Monoclonal antibody or antigen-binding fragment that specifically binds to AXL, and its use

Abstract

The present invention relates to the fields of biotechnology and pharmaceuticals, in particular to monoclonal antibodies or antigen-binding fragments thereof that specifically bind to AXL. The present invention further relates to nucleic acids encoding the antibody, expression vectors, host cells and methods for producing them, methods for producing the antibody according to the present invention, pharmaceutical compositions comprising the antibody according to the present invention, pharmaceutical compositions comprising the antibody according to the present invention and other therapeutically active compounds, methods for treating AXL-mediated diseases or disorders, the use of antibodies or pharmaceutical compositions for treating AXL-mediated diseases or disorders, and the use of antibodies and other therapeutically active compounds for treating AXL-mediated diseases or disorders.

Description

[Technical Field]

[0001] The present invention relates to the fields of biotechnology and pharmaceuticals, in particular to monoclonal antibodies or antigen-binding fragments thereof that specifically bind to AXL. The present invention further relates to nucleic acids encoding the antibody, expression vectors, host cells and methods for producing them, methods for producing the antibody according to the present invention, pharmaceutical compositions comprising the antibody according to the present invention, pharmaceutical compositions comprising the antibody according to the present invention and other therapeutically active compounds, methods for treating AXL-mediated diseases or disorders, the use of antibodies or pharmaceutical compositions for treating AXL-mediated diseases or disorders, and the use of antibodies and other therapeutically active compounds for treating AXL-mediated diseases or disorders. [Background technology]

[0002] Monoclonal antibodies in the form of chimeric, humanized, or fully human molecules have proven effective as pharmaceuticals for treating multiple disorders and diseases. The tyrosine kinase receptor Axl (ARK, TYRO7, and UFO) belongs to the tumor-associated macrophage (TAM) receptor family, which also includes TYRO-3, AXL, and MER. Structurally, the TAM receptor contains two immunoglobulin-like (Ig) domains; the extracellular domain contains two portions of fibronectin type III (FNIII); and its kinase domain contains the conserved amino acid sequence KW(I / L)A(I / L)ES. Gas6 (proliferation arrest-specific 6) is a ligand for Axl. Activated by Gas6 binding, Axl propagates its signal via phosphorylation.

[0003] AXL has been shown to be overexpressed in various malignancies. Overexpression or activation of AXL is closely associated with cell proliferation, survival, migration, and invasion through activation of oncogenic signaling pathways, including the PI3K / Akt and / or MAPK / Erk pathways. Based on the above, AXL is a promising target in antitumor treatment.

[0004] AXL has been found to be overexpressed in many cancers, including lung cancer, liver cancer, kidney cancer, colon cancer, stomach cancer, ovarian cancer, pancreatic cancer, and glioblastoma. Patent documents WO2015193428, WO2015193430, WO2017220695, and WO2017180842 provide various antibodies against AXL.

[0005] It is necessary to create novel antibodies that specifically bind to AXL and have high AXL antigen-binding affinity parameters. [Overview of the project] [Means for solving the problem]

[0006] The group of authors of the present invention has developed an antibody that specifically binds to AXL and has a high AXL binding affinity parameter. Remarkably, the group of authors has developed an antibody that specifically binds to the second immunoglobulin-like domain of AXL (AXL-Ig2), to AXL fragments containing the first and second immunoglobulin-like domains of AXL (AXL-Ig1-Ig2), and to the complete extracellular portion of AXL (ExcAXL), but does not individually bind to the first immunoglobulin-like domain of AXL (AXL-Ig1). The antibody against AXL according to the present invention inhibits the binding of AXL to GAS6, which results in the activation of AXL-mediated cell signaling. The antibody against AXL according to the present invention exhibits antibody-dependent cytotoxicity (ADCC) and antibody-dependent phagocytosis (ADCP) properties. The antibody against AXL according to the present invention has high aggregation stability and high stability in human serum. Furthermore, the antibody against AXL according to the present invention exhibits antitumor activity and does not exhibit toxicity or local irritation effects.

[0007] Definitions and general methods Unless otherwise defined, all technical and scientific terms used in connection with this invention have the same meaning as those commonly understood by those skilled in the art.

[0008] Furthermore, unless otherwise intended by context, singular terms include plural terms, and plural terms include singular terms. Typically, the classifications and methods of the present invention relating to cell culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, organic synthesis chemistry, medical chemistry and medicinal chemistry, as well as the hybridization and chemistry of proteins and nucleic acids described herein, are well known to those skilled in the art and are widely used in such art. Enzymatic reactions and purification methods are carried out in accordance with the manufacturer's guidelines, which is common in such art or described herein.

[0009] In this document, the term "KD" refers to the affinity constant (or equilibrium constant), which is calculated by the ratio of Kd to Ka (i.e., Kd / Ka) and expressed as molar concentration (M). "Binding affinity" typically refers to the sum of the strengths of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, "binding affinity" refers to the intrinsic (characteristic, true) binding affinity that reflects the 1:1 interaction between the members of the binding pair (e.g., antibody and antigen). The affinity of molecule X to its binding partner Y can generally be expressed by an affinity indices (KD). Preferred Kd values ​​are approximately 200 nM, 150 nM, 100 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 8 nM, 6 nM, 4 nM, 2 nM, 1 nM, or less. Affinity can be measured by methods common to the art, including the methods described herein. Low-affinity antibodies typically bind slowly to antigens and tend to dissociate easily, while high-affinity antibodies typically bind quickly to antigens and tend to remain bound for extended periods. Various methods for measuring binding affinity are known in the art, and any one of these methods can be used for the purposes of the present invention.

[0010] The terms "Kd," "koff," or "kdis" refer to the off-rate constant of a specific interaction between a binding molecule and an antigen. The off-rate constant koff can be measured using bio-layer interferometry, for example, using the Octet® system.

[0011] The terms "Ka," "kon," or "on-rate" refer to the association rate constant. The term “R 2 " refers to the coefficient of determination.

[0012] The term "response" refers to the antibody-antigen binding signal. The term "in vitro" refers to a biological entity, process, or reaction outside the body under artificial conditions. For example, in vitro cell proliferation should be understood as cell proliferation in an external environment, such as a test tube, culture vial, or microplate.

[0013] Term "ED" 50 (EC 50 The (50% effective dose / concentration) refers to the concentration of a formulation that produces 50% of the biological effect (which may include cytotoxicity). When used in this application, the “Kabat numbering scheme” or “Kabat numbering” refers to a system for numbering amino acid residues that are more variable (i.e., hypervariable) than other amino acid residues in the variable regions of the heavy and light chains of an antibody (Kabat et al., Ann. NYAcad. Sci., Vol. 190: pp. 382-383 (1971); Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, USD Department of Health and Human Services, NIH Publication No. 91-3242 (1991)).

[0014] As used in the following description and claims, unless the context clearly dictates otherwise, the words "include", "comprise", or variations thereof, such as "includes", "including", "comprises", or "comprising", are to be understood to mean the inclusion of the stated integer or group of integers but not the exclusion of any other integer or group of integers.

[0015] Antibody The present invention relates to a monoclonal antibody or an antigen-binding fragment thereof that specifically binds to AXL.

[0016] The term "monoclonal antibody" or "mAb" refers to an antibody synthesized and isolated as an individual clone population of cells. The antibody of the present invention is a recombinant antibody.

[0017] The term "recombinant antibody" refers to an antibody expressed in a cell or cell line containing a nucleotide sequence encoding the antibody, and the nucleotide sequence is not associated with natural cells. In one aspect, the present invention is a monoclonal antibody or an antigen-binding fragment thereof that specifically binds to AXL, (a) CDR1 having the amino acid sequence of SEQ ID NO: 1, CDR2 having the amino acid sequence of SEQ ID NO: 2, and CDR3 having the amino acid sequence of SEQ ID NO: 3 comprising a light chain variable domain; and (b) CDR1 having the amino acid sequence of SEQ ID NO: 7, CDR2 having the amino acid sequence of SEQ ID NO: 8, and CDR3 having the amino acid sequence of SEQ ID NO: 9 comprising a heavy chain variable domain relates to a monoclonal antibody or an antigen-binding fragment thereof.

[0018] The monoclonal antibodies according to the present invention specifically bind to an AXL fragment containing different Ig-like C2 type 2 domains of AXL (AXL-Ig2) or two Ig-like C2 type 1 and Ig-like C2 type 1 domains (AXL-Ig1-Ig2), as well as the complete extracellular portion of AXL (ExcAXL), but do not bind to an AXL fragment containing a different Ig-like C2 type 1 domain (AXL-Ig1). The monoclonal antibodies against AXL according to the present invention inhibit the binding of AXL and GAS6, which results in the activation of AXL-mediated cell signaling.

[0019] In one aspect of the invention, the antibody according to the invention is an isolated antibody. The term "isolated", as used to describe the various antibodies according to this description, refers to an antibody that has been identified, isolated, and / or reproduced in a cell or cell culture in which it is expressed. Impurities (contaminating components) in the natural environment are materials that typically interfere with the diagnostic or therapeutic use of a polypeptide and can include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. An isolated polypeptide is typically prepared by at least one purification step.

[0020] The term "antibody" or "immunoglobulin" (Ig), as used in this description, includes antibodies in general. The term "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (referred to herein as VH for brevity) and a heavy chain constant region. Each light chain consists of a light chain variable region (referred to herein as VL for brevity) and a light chain constant region. Preferably, the light chain is a kappa (κ) light chain, and the constant domain CL is preferably C kappa (κ).

[0021] The antibodies according to the present invention can be of any class (e.g., IgA, IgD, IgE, IgG, and IgM, preferably IgG), or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2, preferably IgG1).

[0022] The VL and VH regions can be subdivided into hypervariable regions called complementarity-determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen.

[0023] The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or various cells of the immune system (e.g., effector cells) and factors containing the first component (C1q) of the classical complement system.

[0024] The terms “antigen-binding moiety” or “antigen-binding fragment” of an antibody, as used herein, refer to one or more antibody fragments that possess the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments included in the term “antigen-binding moiety” of an antibody include: (i) the Fab fragment, a monovalent fragment consisting of VL, VH, CL, and CH1 domains; (ii) the F(ab')2 fragment, a bivalent fragment containing two Fab fragments linked by disulfide crosslinks in a hinge region; (iii) the Fd fragment, consisting of VH and CH1 domains; (iv) the Fv fragment, consisting of VL and VH domains of a single arm of the antibody; and (v) the dAb fragment, consisting of a VH / VHH domain (Ward et al., (1989) Nature 341: pp. 544-546). In addition, the two regions of the Fv fragment, VL and VH, are encoded by different genes and can be conjugated by recombination using a synthetic linker so that they are received as a single protein chain paired together to form a monovalent molecule (known as single-chain Fv (scFV); see, for example, Bird et al. (1988) Science vol. 242: pp. 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA vol. 85: pp. 5879-5883). Such single-chain molecules are also assumed to be included in the term "antigen-binding portion" of an antibody. Such antibody fragments are produced using conventional techniques known to those skilled in the art, and these fragments are screened in the same manner as intact antibodies.

[0025] The antibodies of the present invention that "specifically bind" to a target antibody refer to antibodies that bind to an antigen with sufficient affinity and cross-react only slightly with other proteins, so that the antibody can be used as a diagnostic and / or therapeutic agent that targets a protein, cell, or tissue expressing the antigen.

[0026] The term “specifically binds” to a particular polypeptide or an epitope of a particular target polypeptide can be described by examples of molecules having a Kd of at least about 200 nM, or at least about 150 nM, or at least about 100 nM, or at least about 60 nM, or at least about 50 nM, or at least about 40 nM, or at least about 30 nM, or at least about 20 nM, or at least about 10 nM, or at least about 8 nM, or at least about 6 nM, or at least about 4 nM, or at least about 2 nM, or at least about 1 nM, or less, relative to the target.

[0027] In one embodiment, the term “specifically bind” refers to a bind in which a molecule binds to a particular polypeptide or a particular target polypeptide epitope without substantially binding to any other polypeptide or polypeptide epitope.

[0028] In some embodiments of the present invention, a monoclonal antibody or its antigen-binding fragment comprises a light chain variable domain containing the amino acid sequence of SEQ ID NO: 13. In some embodiments of the present invention, a monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable domain containing the amino acid sequence of SEQ ID NO: 14.

