased.

[0138] In this application, the term "cancer associated with AXL expression" refers to malignant tumors in which AXL is overexpressed or ectopically expressed on the surface of cancer cells. This expression characteristic can manifest as a significant upregulation of AXL protein levels; preferably, the cancer includes, but is not limited to, solid tumors such as non-small cell lung cancer, breast cancer, large cell lung cancer, and squamous cell lung cancer.

[0139] In this application, the term "subject" generally refers to a human or non-human animal, including but not limited to cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats, or monkeys.

[0140] In this application, the proteins, peptides and / or amino acid sequences involved should also be understood to include at least the following range: variants or homologs that have the same or similar functions as the said protein or peptide.

[0141] In this application, the variant may be, for example, a protein or polypeptide that has undergone substitution, deletion, or addition of one or more amino acids in the amino acid sequence of the protein and / or the polypeptide (e.g., an antibody or fragment thereof that specifically binds to AXL). For example, the functional variant may comprise a protein or polypeptide that has undergone amino acid alterations through substitution, deletion, and / or insertion of at least one, such as 1-30, 1-20, or 1-10, or even 1, 2, 3, 4, or 5 amino acids. 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., antigen-binding capacity) of the protein or polypeptide prior to the alteration. For example, the substitution may be a conserved substitution. In some embodiments, the variant does not involve a change in the CDR.

[0142] In this application, the homolog can be a protein or polypeptide having at least about 85% (e.g., having 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 higher) sequence homology to the amino acid sequence of the protein and / or the polypeptide (e.g., an antibody that specifically binds to AXL or a fragment thereof). In some embodiments, the homolog does not involve changes in CDR.

[0143] In this application, the term "comprising" generally means including, encompassing, containing, or including. In some cases, it also means "to be" or "composed of".

[0144] In this application, the term "about" generally refers to a variation within a range of 0.5% to 10% above or below a specified value, such as 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 a specified value.

[0145] Invention Details

[0146] antigen-binding proteins

[0147] The CDR (Complementarity Determinant Region) of an antibody, also known as the complementarity-determining region, is part of the variable region. Amino acid residues in this region can contact antigens or antigenic epitopes. Antibody CDRs can be determined using various coding systems, such as CCG, Kabat, Chothia, IMGT, AbM, and a combination of Kabat / Chothia. These coding systems are known in the art and can be found, for example, at http: / / www.bioinf.org.uk / abs / index.html#kabatnum. Those skilled in the art can determine the CDR region using different coding systems based on the antibody's sequence and structure. The CDR region may differ when using different coding systems. In this application, the term CDR encompasses CDR sequences partitioned according to any CDR partitioning method; it also encompasses variants of the CDR, including amino acid sequences with substitutions, deletions, and / or additions of one or more amino acids. For example, 1-30, 1-20, or 1-10 amino acid substitutions, deletions, and / or insertions, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions, deletions, and / or insertions; this also covers its homologs, which can be amino acid sequences having at least about 85% (e.g., having 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 higher) sequence homology with the amino acid sequence of the CDR. In some embodiments, the antibody or its antigen-binding fragment described in this application can be defined by the Kabat coding system.

[0148] Furthermore, it should be noted that the antigen-binding protein described in this application may contain heavy and / or light chain sequences with one or more conserved sequence modifications. "Conserved sequence modification" refers to amino acid modifications that do not significantly affect or alter antibody binding properties. Such conserved modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the isolated antigen-binding protein described in this application using standard techniques known in the art, such as point mutations and PCR-mediated mutations. Conserved amino acid substitution involves replacing an amino acid residue with an amino acid residue having a similar side chain. Groups of amino acid residues with 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), nonpolar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In some embodiments, one or more amino acid residues in the CDR region of the isolated antigen-binding protein described in this application may be replaced with other amino acid residues from the same side chain group. Those skilled in the art will know that some conserved sequence modifications do not cause the antigen to lose its binding ability. For example, see Brummell et al., (1993) Biochem 32:1180-8; de Wildt et al., (1997) Prot. Eng. 10:835-41; Komissarov 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.

[0149] The antigen-binding proteins described in this application can be identified, screened, or characterized by various assays known in the art.

[0150] For example, the antigen-binding activity of the antigen-binding protein or fusion protein of this application can be tested by known methods such as enzyme-linked immunosorbent assay (ELISA), immunoblotting (e.g., Western blotting), flow cytometry (e.g., FACS), immunohistochemistry, immunofluorescence, etc.

[0151] In this application, the antigen-binding protein is capable of specifically binding to AXL. In some embodiments, the binding of the antigen-binding protein to AXL can be detected by an ELISA method. For example, the antigen-binding protein of this application can be expressed at EC50 concentrations less than or equal to about 0.10 nM, less than or equal to about 0.09 nM, less than or equal to about 0.08 nM, less than or equal to about 0.07 nM, less than or equal to about 0.06 nM, and less than or equal to about 0.05 nM. 50 The value is combined with AXL.

[0152] polypeptide molecules, nucleic acid molecules, vectors, cells, transformants, fusion proteins, the aforementioned immunoconjugates, and pharmaceutical compositions.

[0153] On the other hand, this application provides isolated nucleic acid molecules that can encode the antigen-binding protein described in this application. For example, they can be produced or synthesized by: (i) in vitro amplification, such as by polymerase chain reaction (PCR); (ii) by clonal recombination; (iii) purification, such as by enzyme digestion and gel electrophoresis fractionation; or (iv) synthesis, such as by chemical synthesis.

[0154] On the other hand, this application provides a vector that may contain the nucleic acid molecule described in this application. Furthermore, the vector may also contain other genes, such as marker genes that allow selection of the vector in appropriate host cells and under appropriate conditions. Additionally, the vector may contain expression control elements that allow the coding region to be correctly expressed in an appropriate host. Such control elements are well known to those skilled in the art and may include, for example, promoters, ribosome binding sites, enhancers, and other control elements regulating gene transcription or mRNA translation. The vector can be transformed, transduced, or transfected into host cells to express the genetic material elements it carries within the host cells. The vector may include, for example, plasmids, granules, viruses, bacteriophages, or other vectors commonly used in, for example, genetic engineering. For example, the vector is an expression vector. Furthermore, the vector may also include components that facilitate its entry into cells, such as viral particles, liposomes, or protein coats, but not only these substances.

[0155] On the other hand, this application provides a cell that may contain the nucleic acid molecules or vectors described in this application. In some embodiments, each or every host cell may contain one or more of the nucleic acid molecules or vectors described in this application. In some embodiments, each or every host cell may contain multiple (e.g., two or more) or more types (e.g., two or more types) of the nucleic acid molecules or vectors described in this application. For example, the vectors described in this application may be introduced into the host cell, such as eukaryotic cells, such as cells from plants, fungi, or yeast cells. In some embodiments, the cell may be a bacterial cell (e.g., Escherichia coli), a yeast cell, or other eukaryotic cells. The vectors described in this application may be introduced into the host cell by methods known in the art, such as electroporation, lipofectine transfection, lipofectamin transfection, etc.

