Antibodies targeting AXL protein and their antigen-binding fragments, methods for their preparation and use.

Abstract

The present invention provides an antibody or antigen-binding fragment thereof targeting AXL protein, a preparation method and use. It further provides an isolated polynucleotide encoding the antibody or antigen-binding fragment thereof targeting AXL protein, and a vector comprising the isolated polynucleotide. The antibody targeting AXL provided by the present invention specifically binds to AXL protein, and can be used to mediate ADCC effect to kill tumors, or as a target recognition domain of CART cells to exert anti-tumor effect and prevent or treat tumors.

Description

[Technical Field]

[0001] The present invention belongs to the field of bioimmunology, and more specifically relates to an antibody and its antigen-binding fragment that can specifically bind to the AXL protein, and further relates to a method for preparing the antibody and its antigen-binding fragment and its use. [Background technology]

[0002] AXL is a 104-140 kDa transmembrane protein belonging to the TAM subfamily of mammalian receptor tyrosine kinases (RTKs) and possessing transformative potential (Paccez et al., 2014). The extracellular domain of AXL consists of a combination of two membrane-distal N-terminal immunoglobulin (Ig)-like domains (Ig1 and Ig2 domains) and two membrane-proximal fibronectin type III (FNIII) repeat sequences (FN1 and FN2 domains) (Paccez et al., 2014). The AXL protein is encoded and expressed by the gene AXL (UFO, ARK, Tyro7, or JTK11), and its receptor is a member of the Tyro-3 family of tyrosine kinases. The AXL protein, after binding to its ligand Gas6 (homologous to anticoagulant protein S, 70-kDa), transmits signals via pathways such as PI-3K / Akt, Ras / Erk, and β-catenin / TCF, and is involved in regulating tumor cell survival, proliferation, migration, and adhesion. AXL was the first transgenic gene identified in chronic myeloid leukemia (CML), and as subsequent studies have revealed, AXL exhibits active expression in many tumor tissues, including lung cancer, breast cancer, prostate cancer, thyroid cancer, endometrial cancer, ovarian cancer, and kidney cancer, compared to normal tissue. In particular, it shows abnormal activation expression in tumor tissues such as highly invasive, highly metastatic basal-like and / or trinegative breast cancer, metastatic lung cancer, and pancreatic cancer, and is involved in regulating tumor cell epithelial-mesenchymal transition, angiogenesis, apoptosis, and immunomodulation, promoting tumor metastasis and invasion, leading to a poor prognosis. On the other hand, in non-tumor diseases, no significant elevation of soluble AXL (sAxl) is observed in the serum of patients with chronic liver diseases (CLD), such as chronic viral hepatitis, autoimmune hepatitis, cholestatic liver disease, or non-alcoholic fatty liver disease. However, sAxl concentrations are specifically increased in the serum of patients with hepatocellular carcinoma (HCC) or advanced liver cancer-related late fibrosis (stage F3) or cirrhosis (stage F4). Therefore, AXL can also be used as a biomarker for the clinical diagnosis of tumors.

[0003] Recent studies have shown that high expression of the AXL protein is closely related to the development of drug resistance in tumors. AXL expression is significantly elevated in cancer cells resistant to chemotherapy and receptor tyrosine kinase inhibitor (TKI) therapy, such as EGFR inhibitor-refractory lung cancer, PI3K inhibitor-resistant head and neck cancer, anti-HER2 antibody-resistant breast cancer, sunitinib-resistant kidney cancer, and ALK inhibitor-resistant neuroblastoma. Inhibiting AXL expression can reverse the sensitivity of drug-resistant tumor cells to cytotoxic drugs and targeted inhibitors. Therefore, AXL kinase inhibitors represent a new policy for cancer treatment. Furthermore, Gas6, the sole ligand of AXL, is also highly expressed in several malignancies, and specifically blocking the AXL-Gas6 interaction can effectively inhibit tumor cell migration, invasion, and drug resistance. Clinical studies have shown that AVB-500 can effectively inhibit the progression of platinum-resistant ovarian cancer by inhibiting the AXL / Gas6 signaling pathway. Currently, this study has already completed Phase I clinical trials in the United States and is on the verge of entering Phase II enrollment clinical research.

[0004] Compared to other targets, antibodies targeting AXL can be rapidly internalized in large quantities, and contribute to binding to the surface of antigen-related tumor target cells in large numbers, binding to non-target cells in small numbers, and entering target cells in large numbers and non-target cells in small numbers. Therefore, the development of antibody-drug conjugates (ADCs) that specifically bind to AXL is attracting attention in this field. Due to enhanced glycolysis, oxygen deficiency, and insufficient tissue perfusion in the tumor microenvironment, a large amount of acidic metabolites accumulate, making the tumor microenvironment acidic. Typically, the pH value is in the range of 5.8 to 7.0, and often between 6.4 and 6.8. Such an acidic tumor microenvironment promotes tumor growth, invasion, and immune escape, posing a challenge to tumor treatment. Therefore, in order to improve the therapeutic effect of tumors, there is a strong need to research and develop AXL-targeting antibodies that are more specific, have high affinity, low immunogenicity, and excellent stability under the conditions of an acidic tumor microenvironment.

[0005] As the data shows, antibody-drug conjugates (ADCs) prepared by combining AXL monoclonal antibodies with small molecule cytotoxic drugs can enhance targeting of tumors and reduce toxic side effects. Compared to fully or partially humanized antibodies or antibody fragment drugs, ADCs effectively inhibit tumor growth and metastasis by releasing highly active cytotoxic drugs within tumor tissue. As is evident from clinical studies, ADCs derived from conventional AXL antibodies are prone to drug resistance due to their single target site for tumor antigens, and their low blood half-life increases toxicity. Therefore, further research, development, and optimization of AXL antibody drugs are necessary. [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] In view of the shortcomings of prior art, the main object of the present invention is to provide an antibody targeting the AXL protein that has higher affinity under acidic conditions, stronger specificity, lower immunogenicity, and superior stability. The present invention further provides a method for preparing the antibody and its applications, and the antibody targeting the AXL protein of the present invention can be used for the detection and / or treatment of tumors. [Means for solving the problem]

[0007] In one embodiment, the present invention provides an antibody or antigen-binding fragment thereof that can specifically bind to the AXL protein, wherein the antibody or antigen-binding fragment thereof (a) Heavy chain variable region (VH) including the following three complementarity-determining regions (CDRs): (i) VH CDR1 consisting of the sequence shown in SEQ ID NO: 9, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared thereto, (ii) VH CDR2 consisting of the sequence shown in SEQ ID NO: 10, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared thereto, and (iii) VH CDR3 consisting of the sequence shown in SEQ ID NO: 11, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared thereto, and / or (b) Light chain variable region (VL) including the following three complementarity-determining regions (CDRs): (iv) VL CDR1 consisting of the sequence shown in SEQ ID NO: 12, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared thereto, (v) VL CDR2 consisting of the sequence shown in SEQ ID NO: 13, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared thereto, and (vi) VL CDR3 consisting of the sequence shown in SEQ ID NO: 14, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared thereto, Includes, Preferably, any one of the substitutions described in (i) to (vi) is a conservative substitution, Preferably, the VH of the antibody or its antigen-binding fragment includes VH CDR1 shown in SEQ ID NO: 9, VH CDR2 shown in SEQ ID NO: 10, and VH CDR3 shown in SEQ ID NO: 11, and the VL of the antibody or its antigen-binding fragment includes VL CDR1 shown in SEQ ID NO: 12, VL CDR2 shown in SEQ ID NO: 13, and VL CDR3 shown in SEQ ID NO: 14.

[0008] In one embodiment, the present invention provides an antibody or antigen-binding fragment thereof that can specifically bind to the AXL protein, wherein the antibody or antigen-binding fragment thereof (a) Heavy chain variable region (VH) including the following three complementarity-determining regions (CDRs): (i) VH CDR1 consisting of the sequence shown in SEQ ID NO: 15, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared thereto, (ii) VH CDR2 consisting of the sequence shown in SEQ ID NO: 16, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared thereto, and (iii) VH CDR3 consisting of the sequence shown in SEQ ID NO: 17, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared thereto, and / or (b) Light chain variable region (VL) including the following three complementarity-determining regions (CDRs): (iv) VL CDR1 consisting of the sequence shown in SEQ ID NO: 18, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared thereto, (v) VL CDR2 consisting of the sequence shown in SEQ ID NO: 19, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared thereto, and (vi) VL CDR3 consisting of the sequence shown in SEQ ID NO: 20, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared thereto, Includes, Preferably, any one of the substitutions described in (i) to (vi) is a conservative substitution, Preferably, the VH of the antibody or its antigen-binding fragment comprises VH CDR1 shown in SEQ ID NO: 15, VH CDR2 shown in SEQ ID NO: 16, and VH CDR3 shown in SEQ ID NO: 17, and the VL of the antibody or its antigen-binding fragment comprises VL CDR1 shown in SEQ ID NO: 18, VL CDR2 shown in SEQ ID NO: 19, and VL CDR3 shown in SEQ ID NO: 20.

[0009] In one aspect, the present invention provides an antibody or an antigen-binding fragment thereof that can specifically bind to the AXL protein, and the antibody or its antigen-binding fragment (a) a heavy-chain variable region (VH) comprising the following three complementarity-determining regions (CDRs), (i) VH CDR1 consisting of the sequence shown in SEQ ID NO: 21, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) as compared thereto, (ii) VH CDR2 consisting of the sequence shown in SEQ ID NO: 22, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) as compared thereto, and (iii) VH CDR3 consisting of the sequence shown in SEQ ID NO: 23, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) as compared thereto, and / or (b) a light-chain variable region (VL) comprising the following three complementarity-determining regions (CDRs), (iv) VL CDR1 consisting of the sequence shown in SEQ ID NO: 24, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) as compared thereto, (v) VL CDR2 consisting of the sequence shown in SEQ ID NO: 25, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared thereto, and (vi) VL CDR3 consisting of the sequence shown in SEQ ID NO: 26, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared thereto, Includes, Preferably, any one of the substitutions described in (i) to (vi) is a conservative substitution, Preferably, the VH of the antibody or its antigen-binding fragment includes VH CDR1 shown in SEQ ID NO: 21, VH CDR2 shown in SEQ ID NO: 22, and VH CDR3 shown in SEQ ID NO: 23, and the VL of the antibody or its antigen-binding fragment includes VL CDR1 shown in SEQ ID NO: 24, VL CDR2 shown in SEQ ID NO: 25, and VL CDR3 shown in SEQ ID NO: 26.

[0010] In one embodiment, the present invention provides an antibody or antigen-binding fragment thereof that can specifically bind to the AXL protein, wherein the antibody or antigen-binding fragment thereof (a) Heavy chain variable region (VH) including the following three complementarity-determining regions (CDRs): (i) VH CDR1 consisting of the sequence shown in SEQ ID NO: 27, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared thereto, (ii) VH CDR2 consisting of the sequence shown in SEQ ID NO: 28, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared thereto, and (iii) VH CDR3 consisting of the sequence shown in SEQ ID NO: 29, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared thereto, and / or (b) Light chain variable region (VL) including the following three complementarity-determining regions (CDRs): (iv) VL CDR1 consisting of the sequence shown in SEQ ID NO: 30, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared thereto, (v) VL CDR2 consisting of the sequence shown in SEQ ID NO: 31, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared thereto, and (vi) VL CDR3 consisting of the sequence shown in SEQ ID NO: 32, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared thereto, Includes, Preferably, any one of the substitutions described in (i) to (vi) is a conservative substitution, Preferably, the VH of the antibody or its antigen-binding fragment includes VH CDR1 shown in SEQ ID NO: 27, VH CDR2 shown in SEQ ID NO: 28, and VH CDR3 shown in SEQ ID NO: 29, and the VL of the antibody or its antigen-binding fragment includes VL CDR1 shown in SEQ ID NO: 30, VL CDR2 shown in SEQ ID NO: 31, and VL CDR3 shown in SEQ ID NO: 32.

[0011] In one embodiment, the present invention provides an antibody or antigen-binding fragment thereof that can specifically bind to the AXL protein, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, where, The heavy chain variable region includes three CDRs included in the heavy chain variable region indicated by any one of SEQ ID NO: 1, 3, 5, and 7, and the light chain variable region includes three CDRs included in the light chain variable region indicated by any one of SEQ ID NO: 2, 4, 6, and 8. Preferably, the three CDRs included in the heavy chain variable region and / or the three CDRs included in the light chain variable region are defined by the Kabat, Chothia, or IMGT numbering system.