[0029] In some aspects of the present invention, a monoclonal antibody or its antigen-binding fragment is (a) Light chain variable domain containing the amino acid sequence of Sequence ID No. 13; and (b) Heavy chain variable domain containing the amino acid sequence of SEQ ID NO: 14 Includes.

[0030] In some embodiments of the present invention, the monoclonal antibody that specifically binds to AXL is a full-length IgG antibody. In some aspects of the present invention, the monoclonal antibody that specifically binds to AXL is a full-length IgG antibody of the human IgG1, IgG2, IgG3, or IgG4 isotype.

[0031] In some aspects of the present invention, the monoclonal antibody that specifically binds to AXL is a full-length IgG antibody of the human IgG1 isotype. In some embodiments of the present invention, the monoclonal antibody contains mutations in the Fc fragment to increase the ADCC, CDC, and / or ADCP properties of the antibody.

[0032] In some embodiments of the present invention, the monoclonal antibody contains mutations S239D and I332E in the Fc fragment according to the EU numbering scheme for the amino acids of the antibody (Edelman GM et al., Proc. Natl. Acad. Sci. USA, Vol. 63 (1969), pp. 78-85; Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991)).

[0033] In some embodiments of the present invention, the monoclonal antibody contains deletions 446G and 447K in the CH3 region within the Fc fragment, according to the EU numbering scheme for the amino acids of the antibody. In some aspects of the present invention, the monoclonal antibody comprises a light chain having the amino acid sequence of SEQ ID NO: 15.

[0034] In some embodiments of the present invention, the monoclonal antibody comprises a heavy chain containing an amino acid sequence selected from the group of SEQ ID NO: 16 or SEQ ID NO: 17. In some aspects of the present invention, a monoclonal antibody is (i)(a) Light chain containing the amino acid sequence of Sequence ID No. 15, and (b) Heavy chain containing the amino acid sequence of SEQ ID NO: 16; or (ii)(a) Light chain containing the amino acid sequence of Sequence ID No. 15, and (b) Heavy chain containing the amino acid sequence of SEQ ID NO: 17 Includes.

[0035] In some aspects of the present invention, the monoclonal antibody that specifically binds to AXL is an antibody selected from the groups 01_004 and 01_004_2. In some aspects of the present invention, the monoclonal antibody that specifically binds to AXL is antibody 01_004.

[0036] Antibody 01_004 is, (a) a light chain containing the amino acid sequence of SEQ ID NO: 15; and (b) Heavy chain containing the amino acid sequence of SEQ ID NO: 16 Includes.

[0037] Antibody 01_004 is, (a) Light chain variable domain containing the amino acid sequence of Sequence ID No. 13; and (b) Heavy chain variable domain containing the amino acid sequence of SEQ ID NO: 14 Includes.

[0038] Antibody 01_004 is, (a)(i) CDR1(Kabat) having the amino acid sequence of Sequence ID No. 1, (ii) CDR2(Kabat) having the amino acid sequence of SEQ ID NO. 2, and (iii) CDR3(Kabat) having the amino acid sequence of SEQ ID NO: 3 Light chain variable domains including Furthermore (b)(i) CDR1(Kabat) having the amino acid sequence of SEQ ID NO: 7, (ii) CDR2(Kabat) having the amino acid sequence of SEQ ID NO: 8, and (iii) CDR3(Kabat) having the amino acid sequence of SEQ ID NO. 9 Heavy chain variable domains including Includes.

[0039] Antibody 01_004 is, (a)(i) CDR1(Chothia) having the amino acid sequence of SEQ ID NO: 4, (ii) CDR2(Chothia) having the amino acid sequence of SEQ ID NO: 5, and (iii) CDR3(Chothia) having the amino acid sequence of SEQ ID NO. 6 Light chain variable domains including Furthermore (b)(i) CDR1(Chothia) having the amino acid sequence of SEQ ID NO: 10, (ii) CDR2(Chothia) having the amino acid sequence of SEQ ID NO: 11, and (iii) CDR3(Chothia) having the amino acid sequence of SEQ ID NO. 12 Heavy chain variable domains including Includes.

[0040] In some aspects of the present invention, the monoclonal antibody that specifically binds to AXL is antibody 01_004_2. Antibody 01_004_2 is, (a) a light chain containing the amino acid sequence of SEQ ID NO: 15; and (b) Heavy chain containing the amino acid sequence of SEQ ID NO: 17 Includes.

[0041] Antibody 01_004_2 is, (a) Light chain variable domain containing the amino acid sequence of Sequence ID No. 13; and (b) Heavy chain variable domain containing the amino acid sequence of SEQ ID NO: 14 Includes.

[0042] Antibody 01_004_2 is, (a)(i) CDR1(Kabat) having the amino acid sequence of Sequence ID No. 1, (ii) CDR2(Kabat) having the amino acid sequence of SEQ ID NO. 2, and (iii) CDR3(Kabat) having the amino acid sequence of SEQ ID NO: 3 Light chain variable domains including Furthermore (b)(i) CDR1(Kabat) having the amino acid sequence of SEQ ID NO: 7, (ii) CDR2(Kabat) having the amino acid sequence of SEQ ID NO: 8, and (iii) CDR3(Kabat) having the amino acid sequence of SEQ ID NO. 9 Heavy chain variable domains including Includes.

[0043] Antibody 01_004_2 is, (a)(i) CDR1(Chothia) having the amino acid sequence of SEQ ID NO: 4, (ii) CDR2(Chothia) having the amino acid sequence of SEQ ID NO: 5, and (iii) CDR3(Chothia) having the amino acid sequence of SEQ ID NO. 6 Light chain variable domains including Furthermore (b)(i) CDR1(Chothia) having the amino acid sequence of SEQ ID NO: 10, (ii) CDR2(Chothia) having the amino acid sequence of SEQ ID NO: 11, and (iii) CDR3(Chothia) having the amino acid sequence of SEQ ID NO. 12 Heavy chain variable domains including Includes.

[0044] The hypervariable regions of all light and heavy chain variable domains (LCDR1, 2, 3 and HCDR1, 2, 3) of the above antibodies are provided according to Kabat and Chothia nomenclature. Those skilled in the art will also understand that the hypervariable regions of the light and heavy chain variable domains (LCDR1, 2, 3 and HCDR1, 2, 3) may also be represented according to other commonly known numbering schemes, e.g., IMGT or AbM. Thus, all of the above antibodies characterized by the hypervariable regions of the light and heavy chain variable domains (LCDR1, 2, 3 and HCDR1, 2, 3) using the IMGT or AbM numbering scheme are also included in the present invention.

[0045] nucleic acid molecule In one aspect, the present invention relates to a nucleic acid encoding any one of the above-mentioned antibodies or antigen-binding fragments that specifically bind to AXL.

[0046] In any one of the above embodiments, nucleic acid molecules can be isolated. The terms “nucleic acid,” “nuclear sequence,” or “nucleic acid sequence,” “polynucleotide,” “oligonucleotide,” “polynucleotide sequence,” and “nucleotide sequence,” as used interchangeably in this description, mean the exact sequence of modified or unmodified nucleotides that determine a fragment or region of nucleic acid, containing or not containing non-natural nucleotides, and which are either double-stranded DNA or RNA, single-stranded DNA or RNA, or transcripts of said DNA.

[0047] Unless otherwise specified, the term nucleotide sequence includes its complement. Therefore, nucleic acids with a particular sequence should be understood to include a complementary strand with a complementary sequence.

[0048] An "isolated" nucleic acid molecule is one that has been identified with at least one nucleic acid molecule-impurity and separated from it. An isolated nucleic acid molecule is different in form or configuration from those found under natural conditions. Therefore, an isolated nucleic acid molecule is different from the nucleic acid molecules present in cells under natural conditions.

[0049] In one aspect, the present invention relates to a nucleic acid molecule comprising a nucleotide sequence encoding an amino acid sequence selected from SEQ ID NOs: 18-22. The nucleic acid molecule may also include any combination of the nucleotide sequences.

[0050] As those skilled in the art will understand, due to the duplication of the genetic code, various different DNA sequences can encode the amino acid sequences of the light or heavy chains (VH, VL, CDR, etc.) of the antibodies or fragments thereof according to the present invention. Creating alternative DNA sequences that encode one identical amino acid sequence is well within the scope of the skills of those skilled in the art. Such variant DNA sequences are within the scope of the present invention.

[0051] In some embodiments of the present invention, nucleic acid is DNA. The nucleic acid molecules of the present invention can be isolated from any source that produces a monoclonal antibody or its antigen-binding fragment that specifically binds to AXL. In certain embodiments of the present invention, the nucleic acid molecules of the present invention can be synthesized by chemical synthesis rather than isolation.

[0052] In some aspects of the present invention, the nucleic acid is a nucleic acid that encodes the amino acid sequences of the light chain variable domains of antibodies 01_004 and 01_004_2 and includes the nucleotide sequence of SEQ ID NO: 18.

[0053] In some aspects of the present invention, the nucleic acid is a nucleic acid that encodes the amino acid sequences of the heavy chain variable domains of antibodies 01_004 and 01_004_2 and comprises the nucleotide sequence of SEQ ID NO: 19.

[0054] In some aspects of the present invention, the nucleic acid is a nucleic acid that encodes the amino acid sequences of the light chains of antibodies 01_004 and 01_004_2 and includes the nucleotide sequence of SEQ ID NO: 20. In some aspects of the present invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the heavy chain of antibody 01_004 and includes the nucleotide sequence of SEQ ID NO: 21.

[0055] In some aspects of the present invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the heavy chain of antibody 01_004_2 and contains the nucleotide sequence of SEQ ID NO: 22. Nucleic acid molecules can be used to express monoclonal antibodies or their antigen-binding fragments that specifically bind to AXL.

[0056] vector In one aspect, the present invention relates to an expression vector comprising any one of the nucleic acid molecules that encode an antibody or a portion thereof (e.g., a heavy chain and / or light chain binding domain sequence) that specifically binds to AXL. The present invention relates to a vector suitable for the expression of any one of the nucleotide sequences described herein.

[0057] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting other nucleic acid molecules to which it is linked. As used in this document, the term “expression” is defined as the transcription and / or translation of a particular nucleotide sequence driven by a promoter.

[0058] In some embodiments of the present invention, the vector is a plasmid, i.e., a circular double-stranded piece of DNA into which additional DNA segments can be inserted. In some embodiments of the present invention, the vector is a viral (expression) vector in which an additional DNA segment can be inserted into the viral genome.

[0059] In some embodiments of the present invention, vectors are capable of autonomous replication within the host cell into which they are introduced (e.g., bacterial vectors having a bacterial site as an origin of replication, and episomal vectors). In further embodiments of the present invention, vectors (non-episomal vectors) can be incorporated into the genome of a host cell by introduction into the host cell, thereby replicating together with host genes. Furthermore, certain vectors can direct the expression of genes that are operablely linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply “expression vectors”).

[0060] In some embodiments of the present invention, expression vectors include plasmids, retroviruses, adenoviruses, adeno-associated viruses (AAVs), plant viruses such as cauliflower mosaic virus, tobacco mosaic virus, cosmids, and YACs. DNA molecules can be inserted into the vector such that transcriptional and translational regulatory sequences within the vector perform the intended function of regulating DNA transcription and translation. Expression vectors and expression regulatory sequences may be selected to be compatible with the expression host cells used.

[0061] In one embodiment of the present invention, DNA molecules that partially or completely encode heavy and light strand sequences can be inserted into different vectors. In one embodiment, any combination of the above DNA molecules is introduced into the same expression vector.

[0062] In one embodiment of the present invention, a DNA molecule can be introduced into an expression vector by a standard method (e.g., ligation of complementary restriction sites in the gene fragments of the antibody and vector, or blunt-end ligation if no restriction sites are present).