[0156] On the other hand, this application also provides immune conjugates that may contain the antigen-binding protein described in this application.

[0157] In some embodiments, the antigen-binding protein described herein can be linked to another reagent, such as a chemotherapeutic agent, toxin, immunotherapeutic agent, imaging probe, spectroscopic probe, etc. This linking can be via one or more covalent bonds, or non-covalent interactions, and may include chelation. Various adapters (which may be known in the art) can be used to form immunoconjugates. Furthermore, immunoconjugates can be provided in the form of fusion proteins, which may be expressed by polynucleotides encoding the immunoconjugate. The immunoconjugates may also comprise, for example, antibody-drug conjugates (ADCs).

[0158] On the other hand, this application also provides pharmaceutical compositions that may comprise the antigen-binding protein described in this application, the polypeptide molecule described in this application, the fusion protein described in this application, the immunoconjugate described in this application, the nucleic acid molecule described in this application, the carrier described in this application, the cell described in this application, and / or the transformant described in this application, and optionally a pharmaceutically acceptable carrier.

[0159] In some embodiments, the pharmaceutical composition may further comprise suitable formulations of one or more (pharmaceutically effective) adjuvants, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and / or preservatives. The acceptable components of the composition are preferably non-toxic to the recipient at the dosage and concentration used. The pharmaceutical compositions of the present invention include, but are not limited to, liquid, freeze-dried, and lyophilized compositions.

[0160] In some embodiments, the pharmaceutical composition may also contain more than one active compound, typically those with complementary activities that do not adversely affect each other. The type and effective amount of such a drug may depend, for example, on the amount and type of antagonist present in the formulation, and on the clinical parameters of the subject.

[0161] In some embodiments, the pharmaceutically acceptable carrier may include any and all solvents, dispersion media, coatings, isotonic agents, and absorption delay agents that are compatible with drug administration and are generally safe and non-toxic.

[0162] In some embodiments, the pharmaceutical composition may be administered parenterally, percutaneously, intracavitarily, intra-arterially, intrathecally, and / or intranasally, or directly injected into tissues. For example, the pharmaceutical composition may be administered to a patient or subject by infusion or injection. In some embodiments, the pharmaceutical composition may be administered in various ways, such as intravenously, intraperitoneally, subcutaneously, intramuscularly, locally, or intradermally.

[0163] Preparation method

[0164] On the other hand, this application provides a method for preparing the antigen-binding protein. The method may include culturing the transformant on a culture medium suitable for its growth under conditions that allow the antigen-binding protein to develop. For example, this can be achieved by using appropriate culture media, appropriate temperatures, and culture times, methods known to those skilled in the art.

[0165] Any method suitable for producing monoclonal antibodies can be used to produce the antigen-binding protein of this application. For example, animals can be immunized with linked or naturally occurring AXL or fragments thereof. Suitable immunization methods can be used, including adjuvants, immunostimulants, repeated booster immunizations, or one or more pathways. In some embodiments, isolated antigen-binding proteins against AXL can be screened and enriched by extracting peripheral blood lymphocytes from immunized alpaca, cloning the cellular nucleic acid fragments into a vector, and using a phage surface display system.

[0166] Any suitable form of AXL can be used as an immunogen (antigen) to generate non-human antibodies specific to AXL and to screen for the biological activity of said antibodies. For example, the stimulating immunogen can be full-length AXL or a peptide containing one or more epitopes. The immunogen can be used alone or in combination with one or more immunogenicity enhancers known in the art.

[0167] The fusion protein can be selected from any type of immunoglobulin, including IgM, IgD, IgG, IgA, and IgE. In this application, the antibody can be an IgG antibody, and the IgG1 subtype can be used. The necessary constant domain sequence can be optimized to produce the desired biological activity by screening antibodies using the biological assays described in the examples below. Similarly, any class of light chains can be used in the compounds and methods of this application. For example, the κ chain or a variant thereof can be used in the compounds and methods of this application.

[0168] Methods and uses

[0169] On the other hand, this application also provides methods for preventing and / or treating diseases and / or conditions, which may include administering to a subject in need the antigen-binding protein, the polypeptide molecule, the nucleic acid molecule, the carrier, the cell, the transformant, the fusion protein, the immunoconjugate, and / or the pharmaceutical composition described in this application.

[0170] In this application, the application can be performed in various ways, such as intravenous, intratumoral, intraperitoneal, subcutaneous, intramuscular, local, or intradermal application.

[0171] On the other hand, this application also provides a method for detecting AXL in a sample, the method comprising administering the antigen-binding protein, the polypeptide molecule, the nucleic acid molecule, the carrier, the cell, the transformant, the fusion protein, the immunoconjugate, and / or the pharmaceutical composition.

[0172] In some cases, the method for detecting AXL in a sample may be an in vitro method. For example, the antigen-binding protein described in this application is brought into contact with an isolated sample to detect the presence and / or content of AXL in the sample. In some cases, the method for detecting AXL in a sample is for non-therapeutic purposes. In some cases, the method for detecting AXL in a sample is not a diagnostic method.

[0173] The embodiments described below are not intended to be limited by any theory, but are merely for illustrating the antigen-binding protein, preparation method and use of this application, and are not intended to limit the scope of the invention.

[0174] Example

[0175] Example 1: Preparation of recombinant protein for mouse immunization, screening, and activity detection of anti-human AXL antibody

[0176] The AXL immunized mouse antigens P00285-Fc (hu-AXL-ECD-Fc, SEQ ID NO: 27), P00285-His (hu-AXL-ECD-his, SEQ ID NO: 28), P00287-his (cyno-AXL-ECD-his, SEQ ID NO: 29), and P00287-Fc (cyno-AXL-ECD-Fc, SEQ ID NO: 30) were all prepared using conventional protein expression and purification methods.

[0177] Antibody screening antigens human AXL P00285-His (SEQ ID NO: 28) and P00285-His-Biotin (SEQ ID NO: 28), recombinant proteins of the extracellular region of Rhesus macaque AXL P00287-his (SEQ ID NO: 29) and P00287-his-Biotin (SEQ ID NO: 29), and mouse AXL screening antigens P00400-his (muAXL-ECD-his, SEQ ID NO: 31) and P00400-his-biotin (muAXL-ECD-his-biotin, SEQ ID NO: 31) were all prepared using conventional protein expression and purification methods.