[0012] An antibody or antigen-binding fragment thereof according to the present invention, wherein the antibody or antigen-binding fragment thereof (a) A heavy chain variable region (VH) containing an amino acid sequence selected from the following: (i) SEQ ID NO: The sequence shown in 1, (ii) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, three, four, or five amino acid substitutions, deletions, or additions) compared to the sequence shown in SEQ ID NO: 1, or (iii) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 1. and / or, (b) A light chain variable region (VL) containing an amino acid sequence selected from the following: (iv) SEQ ID NO: The sequence shown in 2, (v) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, three, four, or five amino acid substitutions, deletions, or additions) compared to the sequence shown in SEQ ID NO: 2, or (vi) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 2, Includes, Preferably, the substitution described in (ii) or (v) is a conservative substitution, Preferably, the antibody or its antigen-binding fragment includes VH having the sequence shown in SEQ ID NO: 1 and VL having the sequence shown in SEQ ID NO: 2.

[0013] An antibody or antigen-binding fragment thereof according to the present invention, wherein the antibody or antigen-binding fragment thereof (a) A heavy chain variable region (VH) containing an amino acid sequence selected from the following: (i) SEQ ID NO: The sequence shown in 3, (ii) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, three, four, or five amino acid substitutions, deletions, or additions) compared to the sequence shown in SEQ ID NO: 3, or (iii) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 3. and / or, (b) A light chain variable region (VL) containing an amino acid sequence selected from the following: (iv) SEQ ID NO: The sequence shown in 4, (v) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, three, four, or five amino acid substitutions, deletions, or additions) compared to the sequence shown in SEQ ID NO: 4, or (vi) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 4, Includes, Preferably, the substitution described in (ii) or (v) is a conservative substitution, Preferably, the antibody or its antigen-binding fragment includes VH having the sequence shown in SEQ ID NO: 3 and VL having the sequence shown in SEQ ID NO: 4.

[0014] An antibody or antigen-binding fragment thereof according to the present invention, wherein the antibody or antigen-binding fragment thereof (a) A heavy chain variable region (VH) containing an amino acid sequence selected from the following: (i) SEQ ID NO: The sequence shown in 5, (ii) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, three, four, or five amino acid substitutions, deletions, or additions) compared to the sequence shown in SEQ ID NO: 5, or (iii) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 5. and / or, (b) A light chain variable region (VL) containing an amino acid sequence selected from the following: (iv) SEQ ID NO: The sequence shown in 6, (v) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, three, four, or five amino acid substitutions, deletions, or additions) compared to the sequence shown in SEQ ID NO: 6, or (vi) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 6, Includes, Preferably, the substitution described in (ii) or (v) is a conservative substitution, Preferably, the antibody or its antigen-binding fragment includes VH having the sequence shown in SEQ ID NO: 5 and VL having the sequence shown in SEQ ID NO: 6.

[0015] An antibody or antigen-binding fragment thereof according to the present invention, wherein the antibody or antigen-binding fragment thereof (a) A heavy chain variable region (VH) containing an amino acid sequence selected from the following: (i) SEQ ID NO: The sequence shown in 7, (ii) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, three, four, or five amino acid substitutions, deletions, or additions) compared to the sequence shown in SEQ ID NO: 7, or (iii) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 7, and / or, (b) A light chain variable region (VL) containing an amino acid sequence selected from the following: (iv) SEQ ID NO: The sequence shown in 8, (v) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, three, four, or five amino acid substitutions, deletions, or additions) compared to the sequence shown in SEQ ID NO: 8, or (vi) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 8, Includes, Preferably, the substitution described in (ii) or (v) is a conservative substitution, Preferably, the antibody or its antigen-binding fragment includes VH having the sequence shown in SEQ ID NO: 7 and VL having the sequence shown in SEQ ID NO: 8.

[0016] An antibody or antigen-binding fragment thereof according to the present invention, wherein the antibody or antigen-binding fragment thereof (a) The heavy chain constant region (CH) of human immunoglobulin or a variant thereof, The variant has one or more amino acid substitutions, deletions, or additions compared to its origin sequence (for example, up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions, or additions, for example, one, two, three, four, or five amino acid substitutions, deletions, or additions). (b) The constant region (CL) of the light chain of human immunoglobulin or a variant thereof, The variant has up to 20 conserved amino acid substitutions compared to its originating sequence (for example, up to 15, up to 10, or up to 5 conserved amino acid substitutions, for example, 1, 2, 3, 4, or 5 conserved amino acid substitutions), It further includes, Preferably, the heavy chain steady region is an IgG heavy chain steady region, for example, an IgG1, IgG2, IgG3, or IgG4 heavy chain steady region, and more preferably, a human IgG1 or human IgG4 heavy chain steady region. Preferably, the light chain steady-state region is the κ-light chain steady-state region.

[0017] An antibody or antigen-binding fragment thereof according to the present invention, wherein the antigen-binding fragment is selected from Fab, Fab', (Fab')2, Fv, Fv linked by disulfide bonds, scFv, diabody, and single-domain antibody (sdAb), and / or the antibody is a mouse-derived antibody, a chimeric antibody, a humanized antibody, a bispecific antibody, or a multispecific antibody, and more preferably the antibody is a fully human antibody.

[0018] In another embodiment, the present invention provides chimeric antigen receptor T cells comprising the antibody or antigen-binding fragment thereof described in the present invention, Preferably, the heavy chain variable region and the light chain variable region in the antibody or antigen-binding fragment are in a series combination or a parallel combination.

[0019] In another embodiment, the present invention provides isolated nucleic acid molecules encoding an antibody or an antigen-binding fragment thereof, or a heavy chain variable region and / or light chain variable region thereof.

[0020] In another embodiment, the present invention provides a vector comprising an isolated nucleic acid molecule described in the present invention, preferably the vector being a cloning vector or an expression vector, and more preferably the vector being a virus. More preferably, the viral skeleton of the viral vector may be a modified or altered vaccinia virus Tian Tan strain, vaccinia virus New York strain, vaccinia virus Copenhagen strain, vaccinia virus Canary strain, vaccinia virus Ankara strain, adenovirus vector, adeno-associated virus vector, herpes simplex virus vector, varicella-zoster virus vector, respiratory syncytial virus, Semryki forest virus, Epstein-Barr virus, giant cell virus, human herpesvirus 6, smallpox virus, molluscum contagiosum virus, eruption virus, reovirus, rotavirus, enterovirus, seneca virus, poliovirus, coxsackievirus, rhinovirus, hepatitis A virus, foot-and-mouth disease virus, togavirus, alphavirus, eastern equine encephalitis virus, Sindbis virus, rubella virus, coronavirus, flavivirus, hepatitis C virus, Japanese encephalitis virus, St. Louis encephalitis virus, Murray Valley fever virus, yellow fever It is derived from viruses, West Nile virus, Zika virus, dengue virus, Ebola virus, Marburg virus, sand grain virus, Lassa fever virus, lymphocytic choriomeningitis virus, Pichinde virus, Junin virus, Machupo virus, Hantavirus, Rift Valley fever virus, paramyxovirus, human parainfluenza virus, mumps virus, simian virus 5, measles virus, varicella stomatitis virus, rabies virus, respiratory syncytial virus, orthomyxovirus, influenza A virus, influenza B virus, influenza C virus, hepatitis D virus, simian immunodeficiency virus, human immunodeficiency virus type 1 and human immunodeficiency virus type 2, Rous sarcoma virus, human T-cell leukemia virus type 1, simian bubble virus, hepatitis B virus, hepatitis E virus, human papillomavirus, or polyomavirus.

[0021] More preferably, the vector is the cloning vector AbVec-hIgKappa (GenBank: FJ475056.1) or the cloning vector AbVec-hIgG1 (GenBank: FJ475055.1).

[0022] In another aspect, the present invention provides a host cell comprising an isolated nucleic acid molecule or vector as described in the present invention. Preferably, the host cell is prokaryotic or eukaryotic, more preferably selected from Escherichia coli cells, yeast cells, mammalian cells, or other cells suitable for preparing antibodies or antigen-binding fragments, multispecific antibodies, even more preferably the host cell is a mammalian cell, even more preferably the host cell is a human, mouse, sheep, horse, dog, or cat cell, and most preferably the host cell is a 293 cell or a CHO cell.

[0023] In a further embodiment, the present invention provides a method for preparing an antibody or antigen-binding fragment according to the present invention, comprising the steps of: culturing host cells under conditions that allow expression of the antibody or antigen-binding fragment according to the present invention; and recovering the antibody or antigen-binding fragment from the cultured host cell culture.

[0024] In a further embodiment, the present invention provides a bispecific or multispecific molecule comprising an antibody or an antigen-binding fragment thereof as described in the present invention, Preferably, the bispecific or multispecific molecule specifically binds to the AXL protein and further specifically binds to one or more other targets. Preferably, the bispecific or multispecific molecule further comprises at least one molecule having a second binding specificity to a second target (e.g., a secondary antibody), Preferably, the bispecific or multispecific molecule further comprises other antibodies or antigen-binding fragments that specifically bind to the AXL protein epitope.

[0025] In a further embodiment, the present invention provides an immunoconjugate comprising an antibody or antigen-binding fragment thereof described in the present invention, and a therapeutic agent linked to the antibody or antigen-binding fragment thereof. Preferably, the therapeutic agent is selected from cytotoxic agents. Preferably, the therapeutic agent is selected from alkylating agents, mitotic inhibitors, antitumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radionuclides, and any combination thereof. Preferably, the immune conjugate is an antibody-drug conjugate (ADC).

[0026] In a further embodiment, the present invention provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof, a bispecific or multispecific molecule or immunoconjugate, and a pharmaceutically acceptable vector and / or excipient, Preferably, the pharmaceutical composition further comprises additional pharmaceutically active agents. Preferably, the additional pharmaceutically active agent is a pharmaceutical product having antitumor activity, such as an alkylating agent, a mitotic inhibitor, an antitumor antibiotic, an antimetabolite, a topoisomerase inhibitor, a tyrosine kinase inhibitor, a radionuclide, a radiosensitizer, angiogenesis inhibitor, a cytokine, a molecular targeted drug, an immune checkpoint inhibitor, or an oncolytic virus. Preferably, the antibody or its antigen-binding fragment, bispecific or multispecific molecule, or immunoconjugate is provided with the additional pharmaceutically active agent as an isolated component or as a component of the same composition.

[0027] In a further embodiment, the present invention provides a kit containing the antibody or antigen-binding fragment thereof described in the present invention, Preferably, the antibody or its antigen-binding fragment has a detectable label, such as an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (e.g., a chemiluminescent substance), or biotin. Preferably, the kit further comprises a secondary antibody that specifically recognizes the antibody or antigen-binding fragment thereof described in the present invention, Preferably, the secondary antibody further comprises a detectable label, such as an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (e.g., a chemiluminescent substance), or biotin.

[0028] In a further embodiment, the present invention provides a chimeric antigen receptor comprising the antigen-binding domain of the antibody or antigen-binding fragment thereof described in the present invention, Preferably, the antigen-binding domain includes the heavy chain variable region and the light chain variable region of the antibody or antigen-binding fragment described in the present invention. Preferably, the antigen-binding domain is scFv, Preferably, the antigen-binding receptor comprises an antigen-binding fragment of the antibody described in the present invention. Preferably, the antigen-binding receptor is expressed by immune effector cells (e.g., T cells).

[0029] In a further embodiment, the present invention provides an isolated nucleic acid molecule encoding the chimeric antigen receptor.

[0030] In a further embodiment, the present invention provides a vector comprising an isolated nucleic acid molecule encoding the chimeric antigen receptor, preferably used for preparing chimeric antigen receptor T cells.

[0031] In a further embodiment, the present invention provides a host cell comprising an isolated nucleic acid molecule or vector encoding the chimeric antigen receptor, Preferably, the host cell is an immune effector cell (e.g., a T cell or an NK cell), Preferably, the host cell is a chimeric antigen receptor T cell (CAR-T).

[0032] The present invention further provides a method for inhibiting tumor cell growth and / or killing said tumor cells, comprising contacting the said tumor cells with an effective amount of the antibody or antigen-binding fragment thereof described in the present invention, or a bispecific or multispecific molecule, or an immunoconjugate, or a pharmaceutical composition, or a chimeric antigen receptor, or a host cell.