[0063] In some embodiments of the present invention, a suitable vector includes a restriction site so that any VH or VL sequence can be readily inserted and expressed, as described above. The recombinant expression vector may also encode a signal peptide that facilitates the secretion of an antibody chain from a host cell. The antibody chain gene may be cloned into the vector so that the signal peptide is in-frame ligated to the amino terminus of the immunoglobulin chain. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

[0064] In some embodiments of the present invention, the vector may include an expression regulatory sequence. The term “expression regulatory sequence,” as used herein, refers to a polynucleotide sequence necessary to bring about the expression and processing of the inserted coding sequence. Those skilled in the art will understand that the design of an expression vector, including the selection of an expression regulatory sequence, may depend on factors such as the selection of the type of host cell to be transformed and the required expression level of the antibody. Expression regulatory sequences include appropriate transcription start, termination, promoter, and enhancer sequences; efficient RNA processing signals, e.g., splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and, if desired, sequences that enhance protein secretion. The nature of such expression regulatory sequences varies depending on the host organism; in prokaryotes, such expression regulatory sequences typically include a promoter, ribosome binding site, and transcription termination sequence, while in eukaryotes, such expression regulatory sequences typically include a promoter and transcription termination sequence. Preferred regulatory sequences for mammalian expression host cells include virus-like elements that ensure high levels of protein expression in mammalian cells, such as retroviral LTRs, cytomegalovirus (CMV) (e.g., DMV promoter / enhancer), simian virus 40 (SV40) (e.g., SV40 promoter / enhancer), adenovirus (e.g., adenovirus major late promoter (AdMLP)), promoters / enhancers derived from polyomaviruses, potent mammalian promoters such as the TTR promoter, undenatured immunoglobulin promoter, or actin promoter. Regulatory sequences encompass at least all components whose presence is important for expression and processing.

[0065] In some embodiments of the present invention, in addition to antibody chain genes and expression regulatory sequences, the recombinant expression vector of the present invention may carry additional sequences, such as sequences that regulate the replication of the vector in host cells (e.g., origin of replication) and the replication of selectable marker genes. The selectable marker genes help in the selection of host cells into which the vector is introduced.

[0066] host cell In one aspect, the present invention relates to a method for producing host cells that produce any of the above antibodies or antigen-binding fragments that specifically bind to AXL, comprising transforming the cells with the above vector.

[0067] In one aspect, the present invention relates to a host cell that produces any of the above antibodies or antigen-binding fragments that specifically bind to AXL, comprising any one of the above nucleic acids. The term “host cell,” as used herein, refers to the cell into which a recombinant expression vector is introduced. The present invention relates, for example, to a host cell that may contain the vector according to the present invention as described above. The present invention further relates to a host cell that contains, for example, a nucleotide sequence encoding a heavy chain or its antigen-binding portion, a nucleotide sequence encoding a light chain or its antigen-binding portion, or both. “Host cell” refers not only to the cell of a particular subject but also to the progeny of such a cell. Such progeny may not actually be identical to the parent cell, as modifications may occur in subsequent generations by either mutation or environmental influence, but such cells are still included within the scope of the term “host cell” as used herein.

[0068] The monoclonal antibodies or antigen-binding fragments that specifically bind to AXL according to the present invention, and nucleic acid molecules encoding vectors containing these nucleic acid molecules, can be used for transfection of mammalian cells, plant cells, bacterial cells, or yeast cells. Transfection can be carried out by any known method for introducing polynucleotides into host cells. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, cationic polymer nucleic acid complex transfection, calcium phosphate precipitation, polybren-mediated transfection, protoplast fusion, liposome-mediated encapsulation of polynucleotides, and direct microinjection of DNA into the nucleus. In addition, nucleic acid molecules can be introduced into mammalian cells by viral (expression) vectors.

[0069] Mammalian cell lines used as host cells for transformation include several well-known and available immortalized cell lines in this technique. These include, for example, Chinese hamster ovary (CHO) cells, NS0 cells, SP2 cells, HEK-293T cells, FreeStyle 293 cells (Invitrogen), NIH-3T3 cells, HeLa cells, baby hamster kidney (BHK) cells, African green monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549, SK-HEP1, HUH7, Hep-RG cells, and numerous other cell lines. Cell lines are selected by determining which cell lines have high expression levels and provide the desired characteristics of the proteins to be produced. Other cell lines that may be used include insect cell lines, e.g., Sf9 or Sf21 cells. When a recombinant expression vector encoding a monoclonal antibody or its antigen-binding fragment that specifically binds to AXL is introduced into a mammalian host cell, the antibody or fragment is produced by culturing the host cell for a sufficient time to allow expression of the antibody or fragment in the host cell, or more preferably, to allow secretion of the antibody or fragment into the culture medium in which the host cell is grown. Monoclonal antibodies or their antigen-binding fragments that specifically bind to AXL can be isolated from the culture medium using standard protein purification techniques. Plant host cells include, for example, tobacco (Nicotiana), Arabidopsis, duckweed, maize, wheat, and potato. Bacterial host cells include Escherichia and Streptomyces species. Yeast hosts include fission yeast (Schizosaccharomyces pombe), Saccharomyces cerevisiae, and Pichia pastoris.

[0070] Furthermore, the production levels of monoclonal antibodies or their antigen-binding fragments that specifically bind to AXL from the producing cell line can be increased using a number of known techniques. For example, glutamine synthase gene expression systems (GS systems) are a common method for increasing expression under certain conditions.

[0071] Monoclonal antibodies or antigen-binding fragments that specifically bind to AXL from various cell lines may have different glycosylation profiles when compared to one another. However, monoclonal antibodies or antigen-binding fragments that specifically bind to AXL, encoded by nucleic acid molecules described herein or nucleic acid molecules containing amino acid sequences provided herein, are part of the present invention, regardless of the glycosylation of the binding molecule and generally with or without post-translational modifications.

[0072] The host cells mentioned above are not related to host cells produced using human embryos. The host cells mentioned above are not related to host cells produced by modifying the genetic integrity of human germ cells.

[0073] Methods for producing antibodies In one aspect, the present invention relates to a method for producing an antibody or an antigen-binding fragment thereof that specifically binds to AXL, comprising culturing the host cells in a growth medium under conditions sufficient to produce the antibody or the antigen-binding fragment, and subsequently isolating and purifying the obtained antibody or the antigen-binding fragment.

[0074] Pharmaceutical composition Another aspect of the present invention is a pharmaceutical composition comprising, as an active ingredient (or as the sole active ingredient), a monoclonal antibody according to the present invention or an antigen-binding fragment thereof that specifically binds to AXL.

[0075] In one aspect, the present invention relates to a pharmaceutical composition comprising any of the aforementioned antibodies or their antigen-binding fragments in combination with one or more other pharmaceutically acceptable excipients. In one aspect, the present invention relates to a pharmaceutical composition used to treat an AXL-mediated disease or disorder, comprising any of the above-mentioned antibodies or their antigen-binding fragments in combination with one or more pharmaceutically acceptable excipients.

[0076] In one aspect, the present invention relates to a pharmaceutical composition used to treat an AXL-mediated disease or disorder, comprising a therapeutically effective amount of any of the above-mentioned antibodies or their antigen-binding fragments in combination with one or more pharmaceutically acceptable excipients.

[0077] "Pharmaceutical composition" means a composition comprising the antibody according to the present invention, and at least one component selected from the group consisting of pharmaceutically acceptable and pharmacologically compatible fillers, solvents, diluents, carriers, auxiliaries, distribution and sensing agents, and delivery agents.

[0078] The term "pharmaceutically acceptable" refers to one or more suitable liquid or solid components suitable for administration to mammals, preferably humans. The term “excipient” is used herein to describe any component other than the antibody according to the present invention. These are inorganic or organic substances used in drug production / manufacturing to impart the physicochemical characteristics required for a drug.

[0079] In some embodiments, the composition is intended to improve, prevent, or treat diseases or disorders that may be mediated by AXL. The term "AXL-mediated disease or disorder" refers to any disease or disorder that is directly or indirectly related to AXL, including its etiology, onset, progression, persistence, or pathogenesis.

[0080] "To treat," "treatment," and "therapy" refer to methods of reducing or suppressing a biological disorder and / or at least one of its accompanying symptoms. Furthermore, references to "treatment" in this specification include references to curative, symptomatic, and preventive treatments.

[0081] The term "disorder" means any condition that would benefit from treatment according to the present invention. The definition of this term includes chronic and acute disorders or diseases, including pathological conditions that make a mammal susceptible to the disorder in question.

[0082] The "therapeutic dose" refers to the amount of therapeutic agent administered during treatment that alleviates, to some extent, one or more symptoms of the disease being treated. The therapeutic dose may vary depending on factors such as the specific condition being treated, the patient's age, sex, and weight, as well as whether the monoclonal antibody or its antigen-binding fragment that specifically binds to AXL is administered as a standalone treatment or in combination with one or more additional drugs or treatments.

[0083] In one aspect, the subject or patient of the treatment is a mammal, preferably a human subject. The subject may be male or female of any age. The pharmaceutical composition and method for preparing the present invention will be undoubtedly obvious to those skilled in the art. The pharmaceutical composition should preferably be manufactured in compliance with GMP (Good Manufacturing Practice) requirements.

[0084] In some embodiments of the pharmaceutical composition, the pharmaceutical composition may include a buffering composition, an isotonic agent (osmotic pressure modifier or osmotic agent), a stabilizer, and / or a solubilizer. The pharmaceutical composition according to the present invention is a stable composition.

[0085] A pharmaceutical composition is “stable” if the activator maintains physical stability and / or chemical stability and / or biological stability over a specific storage life, for example, at a storage temperature of 2–8°C. Preferably, the activator maintains both physical and chemical stability, as well as biological stability. The storage period is adjusted based on the results of stability tests under accelerated or natural degradation conditions.

[0086] The pharmaceutical compositions according to the present invention are suitable for parenteral administration as sterile formulations intended for administration to a target body that bypasses the gastrointestinal tract and penetrates the skin or mucous membrane barrier by injection, infusion, and implantation. In particular, parenteral administration is intended to include, among other things, subcutaneous, intraperitoneal, intramuscular, intravenous, intraarterial, intrathecal, intraventricular, intraurethral, ​​intracranial, intrabursal, percutaneous injection or infusion, and renal dialysis infusion techniques. Intratumoral delivery, for example, intratumoral injection, may also be used. Local perfusion is also intended.

[0087] In some embodiments, the pharmaceutical composition is administered intravenously. In some embodiments, intravenous administration is performed using infusion, extended infusion, or long-term continuous infusion.

[0088] In some embodiments, the pharmaceutical composition is administered subcutaneously. In some embodiments, subcutaneous administration is performed using subcutaneous injection. In some embodiments, the pharmaceutical composition is in an injectable dosage form.

[0089] In some embodiments, the pharmaceutical composition is a liquid preparation for intravenous administration. In some embodiments, the injectable dosage form is an intravenous solution. In some embodiments, the injectable dosage form is a liquid preparation for subcutaneous administration.

[0090] Injectable formulations may be manufactured in unit dosage forms such as ampoules, vials, plastic containers, pre-filled syringes, and auto-injection devices, without limitation. In some embodiments, the pharmaceutical composition is provided in a dry form, i.e., a powder or granular form, which is reconstituted with a suitable solvent (e.g., sterile, pyrogen-free water) before administration. Such pharmaceutical formulations may be prepared, for example, by lyophilization, i.e., by freeze-drying, a method known in the art, which involves freezing the product and then removing the solvent from the frozen material.

[0091] In some embodiments, the pharmaceutical composition is a lyophilized product for preparing an injectable solution. In some embodiments, the pharmaceutical composition is a lyophilized product for preparing a liquid formulation for subcutaneous administration.

[0092] In some embodiments, the pharmaceutical composition is a concentrate for preparing a liquid for injection. In some embodiments, the pharmaceutical composition is a concentrate for preparing a liquid formulation for subcutaneous administration.

[0093] In one aspect, the present invention relates to a pharmaceutical composition comprising a monoclonal antibody or its antigen-binding fragment according to the present invention that specifically binds to AXL, and at least one other therapeutically active compound.

[0094] In one aspect, the present invention relates to a pharmaceutical composition for treating an AXL-mediated disease or disorder, comprising any of the above-mentioned antibodies or their antigen-binding fragments, and at least one other therapeutically active compound.

[0095] In one aspect, the present invention relates to a pharmaceutical composition comprising any of the above-mentioned antibodies or antigen-binding fragments thereof, and further comprising at least one other therapeutically active compound. In one aspect, the present invention relates to a pharmaceutical composition for treating an AXL-mediated disease or disorder, comprising any of the above-mentioned antibodies or their antigen-binding fragments, and further comprising at least one other therapeutically active compound.