[0178] The amino acid sequence of the extracellular domain of human AXL is amino acids 26-451 of Uniprot ID: P30530 (SEQ ID NO: 32), which was prepared using conventional protein expression and purification methods and used for the detection of antibody activity in this application.

[0179] The results of the immunoantigen activity assay are shown in Figures 1A-1C and Tables 1-3, with lpilimumab (purchased from Selleck, catalog number: A2001) used as a negative control antibody.

[0180] Table 1. Immunogeneity assay results of AXL-immunized mouse antigens (antibody: Enapotamb)

[0181] Table 2. Immunogeneity assay results for antibody screening antigens (antibody: Tilvestamab)

[0182] Table 3. Immunogeneity assay results for antibody screening antigens (antibody: Tilvestamab)

[0183] Enapotamab (sequence derived from EP3169706A1, heavy chain sequence SEQ ID NO: 33, light chain sequence SEQ ID NO: 34) and Tilvestamab (sequence derived from CN109311997B, heavy chain sequence SEQ ID NO: 35, light chain sequence SEQ ID NO: 36) were used as AXL control antibodies and prepared using conventional antibody expression and purification methods.

[0184] SEQ ID NO: 27 P00285-Fc

[0185] SEQ ID NO: 28 P00285-his

[0186] SEQ ID NO: 29 P00287-his

[0187] SEQ ID NO: 30 P00287-Fc

[0188] SEQ ID NO: 31 P00400-his

[0189] SEQ ID NO: 32 P00285 (The amino acid sequence of the extracellular domain of human AXL is amino acids 26-451 of Uniprot ID: P30530)

[0190] SEQ ID NO: 33 Enapotamab heavy chain

[0191] SEQ ID NO: 34 Enapotamab Light Chain

[0192] SEQ ID NO: 35 Tilvestamab heavy chain

[0193] SEQ ID NO: 36 Tilvestamab light chain

[0194] Example 2 Construction of hAXL overexpression cell line

[0195] The human AXL overexpression monoclonal cell line HEK-293T-hu-AXL was constructed and its activity was tested by Sanyou Biopharmaceutical (Shanghai) Co., Ltd., catalog number: C2204057. The binding activity of the overexpression cell line is shown in Figure 2A.

[0196] The human AXL overexpression monoclonal cell line CHOK1-hu-AXL was constructed by Pengbo Biotechnology Co., Ltd., catalog number: RD00881. The binding activity of the overexpression cell line is shown in Figure 2B.

[0197] Both hAXL overexpression cell lines expressed amino acids 26-451 of the human AXL extracellular domain amino acid sequence Uniprot ID: P30530 (i.e., P00285, SEQ ID NO: 32).

[0198] Example 3: Phage Display Library Construction

[0199] Balb / c mice were immunized with the AXL immunization mouse antigens P00285-Fc, P00285-His, P00287-Fc(IgG1), and P00287-His antigens prepared in Example 1. The mice were divided into 5 groups of 3 mice each. The initial immunization used Freund's complete adjuvant emulsified antigen (100 μg / mouse), and subsequent immunizations used Freund's incomplete adjuvant (50 μg / mouse). Immunizations were administered via intraperitoneal and subcutaneous injections at multiple sites every two weeks for a total of 5 rounds. Serum titers were measured by ELISA after immunization. The results showed that the serum titers of all immunized mice reached over 40,000, indicating successful immunization. Mouse spleen cells could be used for subsequent phage display library construction.

[0200] RNA was extracted from mouse spleen tissue using the chloroform method, and cDNA was synthesized using the TaKaRa reverse transcription kit. Degenerate primers were designed based on the germline gene sequences of the heavy and light chains, and antibody heavy and light chain variable region gene fragments were amplified by PCR. After fusion PCR ligation, these fragments were digested with the phage display vector PMID-1121B, recovered, and ligated. The ligation product was transformed into competent *E. coli* SS320 cells and plated on ampicillin-resistant 2-YT agar plates. Finally, using VSCM13-assisted phage packaging, a phage display library of 12 anti-human AXL mouse antibody genes was successfully constructed.

[0201] Example 4: Phage Display Library Screening

[0202] Using conventional phage display library technology, and with the screening antigen protein prepared in Example 1 as the screening antigen and HEK-293T-hu-AXL cells prepared in Example 2 as the screening cells, the library was screened, and multiple antibody molecules that specifically bind to human AXL were obtained using the following screening method.

[0203] 4.1. Screening of antibody gene phage display libraries using magnetic bead method

[0204] Magnetic bead screening involves biotin-labeling antigen proteins and then binding them to magnetic beads conjugated with streptavidin. The process involves incubating, washing, and eluting the antigen-bound magnetic beads and a phage display library containing antibody genes. Typically, 3-4 rounds of screening are performed, resulting in a large enrichment of antigen-specific monoclonal antibodies. In this example, biotin-labeled antigen proteins P00285-His-biotin, P00287-his-Biotin, and P00400-his-biotin were used for phage display library screening. Three rounds of screening were performed, with each round using a 3-fold gradient of decreasing antigen concentration to obtain anti-human AXL antibody clones with high affinity.

[0205] 4.2. Screening of antibody gene phage display libraries using the immunotube method

[0206] Immunotube screening involves coating the surface of an immunotube with highly absorbent antigen proteins P00285-His, P00287-his, and P00400-his. A phage-displaying antibody library is then added to the immunotube, followed by incubation, washing, and elution with the adsorbed antigen proteins. After 2-4 rounds of screening, specific monoclonal antibodies against the antigens are finally enriched. In this example, three rounds of screening are performed, with each round using a 3-fold gradient of decreasing antigen concentrations to obtain anti-human AXL antibody clones with high affinity.

[0207] 4.3. Cellular screening of antibody gene phage display libraries

[0208] Cell-binding screening involves adding a phage display antibody library to a cell flask containing HEK-293T-hu-AXL cells, followed by incubation, washing, and elution with target cells adsorbed in the flask. In this embodiment, three rounds of screening were performed, with each round using a three-fold gradient of antigen concentration decreasing to obtain anti-human AXL antibody clones with high affinity.

[0209] Example 5: Monoclonal Antibody Screening

[0210] Based on the detection results of the output set from the initial screening, the phage pools eluted in each round were subjected to ELISA detection to evaluate the enrichment effect. Clones were randomly selected from the phage pools selected in each round for sequence analysis. Combining the enrichment effect and the reproducibility ratio of the measured sequences, an appropriate round was selected for single clone selection.

[0211] Monoclonal antibodies from the second and third rounds of preliminary screening were selected for initial screening using Fab ELISA. The antigens P00285-His, P00287-his, and P00400-his were used for plate coating to screen for monoclonal antibodies, ultimately identifying the specific human AXL-binding monoclonal antibody mA239. The amino acid sequences of the CDR regions of mA239 antibody VH and VL are shown in Table 4. The CDR sequence was determined using the Kabat definition method.