[0033] The present invention further provides a method for preventing and / or treating tumors in a subject (e.g., a human), the method comprising administering to a subject in need an effective amount of the antibody or antigen-binding fragment thereof described in the present invention, or a bispecific or multispecific molecule, or an immunoconjugate, or a pharmaceutical composition, or a chimeric antigen receptor, or a host cell. Preferably, the tumor is selected from B-cell lymphoma, T-cell lymphoma, melanoma, prostate cancer, renal cell carcinoma, sarcoma, gliomas such as high-level glioma, prostate cell tumors such as neuroblastoma, osteosarcoma, plasmacytoma, histiocytic sarcoma, pancreatic cancer, breast cancer, lung cancers such as small cell lung cancer and non-small cell lung cancer, gastric cancer, liver cancer, colon cancer, rectal cancer, esophageal cancer, colorectal cancer, hematopoietic cancer, testicular cancer, cervical cancer, ovarian cancer, bladder cancer, squamous cell carcinoma, adenocarcinoma, AIDS-related lymphoma, bladder cancer, brain cancer, nervous system cancer, head and neck cancer, head and neck squamous cell carcinoma, Hodgkin lymphoma, non-Hodgkin lymphoma, or hematological cancers. Preferably, the subject is a mammal, such as a human. Preferably, the method further comprises administering an additional pharmaceutical having antitumor activity, such as alkylating agents, mitotic inhibitors, antitumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radionuclides, radiosensitizers, angiogenesis inhibitors, cytokines, molecular targeted drugs, immune checkpoint inhibitors, or oncolytic viruses. Preferably, the method further includes administering additional antitumor therapies, such as surgery, chemotherapy, radiotherapy, targeted therapy, immunotherapy, hormone therapy, gene therapy, or palliative therapy.

[0034] The present invention further provides uses for the antibodies or antigen-binding fragments thereof, or bispecific or multispecific molecules, or immunoconjugates, or pharmaceutical compositions, or chimeric antigen receptors, or host cells described in the present invention in pharmaceutical preparations, wherein the pharmaceuticals are used to prevent and / or treat tumors in subjects (e.g., humans). Preferably, the pharmaceutical further comprises additional pharmaceutically active agents. Preferably, the additional pharmaceutically active agent is a pharmaceutical having antitumor activity, such as alkylating agents, mitotic inhibitors, antitumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radionuclides, radiosensitizers, angiogenesis inhibitors, cytokines, molecular targeted drugs, immune checkpoint inhibitors, or oncolytic viruses. Preferably, the tumor is selected from B-cell lymphoma, T-cell lymphoma, melanoma, prostate cancer, renal cell carcinoma, sarcoma, gliomas such as high-level glioma, prostate cell tumors such as neuroblastoma, osteosarcoma, plasmacytoma, histiocytic sarcoma, pancreatic cancer, breast cancer, lung cancers such as small cell lung cancer and non-small cell lung cancer, gastric cancer, liver cancer, colon cancer, rectal cancer, esophageal cancer, colorectal cancer, hematopoietic cancer, testicular cancer, cervical cancer, ovarian cancer, bladder cancer, squamous cell carcinoma, adenocarcinoma, AIDS-related lymphoma, bladder cancer, brain cancer, nervous system cancer, head and neck cancer, head and neck squamous cell carcinoma, Hodgkin lymphoma, non-Hodgkin lymphoma, or hematological cancers. Preferably, the subject is a mammal, such as a human.

[0035] Specifically, the method for preparing an antibody targeting the AXL protein or its antigen binding provided by the present invention is: 1) A step of obtaining a hybridoma strain capable of expressing an antibody that targets the AXL protein or its antigen binding by screening a library of natural phage antibodies, 2) In the hybridoma strain obtained in step 1), the step of cloning an antibody that targets the AXL protein or a gene that expresses its antigen binding, 3) Providing an expression vector containing the gene obtained by cloning in step 2) and an expression regulatory sequence operationally related to the gene, 4) The step of transforming host cells using the expression vector described in step 3), 5) The step of culturing the host cells obtained in step 4), 6) The step of isolating and purifying to obtain a monoclonal antibody, and the method comprises these steps.

[0036] In another embodiment, the present invention provides a hybridoma strain used in the above-described preparation method.

[0037] This invention provides 71 antibody strains targeting AXL by screening a natural phage antibody library. All of these strains can recognize all human antibodies that bind to human AXL, and four of these targeted AXL antibodies (Hu001-5, Hu001-7, Hu001-11, and Hu001-14) specifically recognize the extracellular domain (ECD) protein of human AXL and have the potential to block AXL-Gas6 interaction. These AXL antibodies are expected to exhibit stronger affinity under acidic conditions, exert more precise and effective tumor-killing activity, reduce toxicity to normal tissue, and increase the safety of the clinical use of antibody drugs. The antibodies against AXL described in this invention can mediate ADCC activity, can be administered as a drug alone, and can also be used to prepare CART cells and exert antitumor activity.

[0038] This invention involves preparing a human-mouse hybridoma that secretes an antibody specifically targeting the AXL protein, cloning the heavy and light chain sequences (all human genes) of the antibody using molecular biological techniques, constructing a human monoclonal antibody against the AXL protein, and expressing and producing the antibody using CHO cells. These antibodies, as pharmaceuticals, possess stronger binding affinity and specificity compared to conventional antibodies. As shown in the experimental results, the specific antibody of this invention exhibits good biological efficacy, can recognize the human AXL protein, and has an affinity of 2.92 × 10⁻⁶. -9 M and 1.40×10 -9 The antibody of the present invention is M, possesses chemotaxis in a relatively strong acidic environment, and is very promising as it can be used to prepare therapeutic agents such as CART or monoclonal antibodies for targeting AXL-overexpressing diseases.

[0039] Those skilled in the art will understand that the inventions described herein are easily modified and altered in addition to those specifically described. The present invention includes all of these variations and alterations. The present invention also includes all steps and features mentioned or pointed out individually or in common in the specification, and any and all combinations of steps or features.

[0040] Other features and aspects of the present invention will become apparent from the following detailed description and accompanying drawings. [Brief explanation of the drawing]

[0041] The embodiments of the present invention will be described in detail below in conjunction with the drawings, and here, [Figure 1] Figures 1A to 1G show graphs illustrating the binding of clone supernatants to the antigen hAXL-ECD by ELISA screening. Each of the 71 clones in this application was shown to be able to specifically bind to hAXL-ECD, with clones 5, 7, 11, and 14 showing the highest affinity. Clones 5, 7, 11, and 14 were selected for experiments and named Hu001-5, Hu001-7, Hu001-11, and Hu001-14. [Figure 2] The expression vector maps AbVec-hIgKappa and AbVec-hIgG1 WT constructed for the antibodies used in this invention are shown. [Figure 3] The graphs show the affinity of the anti-AXL antibodies Hu001-5, Hu001-7, Hu001-11, and Hu001-14 of the present invention using flow data. Figure 3A is a schematic diagram showing the change in affinity with respect to the concentration of the positive control CCT301-38 antibody at different pH levels, and Figures 3B to 3E are schematic diagrams showing the change in affinity with respect to the concentration of the anti-AXL antibodies Hu001-5, Hu001-7, Hu001-11, and Hu001-14 at different pH levels. It is clear that the antibodies of the present invention have higher affinity to the positive control and have higher binding ability under acidic conditions than under neutral conditions. [Figure 4]Figure 4 shows CART cells prepared using the anti-AXL antibody Hu001-11 of the present invention and the positive control antibody CCT301-38, respectively, and in vitro activity tests under different conditions. As is clearly evident from Figure 4, the anti-AXL antibody Hu001-11 of the present invention is more strongly activated under acidic in vitro conditions. [Figure 5] This study demonstrates that CART cells prepared using the anti-AXL antibody Hu001-11 of the present invention can mediate higher killing activity in vitro compared to CART cells prepared with the positive control CCT301-38 antibody. [Figure 6] This study demonstrates that the anti-AXL antibodies Hu001-5, Hu001-7, Hu001-11, and Hu001-14 of the present invention can mediate broader antitumor activity in vitro compared to the positive control CCT301-38 antibody. [Figure 7] This shows the sequence scheme for the antibody variable region. [Modes for carrying out the invention]

[0042] The following description of this application is intended solely to illustrate several embodiments of this application. Therefore, the specific modifications discussed herein should not be understood as limiting the scope of the application. Those skilled in the art will readily derive several equivalent forms, variations, and modifications without departing from the scope of this application, and such equivalent embodiments should be understood to be within the scope of the invention. All documents cited in this application, including disclosure publications, patents, and patent applications, are incorporated herein by reference in their entirety.

[0043] definition In this invention, "antibody" includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multispecific antibody, or bispecific (bivalent) antibody capable of binding to a specific antigen. A single naturally occurring intact antibody contains two heavy chains and two light chains. Each heavy chain consists of one variable region and a first, second, and third constant region, and each light chain consists of one variable region and one constant region. Mammalian heavy chains can be classified into α, δ, ε, γ, and μ, and mammalian light chains can be classified into λ or κ. The antibody exhibits a "Y" shape, and the neck of the Y-shaped structure consists of the second and third constant regions of the two heavy chains, which are linked by disulfide bonds. Each arm of the "Y"-shaped structure contains the variable region and first constant region of one of the heavy chains, and is bound to the variable region and constant region of one light chain. The variable regions of the light and heavy chains determine antigen binding. Each chain's variable region contains three hypervariable regions called complementarity-determining regions (CDRs). The CDRs of the light chain (L) include VLCDR1, VLCDR2, and VLCDR3, while the CDRs of the heavy chain (H) include VHCDR1, VHCDR2, and VHCDR3. The CDR boundaries of the antibody-antigen binding fragments disclosed in this invention can be named or recognized by Kabat, Chothia, or Al-Lazikani nomenclature. (AI-Lazikani, B., Chothia, C., Lesk, AM, J.Mol.Biol., 273(4):927(1997); Chothia, C. et al., J.Mol.Biol., 186(3):651-63(1985); Chothia, C. and Lesk, AM, J.Mol.Biol., 196:901(1987); Chothia, C. et al., Nature, 342(6252):877-83(1989); Kabat, EA et al., National Institutes of Health, Bethesda, Md.(1991)). Here, the three CDRs are separated by a lateral continuum called the framework region (FR), which is more conserved than the CDR and forms a stent-supporting hypervariable loop. The constant regions of the heavy and light chains do not participate in antigen binding but have various effector functions. Antibodies can be classified into several types based on the amino acid sequence of their heavy chain constant region.Antibodies can be divided into five main classifications or isomers, IgA, IgD, IgE, IgG, and IgM, depending on whether or not they contain α, δ, ε, γ, and μ heavy chains. Some major antibody classifications can be further divided into subclasses such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain).

[0044] In this application, "antigen-binding fragment" means an antibody fragment formed by an antibody moiety containing one or more CDRs, or by any other antibody fragment that binds to an antigen but does not have a complete antibody structure. Examples of antigen-binding fragments include, but are not limited to, bifunctional antibodies (diabody), Fab, Fab', F(ab')2, Fv fragments, Fv fragments with stable disulfide bonds (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), bifunctional antibodies with stable disulfide bonds (ds diabody), single-chain antibody molecules (scFv), scFv dimers (bivalent bifunctional antibodies), bivalent single-chain antibodies (BsFv), multispecific antibodies, camelized single-domain antibodies, nanoantibodies, domain antibodies, and bivalent domain antibodies. Antigen-binding fragments can bind to the same antigen as the parent antibody. In some embodiments, the antigen-binding fragment comprises one or more CDRs from a specific human antibody and can be transferred to a framework region from one or more different human antibodies.

[0045] The "Fab" fragment of an antibody refers to the portion of the antibody molecule in which one light chain (including the variable and constant regions) and part of the variable and constant regions of one heavy chain are linked by disulfide bonds.

[0046] The "Fab'" fragment refers to a Fab fragment that includes part of the hinge region.

[0047] "F(ab')2" means the dimer of Fab.

[0048] The Fc region of an antibody is responsible for various different effector functions, such as ADCC and CDC, but it is not involved in antigen binding.

[0049] The "Fv" region of an antibody refers to the smallest antibody fragment containing a complete antigen-binding site, and consists of a variable region of one light chain and a variable region of one heavy chain.

[0050] "Single-chain Fv antibody" or "scFv" refers to an engineered antibody in which the light chain variable region and the heavy chain variable region are linked directly or via a single peptide chain (Huston JS et al., Proc Natl Acad Sci USA, 85:5879 (1988)).

[0051] "Single-chain antibody Fv-Fc" or "scFv-Fc" refers to an engineered antibody consisting of scFv linked to a certain antibody Fc region.