[0096] In one aspect, the present invention relates to a pharmaceutical composition for treating an AXL-mediated disease or disorder, comprising any of the above-mentioned antibodies or their antigen-binding fragments, and at least one other therapeutically active compound (which is an antibody, a small molecule, a hormonal therapy agent, or a combination thereof).

[0097] In some embodiments of the pharmaceutical composition, the other therapeutically active compound is an immune checkpoint inhibitor. The term “immune checkpoint inhibitor” (or “checkpoint inhibitor”) refers to a compound that inhibits the activity of an immune checkpoint. Inhibition includes reduction and complete blockade of function. Examples of inhibited checkpoint molecules include B7-H3, B7-H4, BTLA, CTLA-4, KIR, PD-1, PD-L1, PD-L2, LAG-3, TIM-3, TIGIT, and VISTA. In some embodiments of the present invention, an immune checkpoint inhibitor is an antibody that specifically recognizes an immune checkpoint protein. A number of immune checkpoint inhibitors are known, and by analogy with these known immune checkpoint protein inhibitors, alternative immune checkpoint inhibitors may be developed in the near future. Immune checkpoint inhibitors include, but are not limited to, peptides, antibodies, nucleic acid molecules, and low molecular weight compounds.

[0098] In some embodiments of the pharmaceutical composition, the immune checkpoint inhibitor is selected from PD-1 inhibitors, PD-L1 inhibitors, or CTLA-4 inhibitors. In some embodiments of the pharmaceutical composition, a PD-1 inhibitor is an antibody that specifically binds to PD-1.

[0099] In some embodiments of the pharmaceutical composition, the antibody that specifically binds to PD-1 is selected from the group consisting of prolgolimab, pembrolizumab, and nivolumab. In some embodiments of the pharmaceutical composition, a CTLA-4 inhibitor is an antibody that specifically binds to CTLA-4.

[0100] In some embodiments of the pharmaceutical composition, the antibody that specifically binds to CTLA-4 is ipilimumab or nurulimab. In some embodiments of the pharmaceutical composition, a PD-L1 inhibitor is an antibody that specifically binds to PD-L1.

[0101] In some embodiments of the pharmaceutical composition, the antibody that specifically binds to PD-L1 is selected from the group consisting of durvalumab, avelumab, atezolizumab, and manelimab.

[0102] In some embodiments of the pharmaceutical composition, other therapeutically active compounds include EGFR-TKIs, afatinib, erlotinib, gefitinib, osimertinib, doxorubicin, paclitaxel, capecitabine, carboplatin, cisplatin, lapatinib, trastuzumab emtansine, docetaxel, cyclophosphamide, topotecan, niraparib, olaparib, pembrolizumab, platinum compounds, bevacizumab, prorugolimab, nivolumab, vemurafenib, dabrafenib, cobimetinib, trametinib, and The following are selected from the group of therapies: toposide, temozolomide, lomustine, imatinib, dasatinib, vincristine, doxorubicin derivatives, cytarabine, fludarabine, rituximab, venetoclax, PARP inhibitors, irinotecan, trastuzumab, gemcitabine, atezolizumab, fluoropyrimidine, cetuximab, ipilimumab, methotrexate, ifosfamide, doxorubicin, BRAF inhibitors, MEK inhibitors, donepezil, rivastigmine, galantamine, nintedanib, etiotropic therapy, anti-inflammatory therapy, glucocorticoids, antithrombotic therapy, or any combination thereof.

[0103] In some embodiments of the pharmaceutical composition, AXL-mediated diseases or disorders include non-small cell lung cancer, non-small cell lung cancer with EGFR mutations, breast cancer, HER2-positive breast cancer, and four-time-negative breast cancer. The following are selected from the group of cancers: triple-negative breast cancer (TNBC), ovarian cancer, platinum-resistant ovarian cancer, prostate cancer, docetaxel-resistant prostate cancer, endometrial cancer and uterine sarcoma, endometrial adenocarcinoma, serous uterine carcinoma, cutaneous melanoma, neuroblastoma, glioblastoma, squamous cell carcinoma of the head and neck, gastric cancer, renal cell carcinoma, urothelial carcinoma, colorectal cancer, colon cancer, hepatocellular carcinoma, pancreatic cancer, biliary tract cancer, malignant neoplasms of the extrahepatic bile duct, intrahepatic cholangiocarcinoma, malignant neoplasms of the gallbladder, osteosarcoma, Ewing's sarcoma, neoplasms with high levels of AXL expression, chronic myeloid leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, acute lymphoblastic leukemia, chronic liver disease, non-alcoholic steatohepatitis, Alzheimer's disease, or idiopathic pulmonary fibrosis.

[0104] Therapeutic applications of monoclonal antibodies or antigen-binding fragments that specifically bind to AXL In one aspect, antibodies or antigen-binding fragments that specifically bind to AXL are used to treat disorders mediated by AXL activity.

[0105] In one aspect, the subject or patient of the treatment is a mammal, preferably a human subject. The subject may be male or female of any age. In one aspect, the present invention relates to a method for treating an AXL-mediated disease or disorder, comprising administering to a subject in need of such treatment any of the above-mentioned antibodies or their antigen-binding fragments, or the pharmaceutical composition, in a therapeutically effective amount.

[0106] In one aspect, the present invention relates to a method for treating an AXL-mediated disease or disorder, comprising administering to a subject requiring such treatment any of the above-mentioned antibodies or their antigen-binding fragments, and at least one other therapeutically active compound, in a therapeutically effective dose.

[0107] In some aspects of the treatment method, AXL-mediated diseases or disorders include non-small cell lung cancer, non-small cell lung cancer with EGFR mutations, breast cancer, HER2-positive breast cancer, four-time negative breast cancer, triple-negative breast cancer (TNBC), ovarian cancer, platinum-resistant ovarian cancer, prostate cancer, docetaxel-resistant prostate cancer, endometrial cancer and uterine sarcoma, endometrial adenocarcinoma, serous carcinoma of the uterus, cutaneous melanoma, neuroblastoma, glioblastoma, and head and neck tumors. The following are selected from the group of conditions: squamous cell carcinoma, gastric cancer, renal cell carcinoma, urothelial carcinoma, colorectal cancer, colon cancer, hepatocellular carcinoma, pancreatic cancer, biliary tract cancer, malignant neoplasms of the extrahepatic bile duct, intrahepatic cholangiocarcinoma, malignant neoplasms of the gallbladder, osteosarcoma, Ewing's sarcoma, neoplasms with high levels of AXL expression, chronic myeloid leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, acute lymphoblastic leukemia, chronic liver disease, non-alcoholic steatohepatitis, Alzheimer's disease, or idiopathic pulmonary fibrosis.

[0108] In some embodiments of the treatment method, other therapeutically active compounds include antibodies, small molecules, hormonal agents, or combinations thereof. In some embodiments of the treatment method, other therapeutically active compounds are immune checkpoint inhibitors.

[0109] In some embodiments of the treatment method, the immune checkpoint inhibitor is selected from PD-1 inhibitors, PD-L1 inhibitors, or CTLA-4 inhibitors. In some embodiments of the treatment method, PD-1 inhibitors are antibodies that specifically bind to PD-1.

[0110] In some embodiments of the present invention, a PD-1 inhibitor is an antibody that specifically binds to PD-1. Examples of antibodies that specifically bind to PD-1 include pembrolizumab, nivolumab, prorugolimab, tripalimab, semiprimab, and cintilimab. The most preferred are prorugolimab, pembrolizumab, and nivolumab.

[0111] In some embodiments of the treatment method, the antibody that specifically binds to PD-1 is selected from a group that includes prorugolimab, pembrolizumab, and nivolumab. In some embodiments of the treatment method, a CTLA-4 inhibitor is an antibody that specifically binds to CTLA-4.

[0112] In some embodiments of the present invention, a CTLA-4 inhibitor is an antibody that specifically binds to CTLA-4. Examples of antibodies that specifically bind to CTLA4 include ipilimumab, tremelimumab, zarifremab, and nurlimab. The most preferred are ipilimumab or nurlimab.

[0113] In some embodiments of the treatment method, the antibody that specifically binds to CTLA-4 is ipilimumab or nurlimub. In some aspects of the treatment method, PD-L1 inhibitors are antibodies that specifically bind to PD-L1.

[0114] In some embodiments of the treatment method, the antibody that specifically binds to PD-L1 is selected from the group consisting of durvalumab, avelumab, atezolizumab, and manerimab. In one aspect, the present invention relates to the use of the above-mentioned antibody or its antigen-binding fragment, or the above-mentioned pharmaceutical composition, for treating AXL-mediated diseases or disorders in subjects requiring such treatment.

[0115] In one aspect, the present invention relates to the use of the above-mentioned antibody or its antigen-binding fragment, and at least one other therapeutically active compound, in subjects requiring such treatment for AXL-mediated diseases or disorders.

[0116] In some aspects of use, AXL-mediated diseases or disorders include non-small cell lung cancer, non-small cell lung cancer with EGFR mutations, breast cancer, HER2-positive breast cancer, four-time negative breast cancer, triple-negative breast cancer (TNBC), ovarian cancer, platinum-resistant ovarian cancer, prostate cancer, docetaxel-resistant prostate cancer, endometrial cancer and uterine sarcoma, endometrial adenocarcinoma, serous carcinoma of the uterus, cutaneous melanoma, neuroblastoma, glioblastoma, and head and neck cancer. The following are selected from the group of conditions: squamous cell carcinoma, gastric cancer, renal cell carcinoma, urothelial carcinoma, colorectal cancer, colon cancer, hepatocellular carcinoma, pancreatic cancer, biliary tract cancer, malignant neoplasms of the extrahepatic bile duct, intrahepatic cholangiocarcinoma, malignant neoplasms of the gallbladder, osteosarcoma, Ewing's sarcoma, neoplasms with high levels of AXL expression, chronic myeloid leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, acute lymphoblastic leukemia, chronic liver disease, non-alcoholic steatohepatitis, Alzheimer's disease, or idiopathic pulmonary fibrosis.

[0117] In some embodiments for use, other therapeutically active compounds include antibodies, small molecules, hormonal agents, or combinations thereof. In some embodiments of use, other therapeutically active compounds are immune checkpoint inhibitors.

[0118] In some embodiments of use, the immune checkpoint inhibitor is selected from PD-1 inhibitors, PD-L1 inhibitors, or CTLA-4 inhibitors. In some embodiments for use, PD-1 inhibitors are antibodies that specifically bind to PD-1.

[0119] In some embodiments for use, the antibody that specifically binds to PD-1 is selected from the group consisting of prorugolimab, pembrolizumab, and nivolumab. In some embodiments for use, a CTLA-4 inhibitor is an antibody that specifically binds to CTLA-4.

[0120] In some embodiments for use, the antibody that specifically binds to CTLA-4 is ipilimumab or nurlimub. In some embodiments for use, PD-L1 inhibitors are antibodies that specifically bind to PD-L1.

[0121] In some embodiments for use, the antibody that specifically binds to PD-L1 is selected from the group of durvalumab, avelumab, atezolizumab, and manerimab. With respect to antibodies or antigen-binding fragments that specifically bind to AXL, in conjunction with one or more other therapeutic agents, the uses or methods used herein are intended to mean, refer to, and include: 1) When the components are prescribed together in a single dosage form that releases the components to the patient in substantially the same amount of time, simultaneous administration of the combination of an antibody or antigen-binding fragment that specifically binds to AXL and the therapeutic agent to a patient requiring treatment. 2) When the components are prescribed separately to patients in separate dosage forms that are ingested by patients at substantially the same time and thereby released to the patients at substantially the same time, simultaneous administration of a combination of an antibody or antigen-binding fragment that specifically binds to AXL and a therapeutic agent to patients requiring treatment, 3) Sequential administration of a combination of an antibody or antigen-binding fragment that specifically binds to AXL and a therapeutic agent to a patient requiring treatment, in separate dosage forms in which the components are prescribed separately from each other, with a significant time interval between each administration, thereby releasing the components to the patient at substantially different times, and 4) When the components are prescribed together, sequential administration of a combination of an antibody or antigen-binding fragment that specifically binds to AXL and a therapeutic agent to a patient requiring treatment, in which each part controls the release of components that can be administered via the same or different routes, thereby being released simultaneously, sequentially, or together to the patient at the same and / or different times.