[0212] Table 4: CDR and variable region amino acid sequences of AXL-targeting antibodies (KABAT protocol)

[0213] Example 6: Preparation of human-mouse chimeric antibody

[0214] The coding sequences for the heavy chain constant region and the light chain constant region κ of the human-mouse chimeric clone were introduced into the pCDNA3.4 plasmid. The coding sequences for the heavy chain and light chain variable regions of the aforementioned antibody mA239 were synthesized and introduced into the pCDNA3.4 plasmid containing the introduced constant region coding sequences, and the correct clone was confirmed by sequencing. The heavy chain and light chain expression plasmids were mixed and transfected into Expi CHO expression cells to obtain the anti-human AXL human-mouse chimeric antibody A239. The heavy chain (HC) amino acid sequence of the anti-human AXL human-mouse chimeric antibody A239 is SEQ ID NO:18, and the light chain (LC) amino acid sequence is SEQ ID NO:23.

[0215] SEQ ID NO:16 Human clone heavy chain constant region

[0216] SEQ ID NO:17 Human clone light chain constant region κ

[0217] SEQ ID NO:18 A239 heavy chain

[0218] SEQ ID NO:23 A239 Light Chain

[0219] Example 7: Detection of the binding activity of anti-human AXL antibody to target cells

[0220] After trypsin digestion, CHOK1-hu-AXL and MDA-MB-231 cell suspensions (human breast cancer cells) were obtained. Cell density was adjusted, and 100 μL was seeded into each well of a 96-well U-plate (1E5 cells / well). FACS Buffer (PBS + 2% FBS) was prepared using PBS at pH 7.4. Antibody was diluted with FACS Buffer to prepare a 1× antibody solution, with a maximum concentration of 100 nM, and diluted 3-fold. The cell plate was centrifuged, and 100 μL of the 1× antibody solution was added to each well. The plate was incubated at 4°C for 30 min. The plate was washed twice with FACS Buffer. Secondary antibody PE labelled anti-human IgG Fc (purchased from Biolegend; catalog number: 410708) was diluted 1:100 with FACS Buffer and incubated at 4°C in the dark for 30 min. The plate was washed twice with FACS Buffer. Cells were resuspended and analyzed by flow cytometry.

[0221] The results are shown in Figures 3A-3B and Table 5. A239 showed superior binding activity to both MDA-MB-231 and CHOK1-hu-AXL cells compared to the control molecule Tilvestamab.

[0222] Table 5. Binding activity of anti-human AXL antibody to human AXL-overexpressing cells.

[0223] Tumor cell lines with different levels of human AXL expression, including MDA-MB-231, PC-9 (human lung adenocarcinoma cells), Calu-1 (lung squamous cell carcinoma), and LCLC-103H (lung large cell carcinoma), were selected. Their antigen expression levels are shown in Figure 4. Cell suspensions were obtained after trypsin digestion, and the cell density was adjusted to 100 μL per well in 96-well U-type plates (1E5 cells / well). FACS Buffer (PBS + 2% FBS) was prepared using PBS at pH 7.4. Antibody was diluted with FACS Buffer to prepare a 1× antibody solution, with a maximum concentration of 30 nM, and diluted 3-fold. The cell plates were centrifuged, and 100 μL of the 1× antibody solution was added to each well. The plates were incubated at 4°C for 30 min. The plates were washed twice with FACS Buffer. Secondary antibody PE labelled anti-human IgG Fc (purchased from Biolegend; catalog number: 410708) was diluted 1:100 with FACS Buffer and incubated at 4°C in the dark for 30 min. The plates were washed twice with FACS Buffer. After resuspending the cells, flow cytometry was used for detection.

[0224] The results are shown in Figures 5A-5D and Table 6: A239 showed superior binding ability to the control molecule Enapotamab in all four tumor cell lines.

[0225] Table 6. Binding activity of anti-human AXL antibodies to cells with different levels of human AXL expression.

[0226] Example 8: Detection of pH-dependent binding activity of anti-human AXL antibody

[0227] After trypsin digestion, CHOK1-hu-AXL (purchased from: Pengbo Biotechnology, catalog number: RD00881) cell suspension was obtained. The cell density was adjusted and 100 μL was seeded into 96-well U-plates (1E5 cells / well) for later use.

[0228] FACS buffers (PBS + 2% FBS) were prepared using PBS at pH 5.6 and 7.4, respectively. Antibodies were diluted with the two different pH FACS buffers to prepare 1× antibody solutions, with a maximum concentration of 100 nM, and 3-fold dilutions were performed. Cell plates were centrifuged, and 100 μL of 1× antibody solution at different pH values ​​was added to each well. The plates were incubated at 4°C for 30 min. The plates were washed twice with FACS buffer corresponding to the pH of the 1× antibody solution. PE labelled anti-human IgG Fc (purchased from Biolegend; catalog number: 410708) was diluted 1:100 with FACS buffer corresponding to the pH of the 1× antibody solution and incubated at 4°C in the dark for 30 min. The plates were then washed twice with FACS buffer corresponding to the pH of the 1× antibody solution. Cells were resuspended and analyzed by flow cytometry.

[0229] The results are shown in Figures 6A-6B and Table 7: As the pH value decreased, the Max MFI value of A239 decreased slightly, but the EC... 50 No significant changes were observed, indicating that the antibody has stable cell-binding ability.

[0230] Table 7. pH-dependent binding activity of anti-human AXL antibodies Note: NA indicates not detected.

[0231] Example 9: Detection of anti-human AXL antibody endocytosis activity

[0232] After trypsin digestion, CHOK1-hu-AXL (purchased from: Pengbo, catalog number: RD00881) and MDA-MB-231 cell suspensions were obtained. The cell density was adjusted and 50 μL was seeded into each well of a 96-well permeable plate (8000 cells / well). After cross-shading, the cell plate was placed in an incubator at 37°C and 5% CO2.

[0233] Adjust the antibody concentration to 200 nM (final concentration 50 nM) using cell culture medium and vortex to mix. Add the Zenon dye to the cell culture medium. TM pHrodoTM iFL Red (purchased from Invitrogen, catalog number: Z25612) was diluted 12.5 times and mixed thoroughly by pipetting repeatedly. The antibody and dye were mixed separately at a 1:1 ratio and mixed thoroughly by pipetting repeatedly. The mixture was incubated at 37°C for 30 minutes. 50 μL of the antibody-dye mixture was added to each well of the cell culture plate, and the plate was shaken horizontally before being returned to the incubator. Monitoring was performed using an Incucyte instrument for 48 hours.