[0052] "Camelized single-domain antibody," "heavy-chain antibody," or "HCAb (Heavy-chain-only antibody, HCAb)" all refer to antibodies that contain two VH domains but do not contain a light chain (Riechmann L. and Muyldermans S., J Immunol Methods. 231(1-2):25-38 (1999); Muyldermans S., J Biotechnol. 74(4):277-302 (2001); WO94 / 04678; WO94 / 25591; US ​​Patent No. 6,005,079). Heavy-chain antibodies are initially obtained by inducing antibodies from the camelid family (camels, dromedaries, and alpacas). Despite the deletion of the light chain, camelized antibodies have been confirmed to have the full function of antigen binding (Hamers Casterman C. et al., Nature 363(6428):446-8(1993); Nguyen VK. et al., "Heavy-chain antibodies in Camelidae: a case of evolutionary innovation, Immunogenetics. 54(1):39-47(2002); Nguyen VK. et al., Immunology. 109(1):93-101(2003)). The variable region (VH domain) of heavy-chain antibodies is the smallest known antigen-binding unit by acquired immunity (Koch-Nolte F. et al., FASEB J. 21(13):3490-8. Epub(2007)).

[0053] "Nano-antibody" refers to an antibody fragment consisting of one VH domain and two constant regions CH2 and CH3 derived from a heavy-chain antibody.

[0054] A "dual-function antibody (diabody)" contains a small antibody fragment containing two antigen-binding sites, each containing a VH domain and a VL domain, linked to the same polypeptide chain (see Holliger P. et al., Proc Natl Acad Sci USA. 90(14):6444-8(1993);EP404097;WO93 / 11161). The adapter between the two domains is very short, and the two domains on the same chain cannot be paired with each other. Therefore, the two domains are paired with complementary domains on another chain, forming two antibody-binding sites. These two antibody-binding sites can target and bind to the same or different antigens (or antigenic epitopes).

[0055] A "domain antibody" refers to an antibody fragment containing only one heavy chain variable region or one light chain variable region. In some cases, two or more VH domains are covalently linked by a polypeptide adapter to form a bivalent domain antibody. The two VH domains of a bivalent domain antibody can act to target the same or different antigens.

[0056] In some embodiments, "(dsFv)2" comprises three peptide chains, with two VH genes linked by a polypeptide adapter and two VL groups bound by disulfide bonds.

[0057] In some embodiments, the "bi-specific ds bifunctional antibody" contains VL1-VH2 (linked by one polypeptide adapter) and VH1-VL2 (linked by one polypeptide adapter), and the two are linked by a disulfide bond between VH1 and VL1.

[0058] "Dual-specificity dsFv" or "dsFv-dsFv" is a molecule in which, in the VH1-VH2 group, both heavy chains are linked by a polypeptide adapter (e.g., a long elastic adapter), and are then linked to the VL1 and VL2 groups respectively by disulfide bonds, resulting in a molecule containing three polypeptide chains, each paired by disulfide bonds having different antigen specificities.

[0059] In some embodiments, a "scFv dimer" is a bivalent bispecific antibody or a bivalent single-chain antibody (BsFv) that includes two quantified VH-VL (linked by a polypeptide adapter) groups, where the VH of the two groups can cooperate with the VL of the other group to form two binding sites that can bind the same antigen (or antigen epitope) or different antigens (or antigen epitopes). In some other embodiments, a "scFv dimer" is a bispecific bifunctional antibody that includes V L1 -V H2 (linked by a polypeptide adapter) and V H1 -V L2 (linked by a polypeptide adapter), where V H1 and V L1 cooperate such that V H2 and V L2 cooperate, and each cooperating pair has a different antigen specificity.

[0060] When used in an antibody or antigen-binding fragment, the term "fully human" as used in this application means that the antibody or antigen-binding fragment has or consists of an amino acid sequence that corresponds to an amino acid sequence produced from a human or human immune cell, or an amino acid sequence of an antibody of non-human origin, such as a transgenic non-human animal that utilizes, for example, a human antibody library, or another sequence encoding a human antibody. In some embodiments, a fully human antibody does not include amino acid residues (particularly antigen-binding residues) derived from non-human antibodies.

[0061] When used in reference to antibodies or antigen-binding fragments, the term “humanized” as used in this application means an antibody or antigen-binding fragment comprising a CDR derived from a non-human animal, a human-derived FR region, and a human-derived constant region (if applicable). Humanized antibodies or antigen-binding fragments have reduced immunogenicity and are therefore available as human therapeutic agents in certain embodiments. In some embodiments, the non-human animal is a mammal, such as a mouse, rat, rabbit, goat, sheep, guinea pig, or hamster. In some embodiments, the humanized antibody or antigen-binding fragment consists of essentially all human-derived sequences, except that the CDR sequence is of non-human origin. In some embodiments, the human-derived FR region may contain the same amino acid sequence as the human antibody from which it is derived, or it may contain several amino acid changes, for example, changes of 10, 9, 8, 7, 6, 5, 4, 3, 2, or one or fewer amino acids. In some embodiments, such amino acid changes may be present only in the heavy-chain FR region, only in the light-chain FR region, or simultaneously in both chains. In some preferred embodiments, the humanized antibody comprises human FR1-3, human JH, and JK.

[0062] As used in this application, the term "chimera" means an antibody or antigen-binding fragment having a portion of a heavy chain and / or light chain derived from one species, with the remainder of the heavy chain and / or light chain derived from a different species. In exemplary examples, a chimeric antibody may include a constant region derived from a human and a variable region derived from a non-human animal, such as a mouse.

[0063] In this application, "specifically bind" or "specifically bind" means a non-random binding reaction between the two molecules, for example, a reaction between an antibody and an antigen. In certain embodiments, the antibody or its antigen-binding fragment of this application specifically binds to human and / or monkey AXL proteins, and its binding affinity (K D ) is ≤ 10 -6 M is the case in this application. D The ratio of the dissociation rate to the binding rate (k off / k onThis means that it can be measured by the surface plasmon resonance method, for example, using an instrument such as Biacore.

[0064] Unless otherwise specified, the heavy and light chain constant regions of the antibodies described in this invention are human IgG1 and κ chains, respectively.

[0065] In this application, when used in amino acid sequences, "conservative substitution" means replacing one amino acid residue with another amino acid residue having a side chain with similar physicochemical properties. For example, conservative substitutions can be made between hydrophobic side chain amino acid residues (e.g., Met, Ala, VaL, Leu, and Ile), between neutral hydrophilic side chain residues (e.g., Cys, Ser, Thr, Asn, and Gln), between acidic side chain residues (e.g., Asp, Glu), between basic side chain amino acids (e.g., His, Lys, and Arg), or between aromatic side chain residues (e.g., Trp, Tyr, and Phe). In this art, it is known that conservative substitutions usually do not cause significant changes in the protein conformation, and therefore the biological activity of the protein can be preserved.

[0066] When used for amino acid sequences (or nucleic acid sequences), "percent sequence identity" means the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are the same as those in a reference sequence, after sequence alignment and, if necessary, spacing to maximize the number of identical amino acids (or nucleic acids), in the candidate sequence. Conservative substitutions of such amino acid residues may or may not be considered as identical residues. Percent sequence identity of an amino acid (or nucleic acid) sequence can be determined by sequence alignment using the tools disclosed in the Art. Those skilled in the art may use the default parameters of the tools, for example, by selecting an appropriate algorithm, or they may adjust the parameters appropriately as needed for alignment.

[0067] As used in this application, "T cells" include CD4+ T cells, CD8+ T cells, T helper type 1 T cells, T helper type 2 T cells, T helper type 17 T cells, and suppressor T cells.

[0068] As used in this application, “effector function” refers to the biological activity of the Fc region of an antibody binding to its effector, such as a C1 complex and an Fc receptor. Exemplary effector functions include complement-dependent cell-mediated cytotoxicity (CDC) induced by the interaction between the antibody and C1q on the C1 complex, antibody-dependent cell-mediated cytotoxicity (ADCC) induced by the binding of the Fc region of an antibody to an Fc receptor on an effector cell, and phagocytosis.

[0069] In this application, “cancer” or “cancer symptoms” means any medical condition mediated by the growth, proliferation or metastasis of tumors or malignant cells, resulting in solid and non-solid tumors, such as leukemia. In this invention, “tumor” means a physical substance of a tumor and / or malignant cells.

[0070] "Treatment" or "therapy" for a condition includes preventing or mitigating a condition, slowing the onset or progression of a condition, reducing the risk of progression to a condition, preventing or delaying the progression of symptoms associated with a condition, reducing or eliminating symptoms associated with a condition, producing a complete or partial reversal of a condition, curing a condition, or any combination thereof. For cancer, "treatment" or "therapy" may mean inhibiting or delaying the growth, proliferation or metastasis of a tumor or malignant cells, or any combination thereof. For tumors, "treatment" or "therapy" includes removing all or part of a tumor, inhibiting or delaying tumor growth and metastasis, preventing or delaying tumor progression, or any combination thereof.

[0071] An "isolated" substance is one that has been artificially altered from its natural state. When an "isolated" substance or component appears in nature, it has already been altered, deviated from its original state, or both. For example, polynucleotides or polypeptides that naturally occur in the body of a living animal are not isolated, but if these polynucleotides or polypeptides are sufficiently isolated from the substances that coexist with them in nature and exist in a sufficiently pure state, they can be considered "isolated." In certain embodiments, the purity of antibodies and antigen-binding fragments is at least 90%, 93%, 95%, 96%, 97%, 98%, or 99%, and is determined by electrophoresis (e.g., SDS-PAGE, isoelectric focusing, capillary electrophoresis) or chromatography (e.g., ion-exchange chromatography or reverse-phase HPLC).

[0072] In this invention, "vector" refers to a transport medium in which a polynucleotide encoding a protein can be operationally inserted to express that protein. A vector can be used to transform, transduce, or transfect a host cell so that its own genetic material elements are expressed within the host cell. Examples of vectors include plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC), phages such as lambda phage or M13 phage, and animal viruses. Types of animal viruses used as vectors include retroviruses (including lentiviruses, adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40)). A vector may contain multiple expression regulatory elements such as a promoter sequence, a transcription start sequence, an enhancer sequence, a selection element, and a reporter gene. A vector may also further contain a replication start site. The vector may also contain components that facilitate access to cells, such as viral particles, liposomes, or protein exoskeletons, but is not limited to these.

[0073] In this invention, "host cell" refers to a cell into which an exogenous polynucleotide and / or vector has been introduced.

[0074] Regarding antibodies targeting the AXL protein in the present invention, in some embodiments, this application provides exemplary antibodies targeting the AXL protein: Hu001-5 antibody, Hu001-7 antibody, Hu001-11 antibody, and Hu001-14 antibody.

[0075] Those skilled in the art will understand that by modifying the CDR sequence to include one or more amino acid substitutions, improved biological activity, such as improved binding affinity to the AXL protein, can be obtained. For example, a library of antibody variants (e.g., Fab or FcFv variants) can be produced and expressed using phage display technology, and then antibodies with affinity to the AXL protein can be screened. In another example, the binding of the antibody to the AXL protein can be simulated using computer software to identify amino acid residues that form a binding interface with the antibody. Substitutions of these residues may be avoided to prevent a decrease in binding affinity, or these residues may be targeted for substitution to form a stronger bond. In some embodiments, at least one (or all) substitutions of the CDR sequence are conservative substitutions.

[0076] In some embodiments, the antibody or antigen-binding fragment comprises one or more CDR sequences, which have at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity with the sequences of SEQ ID NO: 9-32, and maintain a binding affinity to AXL proteins similar to or higher than that of the parent antibody. The parent antibody has essentially the same sequence, but its corresponding CDR sequence has 100% sequence identity with the sequence described in SEQ ID NO: 9-32.

[0077] In some embodiments, the antibody or antigen-binding fragment described in this application can be measured by surface plasmon resonance to be ≤10 -7 Binding affinity of M (K D It can specifically bind to the AXL protein at (k). The binding affinity is the ratio (k) of the dissociation rate to the binding rate when the binding of the antigen and the antigen-binding molecule reaches a balance. off / k on K calculated by ) D It can be expressed as a value. The antigen binding affinity (e.g., K D This can be appropriately determined by appropriate methods conventionally known in the field, including plasmon resonance coupling methods using instruments such as Biacore.