[0122] Antibodies or antigen-binding fragments that specifically bind to AXL can be administered without further therapeutic intervention, i.e., as a standalone treatment. In some aspects of the treatment or use, an antibody or its antigen-binding fragment that specifically binds to AXL may be administered in combination with an EGFR-TKI agent (afatinib, erlotinib, gefitinib, and / or osimertinib), and / or an immune checkpoint inhibitor selected from PD-1 inhibitors or PD-L1 inhibitors.

[0123] In some aspects of the treatment or use, an antibody or its antigen-binding fragment that specifically binds to AXL may be administered in combination with chemotherapy (doxorubicin, paclitaxel, capecitabine, carboplatin, and / or cisplatin), and / or an immune checkpoint inhibitor selected from PD-1 inhibitors or PD-L1 inhibitors.

[0124] In some aspects of the treatment or use, an antibody or its antigen-binding fragment that specifically binds to AXL may be administered in combination with targeted therapy (lapatinib) and / or cell proliferation inhibitory therapy (trastuzumab emtansine).

[0125] In some aspects of the treatment or use, an antibody or antigen-binding fragment that specifically binds to AXL may be administered in combination with chemotherapy (docetaxel, paclitaxel, cyclophosphamide, and / or topotecan), and / or targeted therapy (niraparib and / or olaparib).

[0126] In some aspects of the treatment or use, an antibody or antigen-binding fragment that specifically binds to AXL may be administered in combination with chemotherapy (docetaxel, paclitaxel, cyclophosphamide, and / or topotecan), as well as / or combined chemohormonal therapy, as well as / or targeted therapy (niraparib and / or olaparib), and / or pembrolizumab.

[0127] In some aspects of the treatment or use, an antibody or antigen-binding fragment that specifically binds to AXL may be administered in combination with chemotherapy (platinum agents, topotecan, and / or paclitaxel), and / or targeted therapy (bevacizumab), and / or pembrolizumab.

[0128] In some aspects of the treatment or use, an antibody or antigen-binding fragment that specifically binds to AXL may be administered in combination with an immune checkpoint inhibitor (prorugolimab, nivolumab, or pembrolizumab) and / or targeted therapy (vemurafenib / dabrafenib and / or cobimetinib / trametinib).

[0129] In some aspects of the treatment or use, an antibody or its antigen-binding fragment that specifically binds to AXL may be administered in combination with cytotoxic chemotherapy (platinum agents, etoposide, and / or temozolomide).

[0130] In some aspects of the treatment or use, antibodies or antigen-binding fragments that specifically bind to AXL may be administered in combination with chemotherapy (temozolomide, platinum agents, etoposide, and / or lomustine), and / or targeted therapy (bevacizumab and / or dabrafenib + trametinib), and / or pembrolizumab.

[0131] In some aspects of the treatment or use, an antibody or its antigen-binding fragment that specifically binds to AXL may be administered in combination with a tyrosine kinase inhibitor (imatinib and / or dasatinib).

[0132] In some aspects of the treatment or use, an antibody or its antigen-binding fragment that specifically binds to AXL may be administered in combination with chemotherapy (vincristine and / or doxorubicin derivatives).

[0133] In some aspects of the treatment or use, an antibody or its antigen-binding fragment that specifically binds to AXL may be administered in combination with chemotherapy (cytarabine and / or doxorubicin derivatives).

[0134] In some aspects of the treatment or use, antibodies or antigen-binding fragments that specifically bind to AXL may be administered in combination with chemoimmunotherapy (fludarabine, cyclophosphamide, and / or rituximab) and / or targeted therapy (venetoclax).

[0135] In some aspects of the treatment or use, an antibody or its antigen-binding fragment that specifically binds to AXL may be administered in combination with an immune checkpoint inhibitor (pembrolizumab) and / or targeted therapy (PARP inhibitor).

[0136] In some aspects of the treatment or use, an antibody or antigen-binding fragment that specifically binds to AXL may be administered in combination with chemotherapy (irinotecan and / or docetaxel), and / or an immune checkpoint inhibitor (nivolumab or pembrolizumab), and / or trastuzumab.

[0137] In some aspects of the treatment or use, an antibody or its antigen-binding fragment that specifically binds to AXL may be administered in combination with an immune checkpoint inhibitor (nivolumab and / or pembrolizumab).

[0138] In some aspects of the treatment or use, an antibody or antigen-binding fragment that specifically binds to AXL may be administered in combination with chemotherapy (cisplatin and / or gemcitabine) and / or an immune checkpoint inhibitor (atezolizumab and / or pembrolizumab).

[0139] In some aspects of the treatment or use, an antibody or antigen-binding fragment that specifically binds to AXL may be administered in combination with chemotherapy (fluoropyrimidine and / or irinotecan), and / or targeted therapy (bevacizumab, cetuximab, and / or dabrafenib + trametinib), and / or immune checkpoint inhibitors (pembrolizumab, nivolumab, and / or ipilimumab).

[0140] In some aspects of the treatment or use, an antibody or its antigen-binding fragment that specifically binds to AXL may be administered in combination with chemotherapy (cisplatin and / or gemcitabine) and / or nivolumab.

[0141] In some aspects of the treatment or use, an antibody or its antigen-binding fragment that specifically binds to AXL may be administered in combination with chemotherapy (methotrexate and / or ifosfamide).

[0142] In some aspects of the treatment or use, an antibody or its antigen-binding fragment that specifically binds to AXL may be administered in combination with chemotherapy (vincristine and / or doxorubicin).

[0143] In some aspects of the treatment or use, an antibody or its antigen-binding fragment that specifically binds to AXL may be administered in combination with chemotherapy (paclitaxel, cisplatin, and / or gemcitabine), and / or pembrolizumab.

[0144] In some aspects of the treatment or use, an antibody or antigen-binding fragment that specifically binds to AXL may be administered in combination with an immune checkpoint inhibitor (nivolumab and / or pembrolizumab), and / or trastuzumab, and / or a targeted therapy (BRAF / MEK inhibitor).

[0145] In some aspects of the treatment or use, an antibody or its antigen-binding fragment that specifically binds to AXL may be administered in combination with a central acetylcholinesterase inhibitor (donepezil, rivastigmine, and / or galantamine).

[0146] In some aspects of the treatment or use, an antibody or its antigen-binding fragment that specifically binds to AXL may be administered in combination with nintedanib. In some embodiments of the method of treatment or use, a preferred dose of the monoclonal antibody or its antigen-binding fragment that specifically binds to AXL according to the present invention is in the range of 0.1 to 200 mg / kg. [Brief explanation of the drawing]

[0147] [Figure 1] Figure 1 shows the gene map of the vector pIntA_CK_01_004, which carries the light chain sequence of the anti-AXL antibody 01_004. [Figure 2] Figure 2 shows the gene map of the vector pIntA_HC_01_004, which carries the heavy chain sequence of the anti-AXL antibody 01_004.

[0148] Figures 1 and 2

[0149] [Table 1] [Figure 3] Figure 3 is the electrophoretic map of candidate 01_004 obtained using vertical gel electrophoresis under non-denaturing, non-reducing conditions in 8% PAAG.

[0150] A buffer solution of the sample to be applied, free of 1-2-mercaptoethanol; 2 - These are molecular protein markers, and the molecular weight (kDa) corresponding to each lane is shown on the left side of the electrophoresis diagram; 3 - 01_004, 10 μg load; 4 - 01_004, 0.1 μg load. [Figure 4] Figure 4 is the electrophoretic map of candidate 01_004 obtained using vertical gel electrophoresis under denaturation reduction conditions in 12.5% ​​PAAG.

[0151] A buffer solution of the sample to be applied, containing 1-2-mercaptoethanol; 2 - These are molecular protein markers, and the molecular weight (kDa) corresponding to each lane is shown on the left side of the electrophoresis diagram; 3 - 01_004, 10 μg load; 4 - 01_004, 0.1 μg load. [Figure 5] Figure 5 is a graph illustrating the results of ELISA measuring the binding of anti-AXL antibodies to different extracellular AXL fragments.

[0152] 10G5 - Control anti-AXL antibody against AXL-Ig1 domain Control - Control without introduction of anti-AXL antibody [Figure 6] Figure 6 is a graph illustrating the inhibitory activity of the anti-AXL antibody in an assay using a reporter cell system. [Figure 7] Figure 7 is a graph illustrating the results of an ELISA measuring the inhibition of GAS6-dependent phosphorylation of AXL by an anti-AXL antibody.

[0153] Blank - Control that does not induce cell lysates, ELISA OD background level pAXL control - Phosphorylated AXL control product obtained using the DuoSet IC ELISA intracellular Human Phospho-Axl kit (R&D Systems) Unstimulated cells - cells incubated in the absence of GAS6 and antibodies. GAS6 - Cells incubated in the presence of GAS6 IgG1-negative control - Cells incubated in the presence of a control (non-anti-AXL) IgG1 antibody. 01_004 - Cells incubated in the presence of antibody 01_004 01_004+GAS6 - Cells incubated in the presence of GAS6 and antibody 01_004 IgG1+GAS6-negative control - Cells incubated in the presence of GAS6 and control (non-anti-AXL) IgG1 antibody [Figure 8] Figure 8 is a graph illustrating antibody-dependent cell-mediated cytotoxicity (ADCC) in an assay using a reporter cell system. [Figure 9] Figure 9 is a graph illustrating antibody-dependent cell-mediated cytotoxicity (ADCC) in assays using PBMCs. [Figure 10] Figure 10 is a graph illustrating antibody-dependent cell phagocytosis (ADCP) in an assay using a reporter cell line. [Figure 11] Figure 11 is a graph illustrating the dose-dependent binding of anti-AXL antibodies to human and cynomolgus monkey AXL. [Figure 12] Figure 12 is a graph illustrating tumor volume as a function of time after the start of administration of anti-AXL drugs to mice in an in vivo study of a subcutaneous xenograft model.

[0154] Note: * p<0.05, significant difference compared to the control (Mann-Whitney test). [Figure 13] Figure 13 is a graph illustrating the changes in the concentration of candidate anti-AXL products in the serum of monkeys after repeated intravenous administration. [Modes for carrying out the invention]

[0155] [Examples]

[0156] The following embodiments are provided for a better understanding of the present invention. These embodiments are for illustrative purposes only and should not be construed as limiting the scope of the invention in any way.

[0157] Materials and general methods General information on the nucleotide sequences of human immunoglobulin light and heavy chains is presented in Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991). The amino acids of antibody chains are numbered according to the EU numbering scheme (Edelman, GM et al., Proc. Natl. Acad. Sci. USA, Vol. 63 (1969), pp. 78-85; Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991)).

[0158] Recombinant DNA technology DNA was manipulated using standard methods as described in Sambrook, J. et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. Molecular biological reagents were used according to the manufacturer's protocol.

[0159] gene synthesis The desired gene segment was prepared from oligonucleotides synthesized by chemical synthesis. Gene segments of 300–1400 bp in length with a single restriction site flanking were assembled by oligonucleotide annealing and ligation, including PCR amplification, and subsequently cloned through the restriction site. The DNA sequences of the subcloned gene fragments were confirmed by DNA sequencing.

[0160] DNA sequencing The DNA sequence was determined by Sanger sequencing. Analysis of DNA and protein sequences, and management of sequence data. Unipro's UGENE suite version 1.29 and SnapGene version 6.1 were used for sequencing, mapping, analysis, annotation, and visualization.

[0161] Expression vector The antibody expression described in this application involved applying a variant of an expression plasmid intended for antibody expression in prokaryotic cells (Escherichia coli), which was transiently expressed in eukaryotic cells (e.g., CHO cells). In addition to the antibody expression cassette, the vector contained a replication origin that enabled replication of the plasmid in E. coli, and genes that confer resistance to various antibiotics (e.g., ampicillin, kanamycin) to E. coli.