[0234] The results are shown in Figures 7A-7D: The endocytic activity of A239 molecule was superior to that of the control molecule in both MDA-MB-231 cells and CHOK1-hu-AXL cells, and the endocytosis results in the tumor cell line MDA-MB-231 showed that the endocytosis rate of A239 was superior to that of the control molecule.

[0235] Example 10: Species Cross-Reaction of Anti-Human AXL Antibody

[0236] Dilute antigen cyno AXL (purchased from R&D, catalog number: 11144-AX-050) with 1×PBS to a concentration of 1 μg / mL, and add 50 μL / well to a 96-well ELISA plate (purchased from Thermo, catalog number: 442404). Incubate overnight at 4°C. Wash the plate three times with 1×PBST, add blocking buffer (PBS + 5% FBS), and block at 37°C for 1 h. After washing three times with 1×PBST, add 100 μL / well of serially diluted antibody solution (maximum concentration 100 nM, 3-fold dilution) in PBS + 2% FBS, and incubate at 37°C for 2 h. Wash the plate three times with 1×PBST, add secondary antibody (Anti-human-IgG-Fc-HRP (purchased from CST; catalog number: 32935S), and incubate at 37°C for 2 hours. Wash the plate three times with 1×PBST, and add 100 μL of TMB to each well. After 5-10 minutes of color development, add 100 μL of stop solution to terminate the reaction, and immediately measure OD450.

[0237] The results are shown in Figure 8 and Table 8, indicating that A239 and cyno AXL have good binding properties.

[0238] Table 8. Species cross-reactivity of anti-human AXL antibodies against antigen cyno AXL.

[0239] Example 11 Detection of Anti-human AXL Antibody Ligand Blocking Activity

[0240] Following the instructions of the biotin-conjugation kit (Abcam, catalog number: ab201795), GAS6 was labeled (R&D, catalog number: 885-GSB) to obtain GAS6-biotin. The binding activity of GAS6-biotin to CHOK1-hu-AXL cells (GenScript, catalog number: RD00881) was detected using FACS. The specific procedures were as follows: GAS6-biotin was diluted with FACS Buffer to prepare a 1×GAS6-biotin solution, with a maximum concentration of 111 nM, diluted 3-fold. The cell plate was centrifuged, and 100 μL of 1×GAS6-biotin solution was added to each well. The plate was incubated at 4°C for 30 min. The plate was washed twice with FACS Buffer. Secondary antibody SA-AF488 (Invitrogen, catalog number: S32354) was diluted 1:1000 with FACS Buffer and incubated at 4°C in the dark for 30 min. The plate was washed twice with FACS Buffer. After resuspending the cells, flow cytometry was used to detect the lowest saturation binding concentration, as shown in Figure 9A. The EC50 was 1.32 nM, so 2 nM GAS6-biotin was selected for subsequent blocking experiments.

[0241] CHOK1-hu-AXL cell suspension was obtained after trypsin digestion. Cell density was adjusted, and 100 μL was seeded into each well of a 96-well U-plate (1E5 cells / well). GAS6-biotin was diluted with FACS Buffer to a final concentration of 2 nM. The cell culture plates were centrifuged, and 100 μL of 2 nM GAS6-biotin solution was added to each well. The plates were incubated at 4°C for 30 min. The plates were washed twice with FACS Buffer. A 1× antibody solution was prepared with FACS Buffer, with a maximum concentration of 100 nM, and diluted 5-fold. 100 μL of the 1× antibody solution was added to each well, and the plates were incubated at 4°C for 30 min. The plates were washed twice with FACS Buffer. Secondary antibody SA-AF488 (purchased from Invitrogen; catalog number: S32354) was diluted 1:1000 with FACS Buffer and incubated at 4°C in the dark for 30 min. The plates were washed twice with FACS Buffer. Cells were resuspended and analyzed by flow cytometry.

[0242] As shown in Figure 9B, A239 can effectively block the binding of GAS6 and AXL, and its blocking activity is superior to that of Tilvestamab.

[0243] Example 12 Detection of Anti-human AXL Antibody Signaling Pathway Blocking Activity

[0244] Calu-1 cell suspension was obtained after trypsin digestion. The cell density was adjusted, and 2 mL was seeded into each well of a 6-well plate (6E5 cells / well). After cross-linking, the cell culture plate was placed in an incubator at 37°C and 5% CO2. After culturing for 6 hours (until the cells adhered), the culture medium was aspirated, the cells were washed twice, and then 2 mL of serum-free culture medium was added for overnight starvation.

[0245] The next day, the serum-free medium was replaced (800 μL of fresh serum-free medium was added to each well). 10× Gas6 (final concentration 0.3 μg / mL, purchased from R&D, catalog number: 885-GSB) and 10× antibody solution (final concentration 50 nM) were prepared using serum-free medium. 100 μL of 10× antibody solution was added to each well and incubated at 37°C for 60 min; then 100 μL of 10× Gas6 solution was added to each well and incubated at 37°C for 60 min. Protein samples were collected and subjected to SDS-PAGE. After transfer, the membrane was blocked with 5% BSA and shaken on a shaker at room temperature for 1 hour. The primary antibody (Phospho-Axl(Tyr702)(D12B2) Rabbit mAb, purchased from CST, catalog number: 5724S; Recombinant Anti-AKT1 (phospho S473) antibody, purchased from Abcam, catalog number: Ab81283; Anti-GAPDH antibody, purchased from Abcam, catalog number: Ab9485) was diluted 1:1000 with 5% BSA solution and incubated overnight at 4°C. It was washed three times with 1×TBST, shaking for 5 minutes each time. The secondary antibody (Anti-rabbit IgG, HRP-linked Antibody, purchased from CST, catalog number: 7074S) was diluted 1:1000 with 5% BSA solution and incubated for 2 hours at room temperature with shaking, washed three times with 1×TBST, shaking for 5 minutes each time. Imaging was performed using ECL developing solution.

[0246] The results are shown in Figures 10A-10C: A239 and Tilvestamab have similar induction levels, but weaker self-activation ability.

[0247] Example 13 Humanization of Anti-human AXL Antibody

[0248] The variable regions of the light and heavy chains of the mA239 antibody were compared with sequences in the human Germline database. The optimal humanization template was selected based on factors including Germline V and J gene similarity, CDR segment structure and conformational matching, Germline expression and stability, and Germline immunogenicity. Specifically, the human Germline template IGHIV1-46*01 was used for the antibody heavy chain, and the human Germline template IGKV1-39*01 was used for the light chain.

[0249] After determining the template, the CDR of the murine antibody was transplanted onto the corresponding human template. Based on this, structural analysis was used to calculate the FR amino acids in the murine antibody that would maintain the original conformation, and these were then reverse-mutated to preserve its affinity. Further simulations of the structures before and after humanization were performed to fine-tune the number of reverse-mutated amino acids to achieve optimal binding activity, low immunogenicity, and high stability. The amino acid sequence of the variable region of the anti-human AXL humanized antibody is shown in Table 9.