[0078] In some embodiments, the antibody or antigen-binding fragment described in this application binds to the AXL protein at an EC50 (i.e., half-binding concentration) of 1 ng / mL to 10 μg / mL. The binding of the antibody or antigen-binding fragment to the AXL protein can be measured by conventionally known methods such as sandwich methods including ELISA, Western blotting, FACS, or other binding tests. In an exemplary example, the antibody to be measured (i.e., primary antibody) is bound to an immobilized AXL protein or cells expressing the AXL protein, the unbound antibody is washed away, and a labeled secondary antibody is introduced so that it can bind to the primary antibody, and thus the bound secondary antibody can be detected. When using an immobilized AXL protein, the detection can be performed using a microplate, or when using cells expressing the AXL protein, the detection can be performed using FACS analysis.

[0079] In some embodiments, the antibody or antigen-binding fragment described in this application binds to the AXL protein at an EC50 (i.e., 50% effective concentration) of 10 ng / mL to 10 μg / mL (measured by FACS analysis).

[0080] In some embodiments, the advantage of the antibodies described in this application is that they can be used in combination with immunogenic substances such as tumor cells, purified tumor antigens, cells transfected with immunostimulators, and tumor vaccines. Furthermore, the antibodies targeting the AXL protein and their antigen-binding fragments may be included in combination therapies including standard chemotherapy and radiotherapy, targeted small molecule therapies, and other emerging immune checkpoint modulator therapies. In some embodiments, the antibodies and their antigen-binding fragments can be used as base molecules for antibody-drug conjugates, bispecific or multivalent antibodies.

[0081] In some embodiments, the antibodies and antigen-binding fragments described in this application are camelized single-chain domain antibodies, diabodies, scFv, scFv dimers, BsFv, dsFv, (dsFv)2, dsFv-dsFv', Fv fragments, Fab, Fab', F(ab')2, ds diabodies, nanoantibodies, domain antibodies, or bivalent domain antibodies.

[0082] In some embodiments, the antibody described in this application includes an immunoglobulin constant region. In some embodiments, the immunoglobulin constant region includes a heavy chain and / or a light chain constant region. The heavy chain constant region includes a CH1, CH1-CH2, or CH1-CH3 region. In some embodiments, the immunoglobulin constant region may further include one or more modifications to obtain desired properties. For example, the constant region may be modified to reduce or eliminate one or more effector functions such that the FcRn receptor binds to or introduces one or more cysteine ​​residues.

[0083] In some embodiments, the antibody and its antigen-binding fragment further comprise a conjugate. The antibody or its antigen-binding fragment in the present invention is thought to be able to be linked to a variety of conjugates (see, for example, "Conjugate Vaccines," Contributions to Microbiology and Immunology, JMCruse and RELewis, Jr. (eds.), Carger Press, New York (1989)). These conjugates can be linked to the antibody or antigen conjugate in other ways, such as by covalent bond, affinity bond, intercalation, coordinate binding, complexation, binding, mixing, or addition. In some embodiments, the antibody and antigen-binding fragment disclosed in the present invention can be engineered to contain specific sites other than the epitope-binding site, which can be used to link one or more conjugates. For example, such sites may include one or more reactive amino acid residues, such as cysteine ​​and histidine residues, and are used to assist in covalent bonding with the conjugate. In some embodiments, the antibody may be indirectly linked to the conjugate or linked via another conjugate. For example, the antibody or its antigen-binding fragment may be linked to biotin, indirectly linked to a second conjugate, and linked to avidin. The conjugate may be a detectable label, a pharmacokinetic modification moiety, a purified moiety, or a cytotoxic moiety. Examples of detectable labels include fluorescent labels (e.g., fluorescein, rhodamine, dansyl, phycoerythrin, or Texas Red), enzyme substrate labels (e.g., horseradish peroxidase, alkaline phosphatase, luciferase, glucoamylase, lysozyme, carbohydrate oxidase, or β-D-galactosidase), stable isotopes or radioisotopes, chromophore moieties, digoxin, biotin / avidin, DNA molecules, or gold for detection. In some embodiments, the conjugate may be a pharmacokinetic modification moiety such as PEG that contributes to extending the half-life of the antibody.Other suitable polymers include, for example, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, and ethylene glycol / propylene glycol copolymers. In some embodiments, the conjugate may be a purified portion, such as magnetic beads. The "cytotoxic activity portion" may be any reagent that is harmful to cells or that may cause necrosis or death of cells. Examples of cytotoxic moieties include paclitaxel, cytochalasin B, gramicidin D, ethyl bromide, erythrin, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracine dione, mitoxantrone, mitramycin, actinomycin D, l-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin and its analogs, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-mercaptoguanine, cytarabine, 5-fluorouracildacarbazine), and alpha Examples of mitotic agents include, but are not limited to, killing agents (e.g., mechloretamine hydrochloride, thiotepachlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C and cis-dichlorodiamine platinum (DDP) cisplatin), anthracycline antibiotics (e.g., daunorubicin (formerly daunomycin) and adriamycin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mitramycin and anthramycin (AMC)), and antimitotic agents (e.g., vincristine and vinblastine).

[0084] Polynucleotides and Recombination Methods Using genetic engineering techniques known in this field, the amino acid sequences of the antibodies and their antigen-binding fragments described in this application can be converted to the corresponding DNA coding sequences. Due to the degenerate nature of the genetic code, the resulting DNA sequences can be perfectly identical, while the encoded protein sequence remains unchanged.

[0085] Using recombinant techniques known in the art, a vector containing the antibody and the polynucleotide encoding its antigen-binding fragment can be introduced into host cells for cloning (DNA amplification) or gene expression. In another embodiment, the antibody and its antigen-binding fragment can be prepared by homologous recombination methods known in the art. Various vectors are selectable. Vector components typically include, but are not limited to, two or more types of elements: a signal sequence, a replication origin, one or more labeled genes, an enhancer sequence, a promoter (e.g., SV40, CMV, EF-1a), and a transcription termination sequence.

[0086] In some embodiments, the vector system includes mammalian, bacterial, and yeast systems, and also includes, but is not limited to, plasmids such as pALTER, pBAD, pcDNA, pCal, pL, pELpGEMEX, pGEX, pCLpCMV, pEGFP, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS420, pLexA, and pACT2, as well as other laboratory-available or commercially available vectors. Suitable vectors may include plasmids or viral vectors (e.g., replication-deficient retroviruses, adenoviruses, and adeno-associated viruses).

[0087] A vector containing the aforementioned antibody and a polynucleotide encoding its antigen-binding fragment can be introduced into a host cell and used for cloning or gene expression. In the present invention, the host cell to which the DNA in the vector is applied for cloning or expression is a prokaryotic cell, yeast, or the above-mentioned higher eukaryotic cell. Prokaryotic cells to which the present invention is applied include fungi, such as Enterobacteriaceae (e.g., Escherichia coli), Enterobacteriaceae, Erwinia, Klebsiella, Proteus, Salmonella such as Salmonella typhimurium, Serratia such as Marcescens, and Racinths such as Shigella, Bacillus subtilis and Bacillus spp., Pseudomonas such as Pseudomonas aeruginosa, and Gram-negative or Gram-positive bacteria such as Streptomyces.

[0088] Besides prokaryotic cells, eukaryotic microorganisms such as filamentous fungi or yeast can also be used as host cells to clone or express antibody-encoding vectors. Saccharomyces cerevisiae, or baker's yeast, are the most commonly used lower eukaryotic host microorganisms. However, many other genera, species, and strains are also relatively commonly used and applicable in this invention, for example, Schizosaccharomyces pombe, Cluyberomyces lactic acid yeast, Cluyberomyces fragilis (ATCC12424), Cluyberomyces bulgaricus (ATCC16045), Cluyberomyces wickarami (ATCC24178), Cluyberomyces warthii (ATCC56500), Cluyberomyces drosophilarum (ATCC36906), and Cluyberomyces sa These include filamentous fungi such as *Motolerance* and *Cluiveromyces marsianus*, *Yarrowia liporitica* (EP402226), *Pichia pastris* (EP183070), *Candida*, *Trichoderma lesei* (EP244234), *Neurocras crassa*, *Schwanniomyces occidentalis*, and *Neurospora*, *Penicillium*, *Culbularia*, and *Aspergillus* (such as *Aspergillus nidurans* and *Aspergillus niger*).

[0089] Host cells suitable for expressing the glycated antibodies or their antigen-binding fragments provided by the present invention are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. Multiple baculovirus strains and their variants, as well as corresponding permissive insect host cells, are commonly found in hosts such as the fall armyworm (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and silkworm. Various virus strains for transfection are publicly available, such as Autographa californica nuclear polyhedrosis virus and the Bm-5 variant of silkworm nuclear polyhedron disease virus, and any of these viruses can be used in the present invention, and in particular can be used to transfect fall armyworm cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be used as hosts.

[0090] However, the most interesting cells are vertebral cells, and culturing (tissue culture) vertebral cells is a standard procedure. Suitable mammalian host cells include SV40-transformed monkey kidney cells CV1 line (COS-7, ATCC CRL 1651), human embryonic kidney cell line (293 or 293 cell subclones in suspension culture, Graham et al., Gen Virol. 36:59 (1977)), baby hamster kidney cells (B blood, ATCC CCL 10), Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)), mouse Sertoli cells (TM4, Ma ther, Biol. Reprod. 23:243-251 (1980)), monkey kidney cells (CV1 ATCC CCL 70), African green monkey kidney cells (VERO-76, ATCC CRL-1587), and human cervical cancer cells (HELA, ATCC CCL 2) Examples include canine kidney cells (MDCK, ATCC CCL 34), buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442), human lung cells (W138, ATCC CCL 75), human hepatocytes (Hep G2, HB 8065), mouse mammary tumor cells (MMT 060562, ATCC CCL 51), TRI cells (Mather et al., Annals NYAcad. Sci. 383:44-68 (1982)), MRC 5 cells, FS4 cells, and human liver cancer cell lineage HepG2). In some preferred embodiments, the host cells are 293F cells.

[0091] Host cells are transformed using an expression or cloning vector that produces the aforementioned antibody and its antigen-binding fragment, and after modification, they are cultured in a standard nutrient medium suitable for promoter induction, selection of transformed cells, or amplification of genes encoding target sequences.

[0092] In the present invention, host cells for producing the antibody and its antigen-binding fragment can be cultured in various media known in the art. The media may further contain any other necessary additives in appropriate concentrations known in the art. The conditions of the media, such as temperature, pH value, and similar conditions, are conditions that have been conventionally used to select host cells for expression and are well known to those skilled in the art.

[0093] When recombinant technology is used, the antibody can be generated intracellularly in the cell wall space or secreted directly into the culture medium. If the antibody is generated intracellularly, the host cell or degradation fragment particle residue can be removed first, for example, by centrifugation or sonication. Carter et al., Bio / Technology 10:163-167 (1992) describes a method for isolating antibodies secreted into the cell wall space of E. coli. Briefly, the cell paste is dissolved for at least 30 minutes under conditions of uranium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF). Cell debris is removed by centrifugation. If the antibody is secreted into the culture medium, the supernatant of the expression system is generally concentrated first using a commercially available protein concentration filter, such as an lAmicon or Millipore Pellicon ultrafiltration unit. In either of the above steps, protease inhibitors such as PMSF to suppress protein degradation and antibiotics to prevent the growth of accidental contaminants can be added.

[0094] The antibodies obtained from the aforementioned cells can be purified using purification methods such as hydroxyapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography column, ammonium sulfate precipitation, salting out, and affinity chromatography, of which affinity chromatography is the preferred purification technique. The type of antibody and the Fc domain in the antibody where any immunoglobulin is present determine whether protein A is suitable as an affinity ligand. Protein A can be used to purify antibodies based on human γ1, γ2, or γ4 heavy chains (Lindmark et al., J. lmmunol. Meth. 62:13 (1983)). Protein G is applicable to all mouse-derived isomers and human γ3 (Guss et al., EMBO J. 5:1567 1575 (1986)). Agarose is the most commonly used affinity ligand substrate, but other substrates may be selected. Substrates that are more mechanically stable than agarose, such as porous glass or poly(styrene)benzene, can achieve faster flow rates and shorter processing times. If the antibody contains a CH3 domain, it can be purified using Bakerbond ABX.TM resin (JTBaker, Phillipsburg, NJ). Other protein purification techniques, such as fractional distillation in ion-exchange columns, ethanol precipitation, reverse-phase HPLC, silica gel chromatography, heparin agarose gel chromatography based on anion or cation exchange resin (e.g., polyaspartate column), chromatography focus, SDS-PAGE, and ammonium sulfate precipitation, can also be determined from the obtained antibody as desired.