[0162] Fusion genes containing the described antibody chains, as described below, were generated by PCR and / or gene synthesis and assembled by known recombination methods and techniques, for example, by linking them according to nucleic acid segments using unique restriction sites in the corresponding vectors. The subcloned nucleic acid sequences were validated by DNA sequencing. For transient transfection, large quantities of plasmids were prepared from plasmid preparations from transformed E. coli cultures.

[0163] Example 1. Design and fabrication of a gene construct for the synthesis of a full-length anti-AXL monospecific antibody. Monospecific anti-AXL antibodies were produced in full-length bivalent IgG1 format. Two C-terminal amino acid GKs were removed from the Fc portion of the heavy chain of the anti-AXL antibody. Removal of two C-terminal amino acid GKs from the heavy chain Fc portion of the sequence results in increased productivity and homogeneity of the antibody product produced by CHO cells, and an improvement in its acid-base profile (ABP), provided that such deletions do not affect the pharmacokinetics, activity, and stability of the antibody.

[0164] Antibodies 01_004 and 01_004_2 have the same variable domains in the light and heavy chains. Antibody 01_004_2 includes additional amino acid substitutions S239D and I332E (according to EU numbering) in the Fc portion of the heavy chain.

[0165] The gene constructs were prepared as follows: PCR products containing sequences of the variable domains of the heavy and light chains of the antibody were generated using PCR primers containing additional sequences complementary to the corresponding sites in the corresponding pIntA expression vectors. The following assembly procedure was performed using a ligation-independent method utilizing the 3'-5' exonuclease activity of Pfu Ultra II DNA polymerase. The nucleotide sequence of the variable domain (VL) of the light chain was inserted into the vector pIntA_CK using a SapI restriction site. Vector pIntA_CK_01_004 (Figure 1) contains the nucleotide sequence encoding the constant portion of the light kappa chain of the IgG1 antibody. The nucleotide sequence of the variable domain (VH) of the heavy chain was inserted into the vector pIntA_HC using a SapI restriction site. Vector pIntA_HC_01_004 (Figure 2) contains the nucleotide sequence encoding the constant portion of the heavy chain of the IgG1 antibody, including a deletion of the C-terminal amino acid GK. In the case of antibody 01-004_2, further nucleotide substitutions are introduced into the nucleotide sequence of the constant portion of the heavy chain to provide amino acid substitutions S239D and I332E.

[0166] The nucleotide sequence of the obtained plasmid construct was confirmed by DNA sequencing. The obtained plasmid was generated in the desired quantity in E. coli cells and purified using a plasmid DNA isolation kit. Subsequently, the plasmid was used to transiently produce the protein in the CHO-K1-S cell line.

[0167] Example 2. Production, isolation, and purification of monoclonal antibodies CHO-K1-S cell lines were used to generate antibodies in a transient expression system. For antibody generation, CHO cell culture medium supplemented with 4 mM glutamine, 10 μg / ml gentamicin, 10 μg / ml ciprofloxacin, and 100 μM deoxy-2-fluoro-L-fucose was used. The cells were cultured in baffled flasks in an orbital shaker incubator at +37°C in the presence of 5% CO2, with constant agitation.

[0168] In transient cases, the response is 1.8–2 × 10⁻⁶. 6 Cell cultures at a concentration of cells / ml were transfected with linear polyethyleneimine. The DNA / PEI ratio was 1:7. On day 7 of culture, the cell suspension was clarified by centrifugation and then filtered through a 0.22 μm filter.

[0169] Seven days after transfection, a cell saturation sample was taken, and the concentration of the generated protein was measured by biolayer interferometry using an OctetRed 96 (ForteBio) instrument with a Protein-A Biosense chip, according to the manufacturer's instructions (Table 1).

[0170] [Table 2]

[0171] While transiently generated, antibodies 01_004 and 01_004_2 demonstrated productivity typical of monoclonal antibodies. Antibody purification was performed by affinity chromatography using a Sepharose-based adsorbent containing immobilized protein A. If necessary, additional chromatographic purification of the product was performed using a Ceramic Hydroxyapatite Type I adsorbent. The quality of the obtained antibodies was analyzed by electrophoresis on denatured (in the presence of sodium dodecyl sulfate) polyacrylamide gels under non-reducing conditions (without additional mercaptoethanol) and reducing conditions (with additional mercaptoethanol) (Figures 3-4).

[0172] Example 3. Production of gene constructs for antigen synthesis, antigen generation, isolation, and purification. To obtain various fragments of the AXL receptor, numerous gene constructs encoding human or cynomolgus monkey AXL domains were created. Expression plasmids that generate antigen proteins in CHO cells contain nucleotide sequences encoding the corresponding AXL receptor fragments fused to a His6 tag or Ig1 antibody Fc moiety tag, based on pEE or pIntA vectors, for convenience in subsequent purification of the generated proteins. Gene constructs were produced using conventional genetic engineering techniques.

[0173] Human AXL amino acid sequence data was obtained from the NCBI database (https: / / www.ncbi.nlm.nih.gov / protein / AAA61243.1?report=genbank&log$=protalign&blast_rank=1&RID=SV8VAB28013). The human AXL extracellular segment sequence (417AA) is represented by Sequence ID No. 23.

[0174] The following antigens were obtained. ExcAXL - A human AXL receptor fragment containing all AXL extracellular domains fused to the His tag (SEQ ID NO: 24).

[0175] AXL-Ig1 - A human AXL receptor fragment containing the extracellular domain Ig-like C2 type 1 sequence of AXL fused to IgG1 Fc (SEQ ID NO: 25) using a linker. AXL-Ig2 - A human AXL receptor fragment containing the extracellular domain Ig-like C2 type 2 sequence of AXL fused to IgG1 Fc (SEQ ID NO: 26) using a linker.

[0176] AXL-Ig1-Ig2 - A human AXL receptor fragment containing sequences of the extracellular domain Ig-like C2 type 1 and 2 of AXL fused to IgG1 Fc (SEQ ID NO: 27) using a linker.

[0177] The gene encoding the cynomolgus monkey AXL (Macaca fascicularis) was amplified from the cDNA product derived from cynomolgus monkey smooth muscle RNA. The gene sequence was then determined by sequencing. Using the resulting extracellular sequence of the cynomolgus monkey AXL, a corresponding expression plasmid construct was created.

[0178] A cynomolgus monkey AXL receptor fragment containing the sequences of all extracellular domains of the cynomolgus monkey AXL receptor fused to the His6 tag and FLAG tag (SEQ ID NO: 28) using the cynoAXL-linker.

[0179] The nucleotide sequences of the obtained plasmid constructs were verified using DNA sequencing. The obtained plasmids were generated in the desired quantity in E. coli cells and purified using a plasmid DNA isolation kit. Subsequently, the plasmids were used to transiently produce the protein in the CHO-K1-S cell line.

[0180] CHO-K1 cell line was used to generate antigens in a transient expression system. The cells were cultured in a baffled flask in an orbital shaker incubator at +37°C in the presence of 5% CO2, with constant stirring.

[0181] In transient cases, the response is 1.8–2 × 10⁻⁶. 6 Cell cultures at a concentration of cells / ml were transfected with linear polyethyleneimine. The DNA / PEI ratio was 1:7. On day 7 of culture, the cell suspension was clarified by centrifugation and then filtered through a 0.22 μm filter.

[0182] Antibodies were similarly purified from antigens containing Fc tags using a protein A affinity adsorbent (see Example 2). Antigens containing Fc tags were purified using affinity chromatography of an adsorbent with immobilized protein A. Antigens containing HIS6 tags were purified using metal chelate chromatography of a Ni-charged adsorbent. The quality of the obtained antigens was analyzed by electrophoresis on denatured 4-15% gradient polyacrylamide gels under non-reducing or reducing conditions. The appropriate quality of the obtained antigens was confirmed by electrophoresis on denatured 4-15% gradient polyacrylamide gels under non-reducing conditions without additional mercaptoethanol and under reducing conditions with additional mercaptoethanol.

[0183] Example 4. Test to evaluate the binding of anti-AXL antibody to human AXL domain using ELISA. Antigens (ExcAXL, AXL-Ig1, AXL-Ig2, AXL-Ig1-Ig2) were immobilized in the wells of a high-absorption 96-well plate. To do this, 100 μl of antigen solution was introduced into each well of the plate at a concentration of 1 μg / ml in PBS, and the plate was incubated at room temperature for 16 hours.

[0184] Next, the liquid was removed from the wells, and the wells were washed with 300 μl of washing buffer (1×PBS pH 7.3-7.5, 0.05% Tween-20), and 300 μl / well of barrier buffer (1×PBS with 1% BSA) was added. The plate was incubated at room temperature for 1 hour. The liquid was removed again, and the plate was washed. Then, 100 μl of test antibody was introduced into the wells of the plate at a concentration of 1 μg / ml in dilution buffer (1×PBS with 0.1% BSA, 0.05% Tween-20). Next, the plate with the antibody was incubated in a thermal shaker at 37°C and 600 rpm for 1 hour. After incubation, the liquid was removed, the plate was washed, and 100 μl of anti-human Fab-HRP antibody in dilution buffer was added. After incubation at 37°C and 600 rpm using a thermal shaker, the plate was washed for 1 hour, and 100 μl of the reaction substrate (0.083 mg / ml TMB, 0.02% H2O2) in acetate buffer (0.05 M, pH 5.5) was added. The plate was incubated at room temperature for 10–15 minutes. The reaction was stopped by adding 50 μl of 10% H2SO4 to each well of the plate. Optical density values ​​were measured at a wavelength of 450 using a plate reader with a 540 nm wavelength correction.

[0185] The ELISA results (Figure 5) show that the reproduced anti-AXL antibody 10G5 binds to AXL fragments containing different Ig-like C2 type 1 domains (AXL-Ig1) or two Ig-like C2 type 1 and Ig-like C2 type 1 domains (AXL-Ig1-Ig2), and binds to the complete extracellular portion of AXL (ExcAXL), but does not bind to AXL fragments containing different Ig-like C2 type 2 domains (AXL-Ig2). Anti-AXL antibodies 01_004 and 01_004_2 bind to AXL fragments containing different Ig-like C2 type 2 domains (AXL-Ig2) or two Ig-like C2 type 1 and Ig-like C2 type 1 domains (AXL-Ig1-Ig2), and bind to the complete extracellular portion of AXL (ExcAXL), but do not bind to AXL fragments containing different Ig-like C2 type 1 domains (AXL-Ig).

[0186] Example 5. Test to determine the blocking activity of anti-AXL antibody using a reporter cell system. The study used a reporter cell line derived from HEK293 cells that stably surface-express AXL and contain a gene encoding firefly luciferase under the control of the STAT3-STAT5-AP1 promoter.

[0187] The assay was performed in a 96-well culture plate designed for luminescence assays. The suspension contained the test antibody at the concentrations shown in the graph, with or without Gas6, in each well of a 30,000-cell reporter cell line. The final volume of the cell suspension in each well was 100 μl, and all components of the suspension were prepared in DMEM medium without fetal bovine serum. After adding all components, the plate was incubated at 37°C and 5% CO2 for 16 hours, and then the luminescence intensity of the wells was measured using a luminescence assay kit. Measurements were performed using a plate reader.

[0188] The test results (Figure 6) show that the anti-AXL antibody 01_004 induces suppression of GAS6-dependent activation of intracellular AXL signaling. Example 6. Test to evaluate the inhibition of GAS6-dependent phosphorylation of AXL AXL phosphorylation was evaluated using ELISA. 3 mL of H1299 cell suspension was placed in complete growth medium (DMEM / F12, 2 mM L-glutamine, 10% FBS HI) at a rate of 1 × 10⁶ 4 cells / cm 2The inoculated dose was introduced into a 6-well flat-bottom plate, and the plate was incubated at 37°C and 5% CO2 for 24 hours. After incubation, the growth medium was removed from the wells, and 2 ml / well of serum-free medium (DMEM / F12, 2 mM L-glutamine) was introduced, and the wells were incubated overnight at 37°C and 5% CO2. After incubation, the serum-free medium was replaced with fresh medium at 2 ml / well, and 500 ml / well of 01_004 or control IgG1 antibody at a concentration of 500 μg / ml was added in a double dose (equal volumes of serum-free medium were added to the wells without antibody), and the wells were incubated at 37°C and 5% CO2 for 1 hour. After incubation, 500 μl of GAS6 was added to one of the replicates of each sample at a concentration of 3 μg / ml, and 500 μl of serum-free medium was added to the other. The samples were incubated at 37°C and 5% CO2 for 30 minutes. After incubation, the medium was removed from the plate wells, the cell monolayer was washed twice with 2 ml / well of ice-cold PBS, 300 μl / well of lysis buffer containing phosphatase and protease inhibitors was introduced, and the plate was incubated on ice for 45 minutes. The total protein concentration in the samples was determined by BCA assay according to the manufacturer's instructions. The cell lysates were stored at -80°C.