[0250] Table 9. Amino acid sequence listing of the variable region of anti-human AXL humanized antibody.

[0251] Example 14 Preparation of anti-human AXL humanized antibody

[0252] The expression and purification methods for the anti-human AXL humanized monoclonal antibody are as described in Example 6. The heavy and light chain sequences of each antibody are shown in Table 10.

[0253] Table 10. Heavy and light chain sequences of humanized antibodies

[0254] SEQ ID NO:19 VH1 heavy chain

[0255] SEQ ID NO:20 VH2 heavy chain

[0256] SEQ ID NO:21 VH3 heavy chain

[0257] SEQ ID NO:22 VH4 heavy chain

[0258] SEQ ID NO:24 VL1 Light Chain

[0259] SEQ ID NO:25 VL2 Light Chain

[0260] SEQ ID NO:26 VL3 light chain

[0261] Example 15 Detection of Cellular Binding of Humanized Anti-human AXL Antibody

[0262] HEK-293T-hu-AXL cell suspension was obtained after trypsin digestion. Cell density was adjusted, and 100 μL was seeded into each well of a 96-well U-plate (1E5 cells / well). FACS Buffer (PBS + 2% FBS) was prepared using PBS at pH 7.4. Antibody was diluted with FACS Buffer to prepare a 1× antibody solution, with a maximum concentration of 20 nM, and diluted 4-fold. The cell plate was centrifuged, and 100 μL of the 1× antibody solution was added to each well. The plate was incubated at 4°C for 30 min. The plate was washed twice with FACS Buffer. Secondary antibody PE labelled anti-human IgG Fc (purchased from Biolegend; catalog number: 410708) was diluted 1:100 with FACS Buffer and incubated at 4°C in the dark for 30 min. The plate was washed twice with FACS Buffer. Cells were resuspended and analyzed by flow cytometry.

[0263] The results are shown in Figure 11 and Table 11. All anti-human AXL humanized antibodies have good cell binding ability.

[0264] Table 11 Cell binding detection of anti-human AXL humanized antibodies

[0265] Example 16: Biacore Affinity Detection of Humanized Anti-Human AXL Antibody

[0266] Prepare the NTA sensor chip and equilibrate the chip surface with HBS-EP+ buffer. Dilute protein P00285-His to 20 μM with HBS-EP+ buffer. After capturing P00285-His on the chip, rinse with HBS-EP+ buffer to stabilize the surface. Dilute each antibody (100 nM, 20 nM, 10 nM, 5 nM, 1 nM, 0 nM) with HBS-EP+ buffer. Set the program, place the sample plate, and monitor the reaction signal in real time at a flow rate of 10 μL / min to obtain binding and dissociation curves. After dissociation is completed in each experimental cycle, regenerate the sensor chip surface with glycine-hydrochloric acid buffer (pH 2.5-3.0). Analyze the antibody-antigen binding kinetics based on the fitted curves.

[0267] The results are shown in Table 12. All six humanized antibodies showed good affinity.

[0268] Table 12. Affinity levels of humanized anti-human AXL antibodies (Biacore)

[0269] Example 17 Detection of ligand blocking activity of humanized anti-human AXL antibody

[0270] The blocking activity of VH1-VL3, VH2-VL3, VH3-VL3, VH4-VL3, VH3-VL1 and VH1-VL2 against ligand GAS6 was detected in HEK-293T-hu-AXL and Calu-1 cells, as described in Example 11.

[0271] The results are shown in Figures 12A-12B and Table 13. There was no significant difference in the blocking effect of the humanized antibody on HEK293T-hu-AXL cells and Calu-1 cells.

[0272] Table 13 Ligand blocking activity of humanized anti-human AXL antibodies

[0273] Example 18: Detection of Signaling Pathway Blocking Activity of Humanized Antibody against Human AXL

[0274] The blocking activity of VH3-VL1, VH4-VL3 and VH2-VL3 against ligand GAS6 was detected on Calu-1 cells, and the specific operation was performed according to Example 12, where Ena represents Enapotamab and Til represents Tilvestamab.

[0275] The results are shown in Figures 13A and 13B. The humanized antibodies of A239 all exhibited autophosphorylation of AXL. Among them, VH2-VL3 showed weaker autophosphorylation of AXL than the parent A239 and also weaker than Enapotamab, indicating that the antigen-binding protein of this application can effectively block the Gas6 / Axl signaling pathway.

[0276] Example 19 Detection of endocytic activity of anti-human AXL humanized antibody

[0277] Calu-1 cell suspension was obtained after trypsin digestion. Cell density was adjusted, and 50 μL was seeded per well into a 96-well permeable plate (8000 cells / well). After cross-linking, the plate was placed in a 37°C, 5% CO2 incubator. pH-sensitive IgG labeling reagents plus powder (purchased from UA-BIO, catalog number: UA070080) was thoroughly dissolved in sterile water to obtain a 6000 nM dye. This dye was then diluted 12.5 times to 480 nM with cell culture medium, and the mixture was repeatedly pipetted to mix. The antibodies were adjusted to a concentration of 160 nM (final concentration 40 nM) with cell culture medium and vortexed to mix. The antibodies and dyes were mixed 1:1, and the mixture was repeatedly pipetted to mix. The plates were incubated at 37°C for 60 minutes. 50 μL of the antibody-dye mixture was added to each well of the cell plate, and the plates were shaken horizontally before being returned to the incubator. Cells were digested with trypsin at 6 hours and 24 hours, respectively. After centrifugation was stopped by culture medium, the cells were resuspended and the FITC channel signal was detected by flow cytometry.

[0278] As shown in Figure 14, the humanized antibody VH2-VL3 exhibited superior endocytic properties compared to the A239 parent antibody and the control antibody.

[0279] Example 20: Identification of Antigen Epitopes of Humanized Anti-Human AXL Antibody

[0280] Label the antibodies VH2-VL3 and Enapotamab according to the AF647 labeling kit instructions (purchased from Invitrogen, catalog number: A20186). Calu-1 cell suspensions were obtained after trypsin digestion, and the cell density was adjusted to 100 μL per well in a 96-well U-plate (1E5 cells / well). FACS Buffer (PBS + 2% FBS) was prepared using PBS at pH 7.4. 1×AF647 labeled antibody was prepared using FACS Buffer (VH2-VL3-AF647 maximum concentration 8.3 nM, 3-fold serial dilution; Enapotamab-AF647 maximum concentration 2.5 nM, 3-fold serial dilution). The cell plates were centrifuged, and 100 μL of the 1×AF647 labeled antibody solution was added to each well. The plates were incubated at 4°C in the dark for 30 min. The plates were washed twice with FACS Buffer. After resuspending the cells, flow cytometry was used to detect the lowest saturation binding concentration, as shown in Figures 15A and 15B. The final concentrations of Enapotamab-AF647 and VH2-VL3-AF647 were selected as 0.7 nM for subsequent antigen epitope competition experiments.