[0095] After any preliminary purification steps, the mixture containing the antibody of interest and impurities can be processed by low-pH hydrophobic interaction chromatography using an elution buffer with a pH of approximately 2.5–4.5, preferably at a low salt concentration (e.g., approximately 0–0.25 M salt concentration).

[0096] kit This application provides a kit comprising the antibody or its antigen-binding fragment. In some embodiments, the kit is used to detect the presence or level of AXL protein in a biological sample. The biological sample may include cells or tissues.

[0097] In some embodiments, the kit includes an antibody conjugated to a detectable label or an antigen-binding fragment thereof. In some embodiments, the kit includes an unlabeled antibody and further includes a labeled secondary antibody conjugable to the unlabeled antibody. The kit may further include instructions for use and packaging in which each component is separated in the kit.

[0098] In some embodiments, the antibody is connected to a substrate or apparatus for sandwich analysis, such as ELISA or immunochromatography. The substrate or apparatus used may be, for example, a microplate and test strip.

[0099] Pharmaceutical composition and therapeutic method This application further provides a pharmaceutical composition comprising the antibody and one or more pharmaceutically acceptable carriers.

[0100] The pharmaceutically acceptable carriers used in the pharmaceutical compositions disclosed in this application may include, for example, pharmaceutically acceptable liquids, gels or solid carriers, aqueous media, non-aqueous media, antimicrobial substances, isotonic substances, buffers, antioxidants, anesthetics, suspensions / dispersants, matching agents, diluents, adjuvants, excipients or non-toxic auxiliary substances, other components known in the art, or combinations thereof.

[0101] Applicable components may include, for example, antioxidants, fillers, adhesives, disintegrants, buffers, preservatives, lubricants, flavoring agents, thickeners, colorants, emulsifiers, or stabilizers such as sugars and cyclodextrins. Applicable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, mercaptoglycerol, thioglycolic acid, mercaptosorbitol, butylmethylanisole, butylated hydroxytoluene, and / or propyl gallate. In a composition containing the antibody disclosed in the present invention, the inclusion of one or more antioxidants such as methionine can reduce the oxidation of the antibody. By reducing the oxidative activity, the stability of the antibody can be improved and the shelf life can be extended by preventing or reducing the decrease in binding affinity.

[0102] Furthermore, pharmaceutically acceptable carriers may include, for example, an aqueous phase medium such as sodium chloride injection, Ringer's solution injection, isotonic glucose injection, sterile water injection, or Ringer's glucose and lactate injection; a non-aqueous phase medium such as plant-derived non-volatile oil, cottonseed oil, corn oil, sesame oil, or peanut oil; an antimicrobial substance at a bacterial or fungal inhibitory concentration; an isotonic agent such as sodium chloride or glucose; a buffer such as phosphate or citrate buffer; an antioxidant such as sodium bisulfate; a local anesthetic such as procaine hydrochloride; a suspending and dispersing agent such as sodium carboxymethylcellulose, hydroxypropyl methylcellulose, or polyvinylpyrrolidone; an emulsifier such as polysorbate 80 (polysorbate-80); and an admixture such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol bis(2-aminoethyl ether)tetraacetic acid), ethanol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid, or lactic acid. Antimicrobial agents as carriers can be added to pharmaceutical compositions in multi-dose containers containing phenols or cresol, mercury preparations, benzyl alcohol, chlorobutanol, methyl and propyl-p-hydroxybenzoic acid esters, thimerosal, chlorphenoxyammonium, and chlorphenethylammonium. Applicable excipients may include, for example, water, salt, glucose, glycerol, or ethanol. Applicable non-toxic auxiliary substances may include, for example, emulsifiers, pH buffers, stabilizers, solubilizers, or substances such as sodium acetate, sorbitan laurate, triethanolamine oleate, or cyclodextrin.

[0103] The pharmaceutical composition may be a liquid solution, suspension, emulsion, pill, capsule, tablet, sustained-release formulation, or powder. Oral formulations may contain pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, polyvinylpyrrolidone, sodium saccharin, cellulose, magnesium carbonate, or other standard carriers.

[0104] In some embodiments, the pharmaceutical composition is formulated into an injectable composition. The injectable pharmaceutical composition can be prepared in any conventional form, for example, a liquid solvent, suspension, emulsifier, or a solid form suitable for producing a liquid solvent, suspension, or emulsifier. The injectable formulation may include a ready-to-use sterile and / or pyrogen-free solution, a sterile-dried soluble product conjugated with a solvent before use, such as a lyophilized powder, a subcutaneous tablet, a sterile suspension ready for injection, a sterile-dried insoluble product conjugated with a medium before use, and a sterile and / or pyrogen-free emulsion. The solvent may be an aqueous or non-aqueous phase.

[0105] In some embodiments, the unit dose of the injectable formulation is contained in an ampoule, tube, or syringe with a needle. In the art, all injectable formulations are known to be sterile and pyrogen-free.

[0106] In some embodiments, a sterile lyophilized powder can be prepared by dissolving the antibody or antigen-binding fragment disclosed herein in a suitable solvent. The solvent can improve the stability of the powder or recombinant solution prepared from the powder, or improve other pharmacological components of the powder or recombinant solution. Applicable excipients include, but are not limited to, water, glucose, sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, brown sugar, or other applicable substances. The solvent may include a buffer, such as citrate buffer, sodium phosphate or potassium phosphate buffer, or other buffers well known to those skilled in the art, and in one embodiment, the pH of the buffer is neutral. The solution is filtered and sterilized under standard conditions known in the art, and then lyophilized to prepare the desired formulation. In one embodiment, the obtained solvent is dispensed into vials and lyophilized. Each vial can contain a single dose or multiple doses of the antibody or antigen-binding fragment or composition targeting the AXL protein. The volume contained in each vial can be slightly higher than the amount required for each dose, or the amount required for multiple doses (e.g., a 10% excess), thereby ensuring sampling accuracy and administration precision. The lyophilized powder can be stored under appropriate conditions, for example, within a range of approximately 4°C to room temperature.

[0107] The lyophilized powder is redissolved in sterile water for injection to obtain a formulation for injection. In one embodiment, the lyophilized powder can be added to sterile pyrogen-free water or other applicable liquid carrier and redissolved. The precise amount is determined by the selected therapy and may be determined based on empirical values.

[0108] The present invention further provides a therapeutic method comprising administering a therapeutically effective amount of the antibody described herein to a subject in need.

[0109] The therapeutically effective dose of the antibody provided in this application depends on various factors known in the art, such as body weight, age, medical history, current treatment, the subject's health status and the possibility of cross-infection, allergies, hypersensitivity and side effects, as well as the route of administration and the degree of tumor progression. Those skilled in the art (e.g., physicians or veterinarians) can proportionally reduce or increase the dose in response to these or other conditions or requirements.

[0110] In some embodiments, the antibodies provided by the present invention can be administered in therapeutically effective doses ranging from approximately 0.0 1 mg / kg to approximately 100 mg / kg. In some embodiments, the antibodies are administered in doses of approximately 50 mg / kg or less, and in some embodiments, the doses are 10 mg / kg or less, 5 mg / kg or less, 1 mg / kg or less, 0.5 mg / kg or less, or 0.1 mg / kg or less. A particular dose can be administered at multiple intervals, for example, once daily, twice daily or more, twice a month or more, once a week, once every two weeks, once every three weeks, once a month, or once every two months or more. In some embodiments, the dose may vary according to the treatment process. For example, in some embodiments, the initial dose may be higher than subsequent doses. In some embodiments, the dose is adjusted in accordance with the patient's response during the treatment process.

[0111] The administration regimen can be adjusted to achieve an optimal response (e.g., therapeutic response). For example, a single dose or multiple divided doses over a period of time may be administered.

[0112] The antibodies disclosed in this invention can be administered by means of administration known in the art, such as injection (e.g., intravenous injection including subcutaneous injection, intraperitoneal injection, intravenous drip infusion, intramuscular injection, or intradermal injection) or non-injection (e.g., oral administration, nasal administration, sublingual administration, rectal administration, or topical administration).

[0113] In some embodiments, the antibodies can be used to treat tumors and cancers selected from, for example, B-cell lymphoma, T-cell lymphoma, melanoma, prostate cancer, renal cell carcinoma, sarcoma, gliomas such as high-level glioma, prostate cell tumors such as neuroblastoma, osteosarcoma, plasmacytoma, histiocytic sarcoma, lung cancers such as pancreatic cancer, breast cancer, small cell lung cancer and non-small cell lung cancer, gastric cancer, liver cancer, colon cancer, rectal cancer, esophageal cancer, colorectal cancer, hematopoietic cancers, testicular cancer, cervical cancer, ovarian cancer, bladder cancer, squamous cell carcinoma, adenocarcinoma, AIDS-related lymphoma, bladder cancer, brain cancer, nervous system cancer, head and neck cancer, head and neck squamous cell carcinoma, Hodgkin lymphoma, non-Hodgkin lymphoma, or hematological cancers, including symptoms related to their molecular mechanisms.

[0114] How to use This application further provides a method for using the antibody.

[0115] In some embodiments, the present application provides a method for treating a condition or symptom in an individual that is related to the antibody mechanism, comprising administering a therapeutically effective dose of the antibody described herein.

[0116] The antibodies disclosed in this invention can be administered alone or in combination with one or more other therapeutic means or substances. For example, the antibodies disclosed in this invention can be used in combination with chemotherapy, radiotherapy, cancer treatment surgery (e.g., tumor resection), antiviral drugs, one or more antiemetics or therapies for complications of other chemotherapy, or therapeutic substances used for any other cancer or virus. In some such embodiments, when the antibodies disclosed in this invention are used in combination with one or more therapeutic substances, they may be administered simultaneously with the one or more therapeutic substances, and in some such embodiments, the antibodies may be administered simultaneously as part of the same pharmaceutical composition. However, antibodies "used in combination" with other therapeutic substances do not need to be administered simultaneously or in the same composition as the therapeutic substance. The meaning of "used in combination" in this invention further includes the fact that even if the antibody and the second substance are administered by different administration methods, an antibody administered before or after the other therapeutic substance is also considered to be "used in combination" with the therapeutic substance. Where possible, other therapeutic substances used in combination with the antibodies disclosed herein may be administered by referring to the methods described in the product description of the other therapeutic substance, or to the Physicians' Desk Reference, 57th Ed; Medical Economics Company; ISBN: 1563634457; 57th edition (November 2002)), or other methods known in the art.

[0117] In some embodiments, the therapeutic substance can induce or enhance an immune response against cancer. For example, tumor vaccines can be used to induce an immune response against certain tumors or cancers. Cytokine therapy can be used to improve the presentation of tumor antigens to the immune system. Examples of cytokine therapy include, but are not limited to, interferons such as interferon-α, β, and γ; colony-stimulating factors such as macrophage CSF, granulocyte-macrophage CSF, and granulocyte CSF; interleukins such as 1L-L, 1L-1a, 1L-2, 1L-3, 1L-4, 1L-5, 1L-6, 1L-7, 1L-8, 1L-9, 1L-10, 1L-II, and 1L-12; and tumor necrosis factors such as TNF-α and TNF-β. Reagents that inactivate immunosuppressive targets, such as PD-1 antibodies, TGF-β inhibitors, IL-10 inhibitors, and Fas ligand inhibitors, can also be used. The other set of reagents includes those that activate the immune response against tumor or cancer cells, such as those that enhance T cell activation (e.g., common T cell signaling pathways, such as CTLA-4, ICOS, OX40, 4-1BB, etc.), and those that enhance dendritic cell function and antigen presentation.

[0118] The following examples are provided to better illustrate the present invention and should not be understood as limiting its scope. All of the specific compositions, materials, and methods described below, in whole or in part, fall within the scope of the present invention. These specific compositions, materials, and methods are not intended to limit the present invention, but merely to illustrate specific embodiments that fall within the scope of the present invention. Those skilled in the art can develop equivalent compositions, materials, and methods without adding inventive step or departing from the scope of the present invention. It should be understood that several modifications made to the methods of the present invention may still fall within the scope of the present invention. The inventors intend to include such variations within the scope of the present invention.