[0189] Next, ELISA was performed using the commercially available kit DuoSet IC ELISA Intracellular Human Phospho-Axl (R&D Systems) according to the manufacturer's instructions. Before introducing the cells into the wells of the experimental plate, the cell lysates were diluted to a concentration of 300 μg / ml.

[0190] The test results (Figure 7) show that the anti-AXL antibody 01_004 induces the suppression of GAS6-dependent phosphorylation of AXL. Example 7. Antibody-dependent cell-mediated cytotoxicity (ADCC) assay using a reporter cell system. The assay used the Jarcutt-NFAT-Luc-CD16 reporter cell line, which is based on the Jarcutt cell line that stably expresses CD16 on its surface and contains a gene encoding firefly luciferase under the control of the NFAT promoter, and NCI-H1299, an AXL expression line, as the target cell. Jarcutt-NFAT-Luc-CD16 and NCI-H1299 cells were cultured in RPMI-1640 nutrient medium supplemented with 10% fetal bovine serum at 37°C with 5% CO2.

[0191] The assay was performed in a 96-well culture plate designed for luminescence assays. Each well containing the suspension contained 25,000 Jarcut-NFAT-Luc-CD16 effector cells, 25,000 targeted NCI-H1299 cells expressing AXL, and the test antibody at the concentrations shown in the graph. The final volume of cell suspension and antibody in each well was 100 μl, and all components of the suspension were prepared in PRMI-1640 medium containing 10% FBS. After adding all components, the plate was incubated at 37°C and 5% CO2 for 16 hours, and then the luminescence intensity of the wells was measured using a luminescence assay kit. Measurements were performed using a plate reader.

[0192] The test results (Figure 8) show that anti-AXL antibodies 01_004 and 01_004_2 possess ADCC activity. Example 8. Antibody-dependent cell-mediated cytotoxicity (ADCC) assay using PBMCs. PBMCs were isolated from whole blood of healthy donors by Ficol density gradient centrifugation. A549 cells were cultured at 37°C in 5% CO2 in DMEM nutrient medium supplemented with 10% fetal bovine serum.

[0193] The assay was performed on a 96-well culture plate. The suspension contained 30,000 A549 target cells expressing AXL, 300,000 newly isolated PBMCs, and the specified concentration of test antibody in each well. The final volume of the cell suspension in each well was 150 ml, and all components were prepared in RPMI-1640 medium containing 10% fetal bovine serum. The plate was incubated at 37°C for 16 hours in 5% CO2. At the end of incubation, a 10× lysis solution (CytoTox96® Non-Radio Cytotoxicity Assay, Promega) was added at a 1× concentration to a control well containing only target cells, and the plate was incubated in a CO2 incubator at 37°C for 30 minutes. The supernatant from all wells was transferred to a V-plate, centrifuged at 300 g, and 50 μl of supernatant was taken from each well and placed into a new plate. Assay buffer and substrate mix (CytoTox96® Non-Radio Cytotoxicity Assay Kit) were mixed, and 15 μl was added to each well containing the supernatant. The plate was incubated in the dark at room temperature for up to 30 minutes. 15 ml of stop solution (CytoTox96® Non-Radioactive Cytotoxicity Assay Kit, Promega) was added to each well. Optical density was measured at a wavelength of 490 nm using a plate reader.

[0194] The assay results (Figure 9) show that anti-AXL antibodies 01_004 and 01_004_2 possess ADCC activity. Example 9. Assay for determining antibody-dependent cell phagocytosis (ADCP) using a reporter cell system. The assay used the Jarcutt-NFAT-Luc-CD64 reporter cell line, which was created based on the Jarcutt cell line that stably expresses CD64 on its surface and contains a gene encoding firefly luciferase under the control of the NFAT promoter, and the A549 cell line as the target cell line. Jarcutt-NFAT-Luc-CD64 and A549 cells were cultured at 37°C in 5% CO2 in appropriate growth medium supplemented with 10% fetal bovine serum. Jarcutt-NFAT-Luc-CD64 was cultured in RPMI-1640 medium, and A549 cells were cultured in DMEM medium.

[0195] The assay was performed in a 96-well culture plate designed for luminescence assays. Each well of the suspension contained 30,000 Jarcut-NFAT-Luc-CD64 reporter cell lines, 30,000 A549 target cells, and the specified concentration of test antibody. The final volume of the suspension per well was 100 μl, and all suspension components were prepared in RPMI-1640 medium containing 10% fetal bovine serum. After adding all components, the suspension was incubated at 37°C and 5% CO2 for 16 hours, and then the luciferase intensity of the wells was measured using a commercially available luminescence assay kit. Measurements were performed using a plate reader.

[0196] The assay results (Figure 10) show that anti-AXL antibodies 01_004 and 01_004_2 induce ADCP-mediated CD64-dependent NFAT signaling activity. Example 10. Anti-AXL antibody antigen binding assay using enzyme-linked immunosorbent assay (ELISA) Antigens (human AXl and cynoAXL) were immobilized in the wells of a high-absorption 96-well plate. To do this, 100 μl of antigen solution was introduced into each well of the plate at a concentration of 1 μg / ml in carbonate buffer (0.1 M NaHCO3, pH 9.5), and the plate was incubated at room temperature for 16 hours.

[0197] Next, the liquid was removed from the wells, and the wells were washed with 300 μl of washing buffer (1×PBS pH 7.3-7.5, 0.05% Tween-20), and 300 μl / well of barrier buffer (1×PBS with 1% BSA) was added. The plate was incubated at room temperature for 1 hour. The liquid was removed again, and the plate was washed. Next, 100 μl of antibody 01_004_2 was added to the wells of the plate at the concentrations shown in the graph in dilution buffer (1×PBS with 0.1% BSA, 0.05% Tween-20). Next, the plate with the antibody was incubated in a thermal shaker at 37°C and 600 rpm for 1 hour. After incubation, the liquid was removed, the plate was washed, and 100 μl of anti-human Fab-HRP antibody in dilution buffer was added. After incubation at 37°C and 600 rpm using a thermal shaker, the plate was washed for 1 hour, and 100 μl of the reaction substrate (0.083 mg / ml TMB, 0.02% H2O2) in acetate buffer (0.05 M, pH 5.5) was added. The plate was incubated at room temperature for 10–15 minutes. The reaction was stopped by adding 50 μl of 10% H2SO4 to each well of the plate. Optical density values ​​were measured at a wavelength of 450 using a plate reader with a 540 nm wavelength correction.

[0198] The assay results (Figure 11) show that the anti-AXL antibody 01_004_2 binds to human AXL and cynomolgus monkey AXL. Example 11. Determination of aggregation stability of anti-AXL antibody after heat stress. The aggregation stability of anti-AXL antibodies was tested using heat stress (50°C, 48 hours). The proportion of monomers in the sample before and after heat stress was determined by size exclusion HPLC.

[0199] The test samples were thermostated at 50°C for 72 hours. After heating, the intact, stressed samples were subjected to analysis by size exclusion HPLC (SEC HPLC) and capillary isoelectric focusing with a UV detector. Chromatography was performed on an Agilent 1100 HPLC system with a Tosoh TSK-Gel G3000SWXL column, with detection at a wavelength of 280 nm.

[0200] [Table 3]

[0201] Conclusion: The antibody against AXL according to the present invention has high aggregation stability. Example 12. Determination of the physicochemical stability of anti-AXL antibodies in human serum. A comparative study was conducted on the stability of anti-AXL antibodies 01_004 and 01_004_2 in human and mouse serum. Pooled serum was obtained from seven healthy donors and seven healthy uninbred mice and mixed in equal volume proportions. The test antibodies were added to the serum with 0.1% sodium meothiolate to a concentration of 25 μg / ml and incubated at 37°C for 14 days. Next, the concentration of the anti-AXL antibodies was determined using enzyme immunoassay in human and mouse serum before and after the 14-day incubation at 37°C, and the stability % was calculated. The results are shown in Table 3.

[0202] [Table 4]

[0203] The detectable concentration of anti-AXL antibody after storage in human serum at a concentration of 25 μg / ml for 14 days at +37°C corresponds to high stability. Normal stability of monoclonal antibodies in human serum is considered to be 60%.

[0204] Example 13. Determination of the binding constant of antibody 01_004 against various antigens. The binding constants of antibody 01_004 against various antigens were determined by SPR (Surface Plasmon Resonance) using a Biacore 8K (Cytiva). The test antigen was immobilized on a Sensor Chip CM5 (Cytiva), and subsequently, the binding to anti-AXL antibody 01_004 was analyzed in PBS with 0.02% Tween-20, pH 7.4. The measurement data was processed using Biacore Insight Evaluation (Cytiva) software.

[0205] The average value of the equilibrium binding constant is shown in Table 4.

[0206]

Table 5

[0207] Conclusion: The antibody against AXL according to the present invention specifically binds to human AXL antigen and similarly to monkey AXL antigen, but does not bind to other closely related antigens tested. Example 14. Determination of the antitumor activity of an anti-AXL product using a subcutaneous xenograft model The antitumor activity of the product was measured using a subcutaneous tumor xenograft model. For this purpose, female BALB / c nude mice were subcutaneously implanted with 5×10 6 tumor A549 strain cells mixed with Matrigel (registered trademark) in a 1:1 ratio. After the average tumor volume reached 95.6±1 mm 3 the mice were divided into three groups of 9 each so that the average tumor volumes between the groups were comparable.

[0208] The 01-004 product and the 01-004_2 product were intraperitoneally administered at a dose of 20 mg / kg twice a week for 4 weeks. The buffer solution of the anti-AXL product was injected into the negative control group in the same manner.

[0209] During the experiment, the linear dimensions of the tumors were determined. The tumor volume was calculated using the formula V = L×W×H×π / 6, where L and W are the major and minor diameters of the tumor, respectively, and H is the height of the tumor.

[0210] The effectiveness of the test product was measured by the tumor growth inhibition (TGI) index calculated according to the following formula: TGI(%)=(V c -V p ) / V c ×100 Wherein, V c and V p are the median tumor volumes of the animals in the negative control group and the group receiving the test product, respectively. The higher the index value, the more significant the antitumor effect.

[0211] On the 32nd day of the experiment, the animals were euthanized, the tumors were excised, and weighed using an analytical balance. Figure 12 shows the dynamics of tumor volume. At the start of product administration, there was no significant difference in tumor volume between the groups. In the group of animals receiving the 01_004_2 product, the tumor volume was significantly smaller than that of the control group on the 25th day of the experiment. In the case of repeated administration of the 01_004 product, the tumor volume was significantly smaller than that of the control group from the 21st day to the 32nd day of the experiment.

[0212] The TGI indices on the 32nd day of the experiment were as follows: 35% in the 01_004_2 product group and 43% in the 01_004 product group. Table 5 shows the median tumor mass at the end of the experiment.

[0213]

Table 6

[0214] This parameter tended to decrease in the experimental group when compared with the control. Example 15. Determination of the toxicity and local irritation effects of an anti-AXL product in cynomolgus monkeys (Macaca fascicularis) following 4 weeks of repeated intravenous administration The toxicity and local irritation effects were measured in cynomolgus monkeys. The 01_004_2 product and the 01_004 product were intravenously administered at a dose of 20 mg / kg once a week for 4 weeks.

[0215] During the experiment, the inventors monitored body weight, motor activity, vegetative reflexes, hair condition, eye changes (palpebral fissure / conjunctiva / sclera condition), oral and nasal mucosa condition, stool parameters, appetite, respiratory parameters, and excretory system function. They also analyzed the cellular composition of peripheral blood, evaluated the coagulation system, protein, carbohydrate, and enzyme liver function (serum biochemical parameters: indicator enzyme activity, total bilirubin, cholesterol, total protein, triglycerides, glucose), and the urinary system condition (urine, creatinine, sodium, and potassium ion levels, as well as urine analysis results) based on the results of biochemical analysis of animal serum, and performed pathological and morphological examinations of the target organs. Local irritant effects were evaluated by visual inspection and histological study of the product administration site and draining lymph nodes.