[0281] VH2-VL3-AF647: Prepare 2× other competitive antibody solutions (maximum final concentration 200 nM, serially diluted 3-fold) with FACS Buffer, add 50 μL to each well of the cell plate, and incubate at 4°C for 30 min; prepare 2× AF647-labeled antibody solutions (final concentration 0.7 nM) with FACS Buffer, add 50 μL to each well of the cell plate, and incubate at 4°C in the dark for 30 min. Wash the plate twice with FACS Buffer. Analyze the cells by flow cytometry after resuspending them.

[0282] Enapotamab-AF647: Prepare 2× other competitive antibody solutions (maximum final concentration 60 nM, serially diluted 3-fold) with FACS Buffer, add 50 μL to each well of the cell plate, and incubate at 4°C for 30 min; prepare 2× AF647-labeled antibody solutions (final concentration 0.7 nM) with FACS Buffer, add 50 μL to each well of the cell plate, and incubate at 4°C in the dark for 30 min. Wash the plate twice with FACS Buffer. After resuspending the cells, perform flow cytometry analysis.

[0283] The results are shown in Figures 15C and 15D. The epitopes of the A239 (VH2-VL3) antibody overlap with those of Tilvestamab, but are completely different from those of Enapotamab.

[0284] While specific embodiments of this application have been described above, those skilled in the art should understand that these are merely illustrative examples, and various changes or modifications can be made to these embodiments without departing from the principles and essence of this application. Therefore, the scope of protection of this application is defined by the appended claims.

Claims

1. An antigen-binding protein capable of specifically binding to AXL protein, said antigen-binding protein comprising a heavy chain variable region VH and a light chain variable region VL, said heavy chain variable region VH comprising HCDR1, HCDR2, and HCDR3, and said light chain variable region VL comprising LCDR1, LCDR2, and LCDR3; wherein, The HCDR1 contains the amino acid sequence shown in SEQ ID NO:1, the HCDR2 contains the amino acid sequence shown in SEQ ID NO:2, and the HCDR3 contains the amino acid sequence shown in SEQ ID NO:3; and / or, the LCDR1 contains the amino acid sequence shown in SEQ ID NO:4, the LCDR2 contains the amino acid sequence shown in SEQ ID NO:5, and the LCDR3 contains the amino acid sequence shown in SEQ ID NO:

6.

2. The antigen-binding protein according to claim 1, wherein the heavy chain variable region VH comprises an amino acid sequence as shown in SEQ ID NO: 7, 8, 9, 10 or 11, or, The heavy chain variable region VH comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 7, 8, 9, 10, or 11; and the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity maintains at least the same antigen-binding function as the original sequence. Preferably, the amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity does not involve any alteration to the CDR sequence.

3. The antigen-binding protein according to any one of claims 1-2, wherein the light chain variable region VL comprises an amino acid sequence as shown in SEQ ID NO: 12, 13, 14 or 15, or, The light chain variable region VL comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 12, 13, 14, or 15; the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity maintains at least the same antigen-binding function as the original sequence, preferably, the amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity does not involve any alteration to the CDR sequence.

4. The antigen-binding protein according to any one of claims 1-3, wherein the heavy chain variable region VH has the amino acid sequence shown in SEQ ID NO:7 and the light chain variable region VL has the amino acid sequence shown in SEQ ID NO:12, or the heavy chain variable region VH has the amino acid sequence shown in SEQ ID NO:8, 9, 10 or 11 and the light chain variable region VL has the amino acid sequence shown in SEQ ID NO:13, 14 or 15; Preferably, the VH contains the amino acid sequence shown in SEQ ID NO:8, and the VL contains the amino acid sequence shown in SEQ ID NO:13; Alternatively, the VH contains an amino acid sequence as shown in SEQ ID NO:9, and the VL contains an amino acid sequence as shown in SEQ ID NO:13; Alternatively, the VH contains an amino acid sequence as shown in SEQ ID NO:10, and the VL contains an amino acid sequence as shown in SEQ ID NO:13; Alternatively, the VH contains an amino acid sequence as shown in SEQ ID NO:11, and the VL contains an amino acid sequence as shown in SEQ ID NO:13; Alternatively, the VH contains an amino acid sequence as shown in SEQ ID NO:8, and the VL contains an amino acid sequence as shown in SEQ ID NO:14; Alternatively, the VH contains an amino acid sequence as shown in SEQ ID NO:9, and the VL contains an amino acid sequence as shown in SEQ ID NO:14; Alternatively, the VH contains an amino acid sequence as shown in SEQ ID NO:10, and the VL contains an amino acid sequence as shown in SEQ ID NO:14; Alternatively, the VH contains an amino acid sequence as shown in SEQ ID NO:11, and the VL contains an amino acid sequence as shown in SEQ ID NO:14; Alternatively, the VH contains an amino acid sequence as shown in SEQ ID NO:8, and the VL contains an amino acid sequence as shown in SEQ ID NO:15; Alternatively, the VH contains an amino acid sequence as shown in SEQ ID NO:9, and the VL contains an amino acid sequence as shown in SEQ ID NO:15; Alternatively, the VH contains an amino acid sequence as shown in SEQ ID NO:10, and the VL contains an amino acid sequence as shown in SEQ ID NO:15; Alternatively, the VH may contain an amino acid sequence as shown in SEQ ID NO:11, and the VL may contain an amino acid sequence as shown in SEQ ID NO:

15.

5. The antigen-binding protein according to any one of claims 1-4, comprising an antibody or an antigen-binding fragment thereof, preferably, the antibody being selected from one or more of the group consisting of: fully human antibodies, humanized antibodies, chimeric antibodies, proantibodies, monospecific antibodies, bispecific antibodies, multispecific antibodies, monoclonal antibodies, and polyclonal antibodies. Preferably, the antigen-binding fragment is selected from one or more of the following group: full-length antibody, Fab, Fab', F(ab')2, F(ab)2, scFv, di-scFv and dAb.

6. The antigen-binding protein according to any one of claims 1-5, wherein the AXL is human AXL.

7. The antigen-binding protein according to any one of claims 1-6, further comprising an antibody heavy chain constant region, preferably a constant region derived from human IgG, more preferably a constant region derived from human IgG1.

8. The antigen-binding protein of claim 7, wherein the antibody heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO:16, or, The constant region of the antibody heavy chain comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO:16; the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity maintains at least the same function as the original sequence.