[0119] Example 1: Construction and screening of a natural human antibody phage display library AXL antigen preparation: Human recombinant AXL protein was purchased from novoprotein, product number: C02B. The AXL whole-human antibody screening was commissioned to Sanyou Bio-Pharmaceutical (Shanghai) Co., Ltd. A total of four screenings were performed, and clones obtained from the fourth screening were selected for ELISA screening of positive clones. Ultimately, 71 positive clones capable of binding to the hAXL-ECD protein were screened from a total of 2304 clones. After sequencing analysis and ELISA binding, the sequences of four clones were selected to construct full-length antibodies for further experiments. The specific procedures are as follows: 1.1 Positive Clone Sequencing and Analysis After completing preliminary screening, 71 positive clones capable of binding to the hAXL-ECD protein were numbered. 2 μL of bacterial suspension was aspirated into 2 mL of 2YT medium and cultured overnight at 37°C and 220 rpm. Plasmids were extracted and bigenerational sequencing was performed. The sequencing results were integrated and compared using SeqMan to remove non-antibody gene sequences, generating an antibody-gene-matched fasta file. Subsequently, the DNA sequences were translated into amino acid sequences using MEGA6. Amino acid sequences containing terminators, atypical sequences, etc., were identified, and a fasta file of the amino acid sequence was derived. The antibody light chain and heavy chain sequences of the positive antibody clones were obtained, and after analysis and comparison, the sequences were found to be novel antibody sequences. 1.2 Binding affinity between clonal supernatant and antigen hAXL-ECD by ELISA screening First, the clones screened in the third step were sorted into a 96-well deep-well plate containing 300 μL of 2-YT medium and incubated overnight at 37°C. The supernatant contained the expressed Fab, which was then taken, gradient diluted, and added to an ELISA plate coated with 2 μg / mL hAXL-ECD. Subsequently, HRP-labeled goat anti-human Fab was detected as a secondary antibody (goat anti-human-HRP, ThermoFisher, 31482, 1:6000 dilution), with higher signal values ​​indicating stronger affinity. The results are shown in Figures 1A-G. Figures 1A to 1G show that the 71 clones of this application can specifically bind to hAXL-ECD. Clones 5, 7, 11, and 14 showed the highest affinity. These clones were selected for experiments and named Hu001-5, Hu001-7, Hu001-11, and Hu001-14. In ELISA assays, the Fabs of the four antibodies (Hu001-5 antibody, Hu001-7 antibody, Hu001-11 antibody, and Hu001-14 antibody) all showed good affinity activity. Sequencing yielded the heavy chain variable region cDNA sequences and light chain variable region cDNA sequences corresponding to the secreted antibodies. The corresponding antibody CDR sequences are shown in Table 1 below. [Table 1] JPEG0007877367000002.jpg177170

[0120] Example 2: Construction, expression, and purification of full-length antibodies In this example, two Fab antibodies with good blocking activity against hAXL-ECD binding obtained in Example 1 were constructed as human IgG1 subtypes. Both of these antibodies have κ-type light chains and are whole-human antibodies. 2.1 Plasmid Construction From bacterial strains containing antibodies obtained through screening, light and heavy chain variable region fragments of the antibodies were obtained by PCR amplification. These were then constructed into eukaryotic expression vector plasmids AbVec-hIgKappa or AbVec-hIgG1 WT (Figure 2), each containing the light and heavy chain constant region fragments, respectively, using homologous recombination. These plasmids were then combined with the full-length light and heavy chain genes of the complete antibody to encode the antibody heavy and light chain amino acid sequences shown in SEQ ID NO1-8. Vectors containing the full-length light and heavy chain genes of the constructed antibodies were used to transform E. coli TOP10 (uniquely DL1010S), which were cultured overnight at 37°C. The antibody light and heavy chain plasmids were then extracted using an endotoxin-free plasmid extraction kit (OMEGA, D6950-01) and subjected to eukaryotic expression. 2.2 Purification of Antibody Expression The candidate antibodies Hu001-5, Hu001-11, Hu001-7, Hu001-14 and the control antibody CCT301-38 (see US10066238) were expressed via the Expi293 transient expression system (Thermo Fisher, product number A1435101). The specific method is as follows: Expi293 cells were cultured in a 250 mL culture flask, and on the day of transfection, the cell density was 7 × 10⁶. 6 The cell viability was confirmed to be approximately 10 cells / mL, with a cell viability of >98%, and the cells were then fed 6 × 10 cells in Expi293 expression medium preheated to 37°C. 6 The final concentration was adjusted to individual cells / mL (final volume 75 mL). 1 mL of Expi293 was pre-cooled at 4°C. (TM) Prepare the expression medium, dilute the target plasmid (50 μg), and add 1 mL of Expi293. (TM) Dilute 75 μL of transfection reagent FectoPro (Polyplus, product number PT-116-001) with expression medium, then mix the two in an equal volume of 1 mL and gently mix until uniform to obtain Expi293. (TM)The expression medium / plasmid DNA mixture was prepared, incubated at room temperature for 15 minutes, slowly added to the prepared cell suspension, placed in a cell culture shaker, and cultured at 37°C under 5% CO2 conditions. 24 hours after transfection, FectoPRO booster was added to the culture system (to a final concentration of 0.6 μL / mL), and the cells were continued to be cultured at 37°C in a shaker under 5% CO2 conditions. On day 5 after transfection, the same volume of Expi293 was added. (TM) The expression medium was slowly added to the culture system, and after transfection for 10 days, the cell culture supernatant was collected and centrifuged at 4000g for 10 minutes. Affinity purification was then performed using a Protein G agarose column (GE, product number 28903134), the target protein was eluted with 100mM sodium acetate (pH 3.0), neutralized with 1M Tris-HCl, and the obtained protein was transferred to PBS buffer through a 30kD ultrafiltration concentrator (Millipore, product number UFC901096). The four candidate antibodies, Hu001-5, Hu001-7, Hu001-11, and Hu001-14, and the control antibody CCT301-38, all had a relative molecular weight of 150 kD and a purity of 90% or higher. The protein concentration of the purified antibodies was measured using an ultra-trace spectrophotometer (Thermo Fisher, product number NanoDrop One C). The measured A280 value was divided by the theoretical extinction coefficient of the antibody to obtain the antibody concentration value, which was then aliquoted and stored at -80°C.

[0121] Example 3: Affinity test of candidate antibodies that specifically bind to hAXL-ECD The affinity of candidate antibodies that specifically bind to hAXL-ECD was detected by flow cytometry. A total of four clones, Hu001-5, Hu001-7, Hu001-11, and Hu001-14, were detected under two conditions: acidic (PH=6.0) and neutral (PH=7.4). CHO-AXL cells (constructed by Shanghai Xinwan Biotechnology Co., Ltd.) were cultured in DMEM / F-12 medium (10% FBS, 1% penicillin / streptomycin) in a cell culture flask. After 48 hours of incubation at 37°C in a 5% CO2 incubator, the supernatant was discarded, and the cells were digested and collected with 0.25% trypsin. The cells were then centrifuged at 800g for 3 minutes. The supernatant was discarded, and the cells were resuspended in 2% FBS wash buffer (10 6 ( / mL). The pH of the buffer was adjusted to separate the cells into pH 6.0 and pH 7.4 groups. Primary antibodies (Hu001-5 antibody, Hu001-7 antibody, Hu001-11 antibody, Hu001-14 antibody, and positive control antibody CCT301-38) were added to each group, and incubated at room temperature for 15 minutes. The supernatant was discarded, and fluorescently labeled secondary antibody IgG-Fc-PE (BioLegend, product number 409304) was added. The cells were incubated in the dark for another 15 minutes at room temperature and then mechanically detected. A higher signal value indicated stronger affinity. The results are shown in Figure 3, which is a graph showing the affinity of the anti-AXL antibodies Hu001-5, Hu001-7, Hu001-11, and Hu001-14 of the present invention in flow data. Here, Figure 3A is a schematic diagram showing the change in affinity with respect to the concentration of the positive control CCT301-38 antibody at different pH levels, and Figures 3B to 3E are schematic diagrams showing the change in affinity with respect to the concentration of the anti-AXL antibodies Hu001-5, Hu001-7, Hu001-11, and Hu001-14 at different pH levels. It is clear that the antibodies of the present invention have a higher affinity for the positive control and have a higher binding ability under acidic conditions than under neutral conditions. As can be seen from the figures, the average fluorescence intensity of the Hu001-5 antibody experimental group and the Hu001-11 antibody experimental group is approximately four times that of the positive control group under both acidic and neutral conditions. The average fluorescence intensity of both the Hu001-5 antibody group and the Hu001-11 antibody group under acidic conditions was higher than the intensity under neutral conditions, approximately 1.5 times higher for the latter. This indicates that Hu001-5 and Hu001-11 have a higher affinity for AXL compared to the positive control, and that they have higher binding ability under acidic conditions than under neutral conditions.

[0122] Example 4: In vitro activity test of CART cells prepared with acid-sensitive antibody Hu001-11 The structures of the CAR cell chimeric antigen receptors for Hu001-11 and the positive control are shown in Figure 4A. Both Hu001-11-CART cells and the positive control CCT301-38-CART cells were prepared by Shanghai Xinwan Bio-Co., Ltd. Flow cytometry was used to measure their CAR expression positivity and mean fluorescence intensity. As shown in Figure 4B, the control antibody CCT301-38 had a positivity rate of 37.4% and a mean fluorescence intensity (MFI) of 7350. Hu001-11-CAR cells had a positivity rate of 46.9% and a mean fluorescence intensity (MFI) of 19571, indicating that the in vitro transduction efficiency of Hu001-11-CART cells is much higher than that of control CART cells. Add CHO-AXL cells as target cells to a 96-well plate (10 4 In the cell / well group, prepared Hu001-11-CART cells were used as effector cells, and the number of target cells was adjusted so that the effector cell to target cell ratio was 2:1. The control effector cells included untransduced T cells (negative control) and CCT301-38-CART cells (positive control). Cell cultures were divided into pH 6.8 and pH 7.4 groups. After co-culturing for 24 hours in a 37°C, 5% CO2 incubator, the content of IL-2 and IFN-γ was detected by ELISA to determine the degree of in vitro activation of CART cells. The protein concentrations of IL-2 (Abclonal, product number RK00002) and IFN-γ (Abclonal, product number RK00015) in the cultured cell supernatant were detected using Elisa. The results are shown in Figures 4C and 4D. In co-culture with CHO-AXL in vitro, the secretion levels of IL-2 and IFN-γ in Hu001-11-CART cells were higher than those of the positive control CCT301-38-CART cells, and the secretion levels were even higher under acidic conditions. This suggests that Hu001-11-CART cells are more strongly activated under in vitro acidic conditions and possess good acidic environment chemotaxis.

[0123] Example 5: In vitro glioma cell toxicization experiment mediated by Hu001-11 antibody In this experiment, the target cells were human glioma cells U251. The effector cells were CD16-CART cells (CD16-T cells, manufactured by Shanghai Xinwan Technology Co., Ltd.). The U251 cell line was cultured in DMEM medium (10% FBS, 1% penicillin / streptomycin) in a 37°C, 5% CO2 incubator, and 10 5 Cells were placed in an 8-well culture chamber at a cell concentration of / mL. After 12 hours, Hu001-11 antibody and CD16-T cells were added, respectively, with an effector cell to target cell ratio of 2:1 in both cases. Untransduced T cells were added as negative controls, and CCT301-38 antibody and CD16-T cells were used as positive controls. The cell index, which is positively correlated with the number of viable cells, was detected in real time using the xCELLigence RTCA S16 instrument. A higher value indicated lower killing activity. Conversely, a higher value indicated higher killing activity. The results are shown in Figure 5. The Hu001-11 and CD16-T combination group showed significant killing of U251 cells at 12 hours and nearly complete killing of target cells over 20 hours. The positive antibody control group showed complete killing effect at 24 hours. This suggests that Hu001-11 mediated better killing activity against glioma U251 cells.