[0216] The experimental results showed that the administration of products 01_004_2 and 01_004 was well tolerated by the animals. Throughout the observation period, the inventors observed no changes in autonomic reflexes, hair condition, eyes (sclera, conjunctiva, lacrimation, secretions), oral and nasal mucosa, stool, respiration, appetite parameters, or animal behavior.

[0217] Products 01_004_2 and 01_004 did not affect body weight, coagulation system, hematological parameters, protein, carbohydrate, and enzyme liver function, or the urinary system in cynomolgus monkeys.

[0218] Pathological examinations showed no abnormalities in the structure of the organs and tissues of the experimental animals in relation to the administration of the test product. After repeated intravenous administration at a dose of 20 mg / kg over four weeks, neither product 01_004_2 nor product 01_004 exhibited any local irritant effect.

[0219] The results indicate that, after repeated intravenous administration over four weeks, products 01_004_2 and 01_004 at doses of 20 mg / kg did not affect the major organs and organ systems of cynomolgus monkeys (Macaca fasciculis).

[0220] Example 16. Determination of the pharmacokinetics of the anti-AXL product after repeated intravenous administration to cynomolgus monkeys (Macaca fascicularis). This study was conducted as part of an analysis of the toxicity of anti-AXL products in primates (see Example 15).

[0221] To assess pharmacokinetic parameters, blood samples were collected from all animals immediately before the first administration of product 01_004_2 or 01_004 (background), then at 0, 2, 4, 8, 24, 48, 72, 120, and 168 hours after the first administration of product 01_004_2 or 01_004, immediately before the fourth administration of the product (504 hours), and at 504, 506, 508, 512, 528, 552, 576, 624, and 672 hours after the fourth administration.

[0222] The concentration of antibodies in serum was measured by solid-phase enzyme immunosorbent assay (ELISA) using horseradish peroxidase as the indicator enzyme. To determine the concentration of anti-AXL antibody in the sample, a calibration curve was constructed using a solution corresponding to anti-AXL antibody at known concentrations.

[0223] Figure 13 shows the mean curves of changes in the concentrations of antibodies 01_004_2 and 01_004 in monkey serum over time. The results showed that the mean PK parameters after repeated intravenous administration of the anti-AXL antibody product at a dose of 20 mg / kg were typical for the therapeutic monoclonal antibody. The obtained data also indicate the presence of test antibody accumulation.

Claims

1. A monoclonal antibody or its antigen-binding fragment that specifically binds to AXL, (a) CDR1 having the amino acid sequence of SEQ ID NO: 1, CDR2 having the amino acid sequence of SEQ ID NO: 2, and CDR3 having the amino acid sequence of SEQ ID NO: 3 Light chain variable domains including; and (b) CDR1 having the amino acid sequence of SEQ ID NO: 7, CDR2 having the amino acid sequence of SEQ ID NO: 8, and CDR3 having the amino acid sequence of SEQ ID NO: 9 Heavy chain variable domains A monoclonal antibody or its antigen-binding fragment, including the above.

2. The monoclonal antibody or its antigen-binding fragment according to claim 1, wherein the light chain variable domain comprises the amino acid sequence of SEQ ID NO:

13.

3. The monoclonal antibody or antigen-binding fragment according to claim 1, wherein the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:

14.

4. (a) The light chain variable domain comprises the amino acid sequence of SEQ ID NO: 13, (b) The heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 14, A monoclonal antibody or its antigen-binding fragment according to claim 1.

5. The monoclonal antibody according to any one of claims 1 to 4, wherein the antibody that specifically binds to AXL is a full-length IgG antibody.

6. The monoclonal antibody according to claim 5, wherein the full-length IgG antibody is a human IgG1, IgG2, IgG3, or IgG4 isotype.

7. The monoclonal antibody according to claim 6, wherein the antibody comprises mutations S239D and I332E according to the EU numbering scheme for the amino acids of the antibody.

8. The monoclonal antibody according to claim 6, wherein the antibody contains deletions 446G and 447K in the CH3 region according to the EU numbering scheme for the amino acids of the antibody.

9. The monoclonal antibody according to claim 1, comprising a light chain containing the amino acid sequence of SEQ ID NO:

15.

10. The monoclonal antibody according to claim 1, comprising a heavy chain containing an amino acid sequence selected from the group of SEQ ID NO: 16 or SEQ ID NO:

17.

11. (i) (a) A light chain containing the amino acid sequence of Sequence ID No. 15, and (b) A heavy chain containing the amino acid sequence of SEQ ID NO: 16; or (ii) (a) A light chain containing the amino acid sequence of Sequence ID No. 15, and (b) Heavy chain containing the amino acid sequence of SEQ ID NO: 17 A monoclonal antibody according to claim 1, comprising:

12. A nucleic acid encoding an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 11.

13. The nucleic acid according to claim 12, wherein the nucleic acid is DNA.

14. An expression vector comprising the nucleic acid according to claim 12 or 13.

15. A method for producing host cells that produce an antibody or antigen-binding fragment according to any one of claims 1 to 11, comprising the step of cell transformation with the vector according to claim 14.

16. A host cell for producing an antibody or antigen-binding fragment according to any one of claims 1 to 11, comprising the nucleic acid according to claim 12 or 13.

17. A method for producing an antibody or antigen-binding fragment according to any one of claims 1 to 11, comprising the steps of culturing a host cell according to claim 16 in a culture medium under conditions sufficient to produce the antibody or antigen-binding fragment, and subsequently isolating and purifying the obtained antibody or antigen-binding fragment.

18. A pharmaceutical composition for treating AXL-mediated diseases or disorders, comprising a therapeutically effective amount of an antibody or antigen-binding fragment according to any one of claims 1 to 11, in combination with one or more pharmaceutically acceptable excipients.

19. The aforementioned AXL-mediated disease or disorder includes non-small cell lung cancer, non-small cell lung cancer with EGFR mutations, breast cancer, HER2-positive breast cancer, four-time negative breast cancer, triple-negative breast cancer (TNBC), ovarian cancer, platinum-resistant ovarian cancer, prostate cancer, docetaxel-resistant prostate cancer, endometrial cancer and uterine sarcoma, endometrial adenocarcinoma, serous uterine carcinoma, cutaneous melanoma, neuroblastoma, glioblastoma, squamous cell carcinoma of the head and neck, gastric cancer, renal cell carcinoma, and urinary tract cancer. The pharmaceutical composition according to claim 18, selected from the group consisting of urothelial carcinoma, colorectal cancer, colon cancer, hepatocellular carcinoma, pancreatic cancer, biliary tract cancer, malignant neoplasm of the extrahepatic bile duct, intrahepatic cholangiocarcinoma, malignant neoplasm of the gallbladder, osteosarcoma, Ewing's sarcoma, neoplasm having a high level of AXL expression, chronic myeloid leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, acute lymphoblastic leukemia, chronic liver disease, non-alcoholic steatohepatitis, Alzheimer's disease, or idiopathic pulmonary fibrosis.

20. A pharmaceutical composition for treating an AXL-mediated disease or disorder, comprising an antibody or antigen-binding fragment according to any one of claims 1 to 11 and at least one other therapeutically active compound.

21. The aforementioned AXL-mediated disease or disorder includes non-small cell lung cancer, non-small cell lung cancer with EGFR mutations, breast cancer, HER2-positive breast cancer, four-time negative breast cancer, triple-negative breast cancer (TNBC), ovarian cancer, platinum-resistant ovarian cancer, prostate cancer, docetaxel-resistant prostate cancer, endometrial cancer and uterine sarcoma, endometrial adenocarcinoma, serous uterine carcinoma, cutaneous melanoma, neuroblastoma, glioblastoma, squamous cell carcinoma of the head and neck, gastric cancer, renal cell carcinoma, and urinary tract cancer. The pharmaceutical composition according to claim 20, selected from the group consisting of urothelial carcinoma, colorectal cancer, colon cancer, hepatocellular carcinoma, pancreatic cancer, biliary tract cancer, malignant neoplasms of the extrahepatic bile duct, intrahepatic cholangiocarcinoma, malignant neoplasms of the gallbladder, osteosarcoma, Ewing's sarcoma, neoplasms with high levels of AXL expression, chronic myeloid leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, acute lymphoblastic leukemia, chronic liver disease, non-alcoholic steatohepatitis, Alzheimer's disease, or idiopathic pulmonary fibrosis.

22. The pharmaceutical composition according to claim 20 or 21, wherein the other therapeutically active compound is an antibody, a small molecule, a hormone therapy agent, or a combination thereof.

23. A method for treating an AXL-mediated disease or disorder, comprising the step of administering to a subject in need of such treatment an effective amount of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, or a pharmaceutical composition according to any one of claims 18 to 22.

24. A method for treating an AXL-mediated disease or disorder according to claim 23, comprising the step of administering to a subject in need of such treatment an effective amount of an antibody or antigen-binding fragment according to any one of claims 1 to 11 and at least one other therapeutically active compound.

25. The aforementioned AXL-mediated disease or disorder includes non-small cell lung cancer, non-small cell lung cancer with EGFR mutation, breast cancer, HER2-positive breast cancer, four-time negative breast cancer, triple-negative breast cancer (TNBC), ovarian cancer, platinum-resistant ovarian cancer, prostate cancer, docetaxel-resistant prostate cancer, endometrial cancer and uterine sarcoma, endometrial adenocarcinoma, serous uterine carcinoma, cutaneous melanoma, neuroblastoma, glioblastoma, squamous cell carcinoma of the head and neck, gastric cancer, renal cell carcinoma, urothelial carcinoma, and A method for treating a disease or disorder according to claim 23 or 24, selected from the group consisting of colorectal cancer, colon cancer, hepatocellular carcinoma, pancreatic cancer, biliary tract cancer, malignant neoplasms of the extrahepatic bile duct, intrahepatic cholangiocarcinoma, malignant neoplasms of the gallbladder, osteosarcoma, Ewing's sarcoma, neoplasms with high levels of AXL expression, chronic myeloid leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, acute lymphoblastic leukemia, chronic liver disease, non-alcoholic steatohepatitis, Alzheimer's disease, or idiopathic pulmonary fibrosis.

26. The method for treating a disease or disorder according to claim 24 or 25, wherein the other therapeutically active compound is an antibody, a small molecule, a hormone therapy agent, or a combination thereof.

27. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, or a pharmaceutical composition according to any one of claims 18 to 22, for treatment of an AXL-mediated disease or disorder in a subject requiring such treatment.

28. The use of an antibody or antigen-binding fragment according to any one of claims 1 to 11, and at least one other therapeutically active compound, according to claim 27, for treatment in subjects requiring treatment of an AXL-mediated disease or disorder.

29. The aforementioned AXL-mediated disease or disorder includes non-small cell lung cancer, non-small cell lung cancer with EGFR mutations, breast cancer, HER2-positive breast cancer, four-time negative breast cancer, triple-negative breast cancer (TNBC), ovarian cancer, platinum-resistant ovarian cancer, prostate cancer, docetaxel-resistant prostate cancer, endometrial cancer and uterine sarcoma, endometrial adenocarcinoma, serous uterine carcinoma, cutaneous melanoma, neuroblastoma, glioblastoma, squamous cell carcinoma of the head and neck, gastric cancer, and renal cell carcinoma. The use according to claim 28, selected from the group consisting of urothelial carcinoma, colorectal cancer, colon cancer, hepatocellular carcinoma, pancreatic cancer, biliary tract cancer, malignant neoplasms of the extrahepatic bile duct, intrahepatic cholangiocarcinoma, malignant neoplasms of the gallbladder, osteosarcoma, Ewing's sarcoma, neoplasms with high levels of AXL expression, chronic myeloid leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, acute lymphoblastic leukemia, chronic liver disease, non-alcoholic steatohepatitis, Alzheimer's disease, or idiopathic pulmonary fibrosis.

30. The use according to claim 28, wherein the other therapeutically active compound is an antibody, a small molecule, a hormone therapy agent, or a combination thereof.

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