9. The antigen-binding protein according to claims 1-8, comprising a heavy chain, wherein the heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 18, 19, 20, 21 or 22.

10. The antigen-binding protein according to any one of claims 1-9, comprising a constant region of an antibody light chain, preferably a constant region derived from human Igκ.

11. The antigen-binding protein of claim 10, wherein the constant region of the antibody light chain comprises the amino acid sequence shown in SEQ ID NO:17, or, The constant region of the antibody light chain comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO:17; the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity maintains at least the same function as the original sequence, preferably, the amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity does not involve any alteration to the CDR sequence.

12. The antigen-binding protein according to any one of claims 1-11, comprising a light chain, wherein the light chain comprises an amino acid sequence as shown in SEQ ID NO: 23, 24, 25 or 26.

13. The antigen-binding protein according to any one of claims 1-12, wherein the amino acid sequence of the heavy chain is as shown in SEQ ID NO:18, and the amino acid sequence of the light chain is as shown in SEQ ID NO:23, or, The amino acid sequence of the heavy chain is as shown in SEQ ID NO:19, 20, 21 or 22, and the amino acid sequence of the light chain is as shown in SEQ ID NO:24, 25 or 26. Preferably, The heavy chain comprises the amino acid sequence shown in SEQ ID NO:19, and the light chain comprises the amino acid sequence shown in SEQ ID NO:24; Alternatively, the heavy chain comprises an amino acid sequence as shown in SEQ ID NO:20, and the light chain comprises an amino acid sequence as shown in SEQ ID NO:24; Alternatively, the heavy chain comprises an amino acid sequence as shown in SEQ ID NO:21, and the light chain comprises an amino acid sequence as shown in SEQ ID NO:24; Alternatively, the heavy chain comprises an amino acid sequence as shown in SEQ ID NO:22, and the light chain comprises an amino acid sequence as shown in SEQ ID NO:24; Alternatively, the heavy chain comprises an amino acid sequence as shown in SEQ ID NO:19, and the light chain comprises an amino acid sequence as shown in SEQ ID NO:25; Alternatively, the heavy chain comprises an amino acid sequence as shown in SEQ ID NO:20, and the light chain comprises an amino acid sequence as shown in SEQ ID NO:25; Alternatively, the heavy chain comprises an amino acid sequence as shown in SEQ ID NO:21, and the light chain comprises an amino acid sequence as shown in SEQ ID NO:25; Alternatively, the heavy chain comprises an amino acid sequence as shown in SEQ ID NO:22, and the light chain comprises an amino acid sequence as shown in SEQ ID NO:25; Alternatively, the heavy chain comprises an amino acid sequence as shown in SEQ ID NO:19, and the light chain comprises an amino acid sequence as shown in SEQ ID NO:26; Alternatively, the heavy chain comprises an amino acid sequence as shown in SEQ ID NO:20, and the light chain comprises an amino acid sequence as shown in SEQ ID NO:26; Alternatively, the heavy chain comprises an amino acid sequence as shown in SEQ ID NO:21, and the light chain comprises an amino acid sequence as shown in SEQ ID NO:26; Alternatively, the heavy chain may contain an amino acid sequence as shown in SEQ ID NO:22, and the light chain may contain an amino acid sequence as shown in SEQ ID NO:

26.

14. A fusion protein or immunoconjugate comprising the antigen-binding protein of any one of claims 1-13.

15. An isolated nucleic acid encoding an antigen-binding protein or a fusion protein or immunoconjugate as described in any one of claims 1-13.

16. A vector comprising the isolated nucleic acid of claim 15, preferably an expression vector.

17. A cell comprising the isolated nucleic acid of claim 15 or the vector of claim 16, wherein the cell is optionally a eukaryotic cell, preferably a mammalian cell, more preferably a CHO cell or a cell line derived therefrom, such as ExpiCHO cells.

18. A transformant comprising the isolated nucleic acid as claimed in claim 15 or the vector as claimed in claim 16, wherein the transformant is preferably a non-animal or non-plant variety; preferably, the host cell of the transformant is a eukaryotic cell, more preferably a mammalian cell, and more preferably a CHO cell or a cell line derived therefrom, such as Expi CHO cells.

19. A method for preparing the antigen-binding protein according to any one of claims 1-13, the method comprising culturing the cells as described in claim 17 or the transformant as described in claim 18 under conditions that cause the antigen-binding protein to be expressed.

20. A pharmaceutical composition comprising the antigen-binding protein of any one of claims 1-13, the fusion protein or immunoconjugate of claim 14, the isolated nucleic acid of claim 15, the carrier of claim 16, the cell of claim 17, and / or the transformant of claim 18, and optionally a pharmaceutically acceptable carrier.

21. A kit comprising the antigen-binding protein of any one of claims 1-13, the fusion protein or immunoconjugate of claim 14, the isolated nucleic acid of claim 15, the vector of claim 16, the cell of claim 17, the transformant of claim 18, and / or the pharmaceutical composition of claim 20.

22. A method for detecting AXL in a sample, the method comprising contacting the sample with the antigen-binding protein of any one of claims 1-13, the fusion protein or immunoconjugate of claim 14, the isolated nucleic acid of claim 15, the vector of claim 16, the cell of claim 17, the transformant of claim 18, the pharmaceutical composition of claim 20, and / or the kit of claim 21, wherein the detection is preferably for non-diagnostic and / or therapeutic purposes, or the method is an ex vivo or in vitro method.

23. Use of the antigen-binding protein of any one of claims 1-13, the fusion protein or immunoconjugate of claim 14, the isolated nucleic acid of claim 15, the vector of claim 16, the cell of claim 17, the transformant of claim 18, the pharmaceutical composition of claim 20, and / or the kit of claim 21 in the preparation of a medicament for the prevention, treatment and / or relief of diseases and / or symptoms; Preferably, the disease and / or condition is a cancer associated with AXL expression; more preferably, the disease and / or condition includes cancers with AXL overexpression and / or ectopic expression, such as breast cancer and / or non-small cell lung cancer; optionally, the drug further includes a second therapeutic agent.

24. A method for diagnosing, preventing, treating, and / or alleviating diseases and / or symptoms, the method comprising administering to a subject in need an antigen-binding protein as described in any one of claims 1-13, a fusion protein or immunoconjugate as described in claim 14, an isolated nucleic acid as described in claim 15, a vector as described in claim 16, a cell as described in claim 17, a transformant as described in claim 18, a pharmaceutical composition as described in claim 20, and / or a kit as described in claim 21; Preferably, the disease and / or condition is a cancer associated with AXL expression; more preferably, the disease and / or condition includes cancers with AXL overexpression and / or ectopic expression, such as breast cancer and / or non-small cell lung cancer; optionally, the method further includes administering a second therapeutic agent.

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