[0124] Example 6: In vitro broad-spectrum antitumor activity experiment mediated by the antibody of the present invention In vitro killing experiments of CD16-T cells mediated by the anti-AXL antibodies Hu001-5, Hu001-7, Hu001-11, and Hu001-14 of the present invention against other solid tumor cells (human ovarian adenoma cells SK-OV3 and human lung cancer cells NCI-H292 tumors, both possessing reporter genes encoding luciferase) were also validated. Unless otherwise noted, all reagents and equipment used were purchased from Promega. Specifically, tumor cells were plated in 96-well plates (10 4The cells were seeded in a well and cultured at 37°C and 5% CO2 for 24 hours. After the culture medium was removed, the anti-AXL antibodies Hu001-5, Hu001-7, Hu001-11, and Hu001-14 of the present invention were added, and the cells were divided into 10 groups. These were: the group with the positive control antibody CCT301-38, the group with the Hu001-5 antibody, the group with the Hu001-7 antibody, the group with the Hu001-11 antibody, the group with the Hu001-14 antibody, the group with the Hu001-5 antibody and CD16-T cells, the group with the Hu001-7 antibody and CD16-T cells, the group with the Hu001-11 antibody and CD16-T cells, the group with the Hu001-14 antibody and CD16-T cells, and the group with the CCT301-38 antibody and CD16-T cells. The ratio of CD16-T cells to target cells was 2:1. After culturing at 37°C for 24 hours, the supernatant was removed, and 50 μL of 1x cell lysate (product no. E1531) was added to each well. The cells were incubated at room temperature for 30 minutes with shaking, and then 30 μL of luciferase analysis substrate (product no. E151A) was added to each well and allowed to develop color for 30 seconds. The fluorescence value was mechanically detected using a GloMax® Navigator Microplate Luminometer. The results are shown in Figure 6. For these two types of tumor cell lines, the in vitro killing efficiency using the anti-AXL antibodies Hu001-5, Hu001-7, Hu001-11, and Hu001-14 of the present invention was at least 1.5 times that of the positive control antibody CCT301-38. This demonstrates a clear killing enhancement effect and shows that the anti-AXL antibodies of the present invention can mediate a broader spectrum of antitumor activity in vitro. Figure 7 shows the sequence of the antibody variable region.

[0125] Although specific embodiments of the present invention have been described in detail above, it should be understood that the present invention is not limited to the above-described embodiments. Various improvements, modifications, and changes can be made to the present invention without departing from the spirit and scope of the invention, and all such improvements, modifications, and changes are within the scope of the present invention.

Claims

1. An antibody or its antigen-binding fragment that can specifically bind to the AXL protein, (a) Heavy chain variable region including the following three complementarity-determining regions, (i) VH CDR1 consisting of the sequence shown in SEQ ID NO: 9, (ii) VH CDR2 consisting of the sequence shown in SEQ ID NO: 10, and (iii) VH CDR3 consisting of the sequence shown in SEQ ID NO: 11, and (b) Light chain variable region including the following three complementarity determination regions, (iv) VL CDR1 consisting of the sequence shown in SEQ ID NO: 12, (v) VL CDR2 consisting of the sequence shown in SEQ ID NO: 13, and (vi) VL CDR3 consisting of the sequence shown in SEQ ID NO: 14, An antibody or its antigen-binding fragment, including the above.

2. An antibody or its antigen-binding fragment that can specifically bind to the AXL protein, (a) Heavy chain variable region including the following three complementarity-determining regions, (i) VH CDR1 consisting of the sequence shown in SEQ ID NO: 15, (ii) VH CDR2 consisting of the sequence shown in SEQ ID NO: 16, (iii) VH CDR3 consisting of the sequence shown in SEQ ID NO: 17, and (b) Light chain variable region including the following three complementarity determination regions, (iv) VL CDR1 consisting of the sequence shown in SEQ ID NO: 18, (v) VL CDR2 consisting of the sequence shown in SEQ ID NO: 19, and (vi) VL CDR3 consisting of the sequence shown in SEQ ID NO: 20, An antibody or its antigen-binding fragment, including the above.

3. An antibody or its antigen-binding fragment that can specifically bind to the AXL protein, (a) Heavy chain variable region including the following three complementarity-determining regions, (i) VH CDR1 consisting of the sequence shown in SEQ ID NO: 21, (ii) VH CDR2 consisting of the sequence shown in SEQ ID NO: 22, and (iii) VH CDR3 consisting of the sequence shown in SEQ ID NO: 23, and (b) Light chain variable region including the following three complementarity determination regions, (iv) VL CDR1 consisting of the sequence shown in SEQ ID NO: 24, (v) VL CDR2 consisting of the sequence shown in SEQ ID NO: 25, (vi) VL CDR3 consisting of the sequence shown in SEQ ID NO: 26, An antibody or its antigen-binding fragment, including the above.

4. An antibody or its antigen-binding fragment that can specifically bind to the AXL protein, (a) Heavy chain variable region including the following three complementarity-determining regions, (i) VH CDR1 consisting of the sequence shown in SEQ ID NO: 27, (ii) VH CDR2 consisting of the sequence shown in SEQ ID NO: 28, (iii) VH CDR3 consisting of the sequence shown in SEQ ID NO: 29, and (b) Light chain variable region including the following three complementarity determination regions, (iv) VL CDR1 consisting of the sequence shown in SEQ ID NO: 30, (v) VL CDR2 consisting of the sequence shown in SEQ ID NO: 31, and (vi) VL CDR3 consisting of the sequence shown in SEQ ID NO: 32, An antibody or its antigen-binding fragment, including the above.

5. The antibody or its antigen-binding fragment is (a) Heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 1, and (b) Light chain variable region containing the amino acid sequence shown in SEQ ID NO: 2, The antibody or antigen-binding fragment thereof according to claim 1, comprising:

6. The antibody or its antigen-binding fragment is (a) Heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 3, and (b) Light chain variable region containing the amino acid sequence shown in SEQ ID NO: 4, The antibody or antigen-binding fragment thereof according to claim 2, comprising:

7. The antibody or its antigen-binding fragment is (a) Heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 5, and (b) Light chain variable region containing the amino acid sequence shown in SEQ ID NO: 6, The antibody or antigen-binding fragment thereof according to claim 3, comprising:

8. The antibody or its antigen-binding fragment is (a) Heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 7, and (b) Light chain variable region containing the amino acid sequence shown in SEQ ID NO: 8, The antibody or antigen-binding fragment thereof according to claim 4, comprising:

9. The antibody or its antigen-binding fragment is (a) A heavy chain constant region of human immunoglobulin or a variant thereof, wherein the variant has one or more amino acid substitutions, deletions or additions compared to its origin sequence. and (b) A light chain constant region of human immunoglobulin or a variant thereof, wherein the variant has a maximum of 20 conserved amino acid substitutions compared to its origin sequence. It further includes, The heavy chain steady-state region is the IgG1, IgG2, IgG3, or IgG4 heavy chain steady-state region. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, wherein the light chain constant region is a κ-light chain constant region.

10. The antigen-binding fragments are Fab, Fab', (Fab') 2 Selected from Fv, Fv connected by disfield bonds, scFv, and diamond bodies, and / or, The antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, wherein the antibody is a chimeric antibody, a humanized antibody, a bispecific antibody, a multispecific antibody, or a fully human antibody.

11. A chimeric antigen receptor T cell comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 10, A chimeric antigen receptor T cell in which the heavy chain variable region and light chain variable region in the antibody or antigen-binding fragment are combined in series or in parallel.

12. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, or an isolated nucleic acid molecule encoding a heavy chain variable region and a light chain variable region thereof.

13. A vector comprising an isolated nucleic acid molecule as described in claim 12, The aforementioned vector is a virus, The viral skeleton of the aforementioned viral vector is modified or engineered. Vaccinia virus Tian Tan strain, Vaccinia virus New York strain, Vaccinia virus Copenhagen strain, Vaccinia virus Canary strain, Vaccinia virus Ankara strain, adenovirus vector, adeno-associated virus vector, herpes simplex virus vector, varicella-zoster virus vector, respiratory syncytial virus, Semryki forest virus, EB virus, giant cell virus, human herpesvirus 6, smallpox virus, molluscum contagiosum virus, eruption virus, reovirus, rotavirus, enterovirus, seneca virus, poliovirus, coxsackievirus, rhinovirus, hepatitis A virus, foot-and-mouth disease virus, togavirus, alphavirus, eastern equine encephalitis virus, Sindbis virus, rubella virus, coronavirus, flavivirus, hepatitis C virus, Japanese encephalitis virus, St. Louis encephalitis virus, Murray Valley fever virus, yellow Derived from fever viruses, West Nile virus, Zika virus, dengue virus, Ebola virus, Marburg virus, sand grain virus, Lassa fever virus, lymphocytic choriomeningitis virus, Pichinde virus, Junin virus, Machupo virus, Hantavirus, Rift Valley fever virus, paramyxovirus, human parainfluenza virus, mumps virus, simian virus 5, measles virus, varicella stomatitis virus, rabies virus, respiratory syncytial virus, orthomyxovirus, influenza A virus, influenza B virus, influenza C virus, hepatitis D virus, simian immunodeficiency virus, human immunodeficiency virus type 1 and human immunodeficiency virus type 2, Rous sarcoma virus, human T-cell leukemia virus type 1, simian bubble virus, hepatitis B virus, hepatitis E virus, human papillomavirus or polyomavirus, Alternatively, the vector is the cloning vector AbVec-hIgKappa shown in SEQ ID NO: 33 or the cloning vector AbVec-hIgG1 shown in SEQ ID NO:

34.

14. A host cell comprising an isolated nucleic acid molecule according to claim 12 or a vector according to claim 13, The host cell is selected from Escherichia coli cells, yeast cells, mammalian cells, or other cells suitable for preparing antibodies or antigen-binding fragments, multispecific antibodies.

15. A method for preparing an antibody or antigen-binding fragment according to any one of claims 1 to 10, comprising the steps of: culturing a host cell according to claim 14 under conditions that allow expression of the antibody or antigen-binding fragment according to any one of claims 1 to 10; and recovering the antibody or antigen-binding fragment from the cultured host cell culture.

16. A bispecific or multispecific molecule comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, The aforementioned bispecific or multispecific molecule is a bispecific or multispecific molecule that specifically binds to the AXL protein and further specifically binds to one or more other targets.

17. A bispecific or multispecific molecule according to claim 16, The bispecific or multispecific molecule further comprises at least one molecule having a second binding specificity to a second target, or further comprises another antibody or antigen-binding fragment that specifically binds to the AXL protein epitope.

18. An immunoconjugate comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, and a therapeutic agent linked to the antibody or antigen-binding fragment, The therapeutic agent is an immunoconjugate selected from alkylating agents, mitotic inhibitors, antitumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radionuclides, and any combination thereof.

19. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, a bispecific or multispecific molecule according to claim 16 or 17, or an immunoconjugate according to claim 18, and a pharmaceutically acceptable vector and / or excipient.

20. A pharmaceutical composition according to claim 19, The aforementioned pharmaceutical composition further comprises an additional pharmaceutically active agent, The aforementioned additional pharmacoactive agent is a pharmaceutical composition having antitumor activity.

21. A kit for detecting the presence or level of AXL protein, comprising an antibody or antigen-binding fragment thereof as described in any one of claims 1 to 10, The antibody or its antigen-binding fragment is a kit comprising a detectable label, radionuclide, fluorescent dye, luminescent substance, or biotin.

22. A kit according to claim 21, wherein the kit further comprises a secondary antibody that specifically recognizes an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 10, The kit further comprises a detectable label, a radionuclide, a fluorescent dye, a luminescent substance, or biotin as the secondary antibody.

23. A chimeric antigen receptor comprising the antigen-binding domain of an antibody or antigen-binding fragment thereof as described in any one of claims 1 to 10, Alternatively, a chimeric antigen receptor in which the antigen-binding domain includes a heavy chain variable region and a light chain variable region of the antibody or antigen-binding fragment described in any one of claims 1 to 10.

24. An isolated nucleic acid molecule encoding a chimeric antigen receptor according to claim 23.

25. A vector comprising the isolated nucleic acid molecule described in claim 24.

26. A host cell comprising an isolated nucleic acid molecule according to claim 24 or a vector according to claim 25, The aforementioned host cells are host cells that are immune effector cells.

27. The use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, or a bispecific or multispecific molecule according to claim 16 or 17, or an immunoconjugate according to claim 18, or a pharmaceutical composition according to claim 19 or 20, or a chimeric antigen receptor according to claim 23, or a host cell according to claim 26, wherein the pharmaceutical is used to prevent and / or treat a tumor in a subject. The tumors used are selected from B-cell lymphoma, T-cell lymphoma, melanoma, prostate cancer, renal cell carcinoma, sarcoma, glioma, preferably high-level glioma, maternal cell tumor, preferably neuroblastoma, osteosarcoma, plasmacytoma, histiocytic sarcoma, pancreatic cancer, breast cancer, lung cancer, preferably small cell lung cancer and non-small cell lung cancer, gastric cancer, liver cancer, colon cancer, rectal cancer, esophageal cancer, colorectal cancer, hematopoietic cancer, testicular cancer, cervical cancer, ovarian cancer, bladder cancer, squamous cell carcinoma, adenocarcinoma, AIDS-associated lymphoma, bladder cancer, brain cancer, nervous system cancer, head and neck cancer, head and neck squamous cell carcinoma, Hodgkin lymphoma, non-Hodgkin lymphoma, or hematological cancers.

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